ALBERT

Description: Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB -deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1 -encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum , and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum . These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.

Description: Aims Afforestation causes important alterations in SOM content and composition that affect the soil functions and C balance. The aim of this study was to identify the mechanisms that determine the changes in SOM composition following afforestation of grasslands. Methods The study included 4 chronosequences and 5 paired plots comprising pastures and land afforested with Pinus radiata . The SOM was characterized by 13 C CP-MAS NMR spectroscopy and differential scanning calorimetry. Results During the first 10–20 year after afforestation, the changes in SOM content varied from slight gains to large losses (〉40 %). The analyses revealed that even SOM compounds considered resistant to decomposition were degraded during this time. The SOM gains, observed 20 year after stand establishment, were favoured by the higher recalcitrance of pine litter and possibly by soil acidification. The concentrations of most SOM compounds, particularly the stable compounds, were higher at the end of the rotation. The low degree of protection, along with the favourable climatic conditions, may also explain the rapid decomposition of SOM, including resistant compounds, in these soils. DSC analysis complemented the information about SOM composition provided by other techniques. Conclusions The accumulation of stable SOM compounds at the end of the rotation suggests a longer soil C turnover in these afforested soils, which may alleviate the gradual loss of SOC in intensively managed forest soils.

Description: Background and aims Plant nutrient uptake from coarse soil (2–4 mm diameter) has been demonstrated for only a limited number of nutrients, and the nutritional contribution of coarse soil when present with fine soil (material 〈2 mm diameter) in realistic ratios is unknown. We conducted a seedling pot trial to investigate the functional relevance of this soil fraction to plant nutrition. Methods Fine soil was mixed with either coarse soil, or the equivalent volume of inert glass chips, in ratios identical to those occurring naturally in soil sampled from two depths at each of two sites. Seedlings of Nothofagus solandri var. cliffortioides and Weinmannia racemosa were planted in the soil mixtures and harvested after 9 months. Results The content of nitrogen, phosphorous, potassium, magnesium and other elements in the above ground seedling tissue was significantly increased by the presence of coarse soil. The coarse soil fraction also contributed proportionally much more to plant nutrient uptake than fine soil on a mass per mass basis. Conclusions Coarse soil is excluded from conventional soil analysis, so is possible that soil nutrient capital is systematically underestimated. This has implications for land management and studies of plant dynamics in relation to nutrient supply.

Description: Background and aims Lateral tree-scale variability in plantations should be taken into account when scaling up from point samples, but appropriate methods for sampling and calculation have not been defined. Our aim was to define and evaluate such methods. Methods We evaluated several existing and new methods, using data for throughfall, root biomass and soil respiration in mature oil palm plantations with equilateral triangular spacing. Results Three ways of accounting for spatial variation within the repeating tree unit (a hexagon) were deduced. For visible patch patterns, patches can be delineated and sampled separately. For radial patterns, measurements can be made in radial transects or a triangular portion of the tree unit. For any type of pattern, including unknown patterns, a triangular sampling grid is appropriate. In the case studies examined, throughfall was 79 % of rainfall, with 95 % confidence limits being 62 and 96 % of rainfall. Root biomass and soil respiration, measured on a 35-point grid, varied by an order of magnitude. In zones with steep gradients in parameters, sampling density has a large influence on calculated mean values. Conclusions The methods defined here provide a basis for representative sampling and calculation procedures in studies requiring scaling up from point sampling, but more efficient methods are needed.

Description: Disruption of spatiotemporal behavior of intracellular signaling cascades including tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-mediated signaling in prostate cancer has gained tremendous attention in the past few years. There is an increasing effort in translating the emerging information about TRAIL-mediated signaling obtained through experimental and preclinical data to clinic. Fascinatingly, novel targeting approaches are being developed to enhance the tissue- or subcellular-specific delivery of drugs with considerable focus on prostate cancer. These applications have the potential to revolutionize prostate cancer therapeutic strategies and include the accumulation of drugs in target tissue as well as the selection of internalizing ligands for enhanced receptor-mediated uptake of drugs. In this mini-review, we outline outstanding developments in therapeutic strategies based on the regulation and/or targeting of TRAIL pathway for the treatment of prostate cancer. Moreover, microRNAs (miRNAs), with potential transcriptional and posttranscriptional regulation of gene expression, will be presented for their potential in prostate cancer treatment. Emphasis has been given to the use of delivery approaches, especially based on nanotechnology. Considerably, enhanced information regarding miRNA regulation of TRAIL-mediated signaling in prostate cancer cells may provide potential biomarkers for the characterization of patients as responders and nonresponders of TRAIL-based therapy and could provide rationalized basis for combination therapies with TRAIL death receptor-targeting drugs.

Description: The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase ( celA ), cellobiohydrolase ( celK ), and β-glucosidase ( gls ) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli , each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose–hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.

Description: Background Plants must acquire at least 14 mineral nutrients from the soil to complete their life cycles. Insufficient availability or extreme high levels of the nutrients significantly affect plant growth and development. Plants have evolved a series of mechanisms to adapt to unsuitable growth conditions where nutrient levels are too low or too high. microRNAs (miRNAs), a class of small RNAs, are known to mediate post-transcriptional regulation by transcript cleavage or translational inhibition. Besides regulating plant growth and development, miRNAs are well documented to regulate plant adaptation to adverse environmental conditions including nutrient stresses. Scope In this review, we focus on recent progress in our understanding of how miRNAs are involved in plant response to stresses resulting from deficiency in nutrients, such as nitrogen, phosphorus, sulfur, copper and iron, as well as toxicities from heavy metal ions. Conclusions Accumulated evidence indicates that miRNAs play critical roles in sensing the abundance of nutrients, controlling nutrient uptake and phloem-mediated long-distance transport, and nutrient homeostasis. miRNAs act as systemic signals to coordinate these physiological activities helping plants respond to and survive nutrient stresses and toxicities. Knowledge about how miRNAs are involved in plant responses to nutrient stresses promise to provide novel strategies to develop crops with improved nutrient use efficiency which could be grown in soils with either excessive or insufficient availability of nutrients.

Description: Background and aims (i) compare the concentrations of total polyphenols (TP) and condensed tannins (CT), and CT profiles in different organs of mature trees and seedlings of eight true mangrove species in Hong Kong; (ii) examine the antioxidant activities of CT and (iii) relate the non-enzymatic antioxidative defence system with the vertical zonation pattern of mangrove species. Methods Mature trees and seedlings of eight species were collected from a Hong Kong mangrove swamp to determine TP and CT concentrations and the antioxidant activities of CT. Results According to TP concentrations, the true mangrove species could be broadly classified into three groups, (i) Lumnitzera racemosa and Aegiceras corniculatum 〉 (ii) Heritiera littoralis , Excoecaria agallocha , Bruguiera gymnorrhiza and Kandelia obovata 〉 (iii) Acanthus ilicifolius and Avicennia marina . The last two are pioneer species in the most foreshore location. They also had significantly lower antioxidant activities, CT concentrations and different CT profiles than the other six species in mid- and low-tides. Conclusions Classification of the eight true mangrove species into three groups based on polyphenols was similar to their vertical zonation from land to sea. The relationships between these antioxidants and zonation should be further verified by transplantation studies.

Description: Background and aims Soil phosphorus (P) indices that have been originally developed and applied to agricultural soils for predicting P uptake by plants were examined in a pot experiment to determine the most suitable index for P availability in bauxite-processing residue sand (BRS). Methods Pot trials with ryegrass were established using BRS that had been amended with various organic (greenwaste compost, biochar and biosolids) and inorganic (zeolite) materials and different levels of di-ammonium phosphate fertiliser. Soil P availability indices tested included anion-exchange membrane (AEM-P), 0.01 M calcium chloride (CaCl 2 -P), Colwell-P, and Mehlich 3-P. Results AEM-P was found to most closely reflect the available P status in BRS across all treatments, and had the strongest associations with plant P uptake compared to Colwell-P, Mehlich 3-P and CaCl 2 -P. AEM-P was more closely correlated with P uptake by ryegrass than other P indices, while Colwell-P was closely related to leaf dry matter. Interestingly, a strong inverse relationship between plant indices and pH in BRS growth media was observed, and an adequate level of plant P uptake was found only in 15 year-old rehabilitated BRS with pH 〈 8.0. Conclusions AEM-P was found to be the most suitable index for evaluating P availability in highly alkaline BRS and pH was an important parameter affecting uptake of P by ryegrass. Importantly, time is required (〉 5 years) before improved uptake of P by plants can be observed in rehabilitated residue sand embankments.

Description: Background & aims Herbivore-driven changes to soil properties can influence the decomposition rate of organic material and therefore soil carbon cycling within grassland ecosystems. We investigated how aboveground foraging mammalian and invertebrate herbivores affect mineral soil decomposition rates and associated soil properties in two subalpine vegetation types (short-grass and tall-grass) with different grazing histories. Methods Using exclosures with differing mesh sizes, we progressively excluded large, medium and small mammals and invertebrates from the two vegetation types in the Swiss National Park (SNP). Mineral soil decomposition rates were assessed using the cotton cloth (standard substrate) method between May and September 2010. Results Decomposition displayed strong spatio-temporal variability, best explained by soil temperature. Exclusion of large mammals increased decomposition rates, but further exclusion reduced decomposition rates again in the lightly grazed (tall-grass) vegetation. No difference among treatments was found in the heavily grazed (short-grass) vegetation. Heavily grazed areas had higher decomposition rates than the lightly grazed areas because of higher soil temperatures. Microbial biomass carbon and soil C:N ratio were also linked to spatio-temporal decomposition patterns, but not to grazing history. Conclusions Despite altering some of the environmental controls of decomposition, cellulose decomposition rates in the SNP’s subalpine grasslands appear to be mostly resistant to short-term herbivore exclusion.

Description: Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments.

Description: A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH 6.5, 30 °C, 15 g l −1 ricinoleic acid, 9 mg ml −1 of enzyme, and 4 % ( v / v ) methanol. Under the optimized conditions, the enzyme produced 13.5 g l −1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % ( w / w ) and a productivity of 4.5 g l −1  h −1 . The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.

Description: Background Our recent publication (Warren et al., Plant Soil 366:683–693, 2013 ) described how pulses of deuterium oxide (D 2 O) or H 2 O combined with neutron radiography can be used to indicate root water uptake and hydraulic redistribution in maize. This technique depends on the large inherent differences in neutron cross-section between D and H atoms resulting in strong image contrast. Scope and Conclusions However, as illustrated by Carminati and Zarebanadkouki ( 2013 ) there can be a change in total water content without a change in contrast simply by a change in the relative proportions of D 2 O and H 2 O. We agree with their premise and detailed calculations (Zarebanadkouki at al. 2012 , 2013 ), and present further evidence that mixing of D 2 O and H 2 O did not confound evidence of hydraulic redistribution in our study.

Description: Background and aims The growth of green plants depends not only on photosynthesis, but also on the successful remobilization and translocation of seed phosphorus (P) reserves to the vegetative parts of the developing seedling during early growth. Remobilization and photosynthesis are therefore two parallel and co-coinciding processes involved in better seedling establishment and early growth. Methods A study was conducted to evaluate the priority of developing maize seedlings to translocate the remobilized seed P reserves and external P uptake to seedling root and shoot sinks during 4 weeks of early growth. Two fluxes of P in growing seedlings, one from seed remobilized P reserves and one from external P uptake, were distinguished by labelling external nutrient solution P with 32 P. Results The seedling phytomass was equally distributed between seedling roots and shoots for 530 cumulated degree days after sowing. Seedlings partitioned up to 71 % of P from seed reserves and up to 68 % of P acquired from the nutrient solution, to the shoots, depending on the seed P content and P concentration in the nutrient solution. It appears that accumulation of P slows down in seedling roots corresponds to the translocative functions of root P towards shoots for start of photosynthesis. Conclusions Our results suggest that the major part of seed P reserves and external P uptake were used in early development of the seedling and the preferred sink was seedling shoots.

Description: Background and aims Nickel (Ni) has become a major heavy metal contaminant. The form of nitrogen nutrition remarkably affects IRT1 expression in roots. IRT1 has an activity of transporting Ni 2+ into root cells. Therefore, nitrogen-form may affect Ni accumulation and toxicity in plants. The assumption was investigated in this study. Methods The Arabidopsis plants were treated in Ni-contained growth solutions with either nitrate (NO 3 − ) or ammonium (NH 4 + ) as the sole N source. After 7-day treatments, Ni concentration, IRT1 expression, Ni-induced toxic symptoms and oxidative stress in plants were analyzed. Results The NO 3 − -fed plants contained a higher Ni concentration, had a greater IRT1 expression in roots, and developed more severe toxic symptoms in the youngest fully expanded leaves, compared with the NH 4 + -fed plants. The Ni-induced growth inhibition was also more significant in NO 3 − -fed plants. Interestingly, Ni exposure resulted in greater hydrogen peroxide (H 2 O 2 ) and superoxide radical (O 2 . − ) accumulations, more severe lipid peroxidation and more cell death in NO 3 − -fed plants, whereas the opposite was true for NH 4 + -fed plants. Furthermore, the Ni-enhanced peroxidase (POD) and superoxide dismutase (SOD) activities were greater in NO 3 − -fed plants Conclusion NO 3 − nutrition promotes Ni uptake, and enhances Ni-induced growth inhibition and oxidative stress in plants compared with NH 4 + nutrition.

Description: Background and aims Brassica napus has high boron (B) demand, but significant genotype differences exist with respect to B deficiency. The aim of this research was to elucidate the relationship between the different sensitivities of Brassica napus cultivars to low B stress and the characteristics of B uptake and transport to characterise the regulation of B efficiency in Brassica napus . Methods B-efficient and B-inefficient Brassica napus cultivars were used to compare the uptake and transport of B using the stable isotope 10 B tracer and grafting experiments, as well as expression of B transporters by RT-PCR. Results B-efficient cultivars have significant advantages with regard to B limitation. The B-efficient cultivar HZ showed less severe B deficiency symptoms and higher dry biomass than the B-inefficient cultivars LW and LB. Both the amount of total B and the 10 B concentration and accumulation in the shoots and roots of B-efficient HZ were higher than those of B-inefficient cultivars. In B-inefficient LW, the amount of total B and the 10 B that was transported into shoots was less than in the other three cultivars and the content and accumulation of total B and 10 B in the roots of B-inefficient LB were the lowest among all of the cultivars. When the roots of B-efficient HZ were used as stocks, the grafted plants showed B-efficient characteristics, such as mild B deficiency symptoms, and higher dry biomass and B accumulation, regardless of whether they originated from B-efficient or B-inefficient cultivars. In contrast, the grafted plants with B-inefficient LW used as stocks were B-inefficient. The expressions of BnBOR1;1c , BnBOR1;2a and BnNIP5;1 were up-regulated in roots under low B stress compared with the normal B condition. However, there was no obvious difference in the expressions of the three genes or of four other BnBOR1s between B-efficient and B-inefficient cultivars in low or normal B environments. Conclusions These results indicate that the B efficiency of Brassica napus is controlled primarily by roots, which allow more uptake and accumulation of B in B-efficient cultivars than B-inefficient cultivars in a low B environment. However the molecular mechanism regulating B efficiency in Brassica napus remains to be determined.

Description: Aims Application of carbon (C) and nitrogen (N) isotopes is an essential tool to study C and N flows in plant-soil-microorganisms systems. When targeting single plants in a community the tracers need to be added via e.g., leaf-labeling or stem-feeding approaches. In this study we: (i) investigated if bicarbonate can be used to introduce 14 C (or 13 C) into white clover and ryegrass, and (ii) compared the patterns of 14 C and 15 N allocation in white clover and ryegrass to evaluate the homogeneity of tracer distribution after two alternative labeling approaches. Methods Perennial ryegrass and white clover were pulse labeled with 15 N urea via leaf-labeling and 14 C either via a 14 CO 2 atm or with 14 C bicarbonate through leaf-labeling. Plants were sampled 4 days after labeling and prepared for bulk isotope analysis and for 14 C imaging to identify plant parts with high and low 14 C activity. Subsequently, plant parts with high and low 14 C activity were separated and analyzed for 15 N enrichment. Results Bicarbonate applied by leaf-labeling efficiently introduced 14 C into both white clover and ryegrass, although the 14 C activity in particular for white clover was found predominantly in the labeled leaf. Using 14 C imaging for identification of areas with high (hotspots) and low 14 C activity showed that 14 C was incorporated very heterogeneously both when using bicarbonate and CO 2 as expected when using pulse labeling. Subsequent analysis of 15 N enrichment in plant parts with high and low 14 C activity showed that 15 N also had a heterogeneous distribution (up to two orders of magnitude). Conclusion Bicarbonate can efficiently be used to introduce 14 C or 13 C into plant via the leaf-labeling method. Both 14 C and 15 N showed heterogeneous distribution in the plant, although the distribution of 15 N was more even than that of 14 C.

Description: Background and aim In numerous areas, rice cultivated under flooded conditions is exposed simultaneously to iron excess and arsenic contamination. The impact of these combined stresses on yield-related parameters and As distribution and speciation in various plant parts remains poorly documented. Methods Rice (cv I Kong Pao) was exposed to iron excess (125 mg L −1 Fe 2 SO 4 ), arsenic (50 and 100 μM Na 2 HAsO 4 .7H 2 O) or a combination of those stressing agents in hydroponic culture until harvest. Plant growth, yield-related parameters, non protein thiols concentration and mineral nutrition were studied in roots and shoots. Arsenic speciation was determined by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. Key Results Iron excess increased As retention by the roots in relation to the development of the root iron plaque but decreased As accumulation in the shoot. Arsenic concentration was lower in the grains than in the shoots. Iron stress reduced As accumulation in the husk but not in the dehusked grains. Iron excess decreased the proportion of extractable As(III) and As(V) in the grain while it increased the proportion of extractable As(III) in the shoot. Combined stresses (Fe+As) affected plant nutrition and significantly reduced the plant yield by limiting the number of grains per plant and the grain filling. Conclusions Fe excess had an antagonist impact on shoot As concentration but an additive negative impact on several yield-related parameters. Iron stress influences both As distribution and As speciation in rice.

Description: Background and aims Intermittently frozen ground in winter is expected to disappear over large areas in the temperate zone due to ongoing climate warming. The lack of soil frost influences plant soil interactions and needs to be studied in more detail. Methods Winter soil frost was avoided by belowground heating wires in a field experiment over two subsequent winters in a temperate grassland. Soil respiration, soil nitrogen availability and plant performance (aboveground biomass, root length at two depth levels, greenness, nutrient content) were compared between “no-frost” and reference plots which underwent repeated freeze-thaw cycles in both winters. Results Soil respiration increased in the “no-frost” treatment during the warming phase (+291 %). N-availability in the upper 10 cm of the soil profile was not affected, possibly due to increased plant N accumulation during winter (+163 %), increased plant N concentration (+18 %) and increased biomass production (+31.5 %) in the growing season. Translocation of roots into deeper soil layers without changes in total root length in response to the “no-frost” treatment, however, may be a sign of nutrient leaching. Conclusions The cumulative effect on carbon cycling due to warmer soils therefore depends on the balance between increased winter carbon loss due to higher soil biotic activity and enhanced plant productivity with higher nutrient accumulation in the growing season.

Description: Aims The aims of this work were to investigate the aluminum (Al) and phosphate (P) interactions in the regulation of root system architecture of Arabidopsis thaliana seedlings and the contribution of auxin signaling in primary and lateral root growth in response to Al toxicity. Methods Detailed analyses of root system architecture and cell division were performed in Arabidopsis WT seedlings and in low phosphorus insensitive mutants lpi1 - 3 and lpr1 - 1 lpr2 - 1 in response to Al. Expression studies of P-deficiency regulated phosphate transporter AtPT2 were also conducted. The role of auxin as a mediator of root morphogenetic changes by Al was evaluated by using the auxin-signaling mutants tir1 , tir1 afb2 afb3 , and arf7 arf19 . Results Al inhibited primary root growth by affecting cell cycle progression and causing differentiation of cells in the root meristem. These effects were reduced in low phosphorus insensitive lpi1 - 3 and low phosphate resistant lpr1 - 1 lpr2 - 1 Arabidopsis mutants. Al also activated the expression of the low phosphate-induced P transporter AtPT2 in roots. Lateral root formation by Al decreased in tir1 afb2 afb3 while arf7 arf19 mutants were highly resistant to Al in both primary root inhibition and lateral root induction. Conclusions Our results suggest that lateral root formation in response to Al toxicity and P deficiency may involve common signaling mechanisms, while a pathway involving ARF7 and ARF19 is important for primary root growth inhibition by Al.

Description: Aims Litter decomposition and subsequent nutrient release play a major role in forest carbon and nutrient cycling. To elucidate how soluble or bulk nutrient ratios affect the decomposition process of beech ( Fagus sylvatica L.) litter, we conducted a microcosm experiment over an 8 week period. Specifically, we investigated leaf-litter from four Austrian forested sites, which varied in elemental composition (C:N:P ratio). Our aim was to gain a mechanistic understanding of early decomposition processes and to determine microbial community changes. Methods We measured initial litter chemistry, microbial activity in terms of respiration (CO 2 ), litter mass loss, microbial biomass C and N (C mic and N mic ), non purgeable organic carbon (NPOC), total dissolved nitrogen (TDN), NH 4 + , NO 3 - and microbial community composition (phospholipid fatty acids – PLFAs). Results At the beginning of the experiment microbial biomass increased and pools of inorganic nitrogen (N) decreased, followed by an increase in fungal PLFAs. Sites higher in NPOC:TDN (C:N of non purgeable organic C and total dissolved N), K and Mn showed higher respiration. Conclusions The C:N ratio of the dissolved pool, rather than the quantity of N, was the major driver of decomposition rates. We saw dynamic changes in the microbial community from the beginning through the termination of the experiment.

Description: Background and aims Grazing may influence nutrient cycling in several ways. In productive mountain grasslands of central Argentina cattle grazing maintain a mosaic of different vegetation patches: lawns, grazed intensively and dominated by high quality palatable plants, and open and closed tussock grasslands dominated by less palatable species. We investigated if differences in the resources deposited on soil (litter and faeces) were associated with litter decomposition rates and soil nitrogen (N) availability across these vegetation patches. Methods We compared the three vegetation patches in terms of litter and faeces quality and decomposability, annual litterfall and faeces deposition rate. We determined decomposition rates of litter and faces in situ and decomposability of the same substrates in a common garden using “litter bags”. We determined soil N availability (with resin bags) in the vegetation patches. Also, we performed a common plant substrates decomposition experiment to assess the effect of soil environment on decomposition process. This technique provides important insights about the soil environmental controls of decomposition (i.e. the sum of soil physicochemical and biological properties, and microclimate), excluding the substrate quality. Results The litter quality and faeces deposition rate were higher in grazing lawns, but the total amounts of carbon (C) and nitrogen (N) deposited on soil were higher in tussock grasslands, due to higher litterfall in these patches. The in situ decomposition rates of litter and faeces, and of the two common plant substrates were not clearly related to either grazing pressure, litterfall or litter quality (C, N, P, lignin, cellulose or hemicellulose content). In situ litter decomposition rate and soil ammonium availability were correlated with the decomposition rates of both common plant substrates. This may suggest that difference in local soil environment among patch types is a stronger driver of decomposition rate than quality or quantity of the resource that enter the soil. Conclusions Our results show that, although high grazing pressure improves litter quality and increases faeces input, the reduction in biomass caused by herbivores greatly reduces C and N input for the litter decomposition pathway. We did not find an accelerated decomposition rate in grazing lawns as proposed by general models. Our results point to soil environment as a potential important control that could mask the effect of litter quality on field decomposition rates at local scale.

Description: Background and aims Cd uptake has been shown to increase during conditions of Fe deficiency. This study tested the hypothesis that Fe-deficiency-responsive genes, particularly OsNRAMP1, play a role in the increased Cd uptake that occurs when rice is grown in aerobic soil conditions. Methods Plants were grown in aerobic or flooded soil conditions. Uptake of Cd was compared to levels of expression of candidate metal transporters and to metal ion availability in soil. Results Plants grown with intermittent soil flooding experienced a predominantly aerobic root environment and had the highest plant Cd uptake. Stronger upregulation of OsNRAMP1 was detected in plants grown in unflooded soil than in flooded soil. However, these transcriptional responses were not linked to an increase in Cd uptake. Overexpression of OsNRAMP1 was not found to increase the uptake of Cd in rice in soil or solution culture. In contrast, there were large differences in availability of Cd, Fe and Mn between flooded and aerobic soils, which were linked to changes in Cd uptake. Conclusions Aerobic soil conditions favour Cd uptake through increased Cd availability and decreased competition between Cd and Fe rather than through the increased expression of the Fe transporters themselves.

Description: Background and aims Intensive land use has led to degradation and abandonment of Portuguese oak woodlands, and subsequent shrub encroachment may have altered the spatial heterogeneity of soil C and N pools. The aim of this study was to evaluate the effects of shrub invasion on soil C and N dynamics in an oak woodland in Southern Portugal. Methods Soil was sampled beneath and outside scattered Quercus suber L. canopies, considering non-encroached areas and areas encroached by shrubs ( Cistus ladanifer L. or Cistus salviifolius L.). Results The spatial heterogeneity of soil C and N contents was mainly associated with tree presence. Outside tree canopies, the labile C pools were larger (mainly beneath C . ladanifer ) and C cycling was faster in encroached areas than in non-encroached areas. Net and gross N mineralization and urease and protease activities were also higher in encroached than in non-encroached areas; however, the metabolic quotient and the Cmicrobial/Corganic ratio were not significantly affected. Beneath the tree canopy, significant effects of encroachment included a small increase in soil labile C and the enzymatic activity beneath C . ladanifer . Conclusions The results indicate the potential capacity of shrub encroachment to accumulate soil organic C in the long term. The rate of soil C and N turnover promoted by shrub encroachment may depend on the Cistus species present.

Description: Aims Decreased expression of TaNAM genes by RNAi results in delayed senescence and decreased grain protein, iron, and zinc concentrations. Here, we determined whether NAM expression level alters onset of senescence under stress conditions, whether delayed senescence in the TaNAM -RNAi line resulted in improved tolerance to post-anthesis abiotic stress, and determined the effects of post-anthesis abiotic stress on N and mineral remobilization and partitioning to grain. Methods Greenhouse-grown WT and TaNAM -RNAi wheat were characterized in two studies:three levels of N fertility or water limitation during grain fill. Studies were conducted under both optimal and heat stress temperatures. Senescence onset was determined by monitoring flag leaf chlorophyll. Results Under optimal tempertures, TaNAM -RNAi plants had a yield advantage at lower N. TaNAM -RNAi plants had delayed senescence relative to the WT and lower grain protein and mineral concentrations, N remobilization efficiency, and partitioning of N and most minerals to grain. Conclusions Nutritional quality of TaNAM -RNAi grain was consistently lower than WT. Delayed senescence of TaNAM -RNAi plants provided a yield advantage under optimal temperatures but not under water or heat stress. Discovery of specific NAM protein targets may allow separation of the delayed senescence and nutrient partitioning traits, which could be used for improvement of wheat.

Description: Aims Nitrification inhibitors (NI) formulated on granulated ammonium sulphate nitrate (ASN) are an option to minimize nitrate leaching into ground waters and emissions of the greenhouse gas N 2 O. This paper focuses (a) on the development of an analytic enabling to extract and quantify the NI 3,4-dimethylpyrazolephosphate (DMPP), marketed since 1999. The efficiency of DMPP has been studied in laboratory and field soils. Here the DMPP analytic and the behaviour of a nitrifying bacterial consortium enriched from a field soil and exposed to zero, field applied and a 10 fold higher DMPP concentration than the recommended one for field application are in the focus. Methods For extracting DMPP quantitatively from soils a method connected to a HPLC analytic has been developed by us and was standardized in laboratory experiment with a silt clay field soil (allochtone Vega). The method is detailed described here. Its reliability has been tested in a 3 years field trial under varying cropping systems and climatic conditions asides the influence of DMPP on CO 2 −, CH 4 − and N 2 O- emissions, measured by the closed chamber method. Parallel a nitrifying bacterial consortium of the silty clay field soil was enriched and subjected to 0, the recommended DMPP concentration for field applications and a 10 times higher one. In incubation experiments the conversion of ammonium to nitrite and nitrate in presence and absence of DMPP was spectrophotometer determined and pH-shifts with a scaled litmus paper. In sacrificed flasks at the end of incubation morphological changes of the bacteria involved were studied by transmission electron microscope (TEM). Results The ammonium, nitrite and nitrate determinations and the TEM pictures show that in presence of the field applied DMPP concentration the nitrifying activity returned around 30 days later than in the control and the cells were slightly enlarged. In presence of a 10 times higher DMPP concentration a recovery was prevented. DMPP prolongs, compared with dicyandiamide (DCD), the period of nitrifiers’ inhibition and reduced N 2 O− and CO 2 − the emissions (Weiske et al., Biol Fertil Soils 34:109–117, 2001a , Nutr Cycl Agroecosys 60:57–64, b ). Conclusions With the method developed by us the stability of DMPP in agricultural soils can be satisfyingly and reproducible studied down to a detection limit of 0.01 μg DMPP g −1 dry soil. The morphological changes in the nitrifying consortium due to DMPP concentrations are in agreement with the recovery rate found by nitrite and nitrate formation.

Description: Background and aims Adequate zinc (Zn) in maize ( Zea mays L.) is required for obtaining Zn-enriched grain and optimum yield. This study investigated the impact of varying Zn fertilizer placements on Zn accumulation in maize plant. Methods Two pot experiments with same design were conducted to investigate the effect of soil Zn heterogeneity by mixing ZnSO 4 ·7H 2 O (10 mg Zn kg −1 soil on an average) in 10–15, 0–15, 25–30, 0–30, 30–60 and 0–60 cm soil layers on maize root growth and shoot Zn content at flowering stage in experiment-1, and assessing effects on grain Zn accumulation at mature stage in experiment-2. Results In experiment-1, Zn placements created a large variation in soil DTPA-Zn concentration (0.3–29.0 mg kg −1 ), which induced a systemic and positive response of root growth within soil layers of 0–30 cm; and shoot Zn content was increased by 102 %–305 % depending on Zn placements. Supply capacity of Zn in soil, defined as sum of product of soil DTPA-Zn concentration and root surface area at different soil layers, was most related to shoot Zn content ( r  = 0.82, P  〈 0.001) via direct and indirect effects according to path analysis. In experiment-2, Zn placements increased grain Zn concentration by up to 51 %, but significantly reduced the grain Zn harvest index from 50 % by control to about 30 % in average. Conclusion Matching the distribution of soil applied Zn with root by Zn placement was helpful to maximize shoot Zn content and grain Zn concentration in maize.

Description: Background and Aims Wetting-drying cycles are important environmental processes known to enhance aggregation. However, very little attention has been given to drying as a process that transports mucilage to inter-particle contacts where it is deposited and serves as binding glue. The objective of this study was to formulate and test conceptual and mathematical models that describe the role of drying in soil aggregation through transportation and deposition of binding agents. Methods We used an ESEM to visualize aggregate formation of pair of glass beads. To test our model, we subjected three different sizes of sand to multiple wetting-drying cycles of PGA solution as a mimic of root exudates to form artificial aggregates. Water stable aggregate was determined using wet sieving apparatus. Results A model to predict aggregate stability in presence of organic matter was developed, where aggregate stability depends on soil texture as well as the strength, density and mass fraction of organic matter, which was confirmed experimentally. The ESEM images emphasize the role of wetting-drying cycles on soil aggregate formation. Conclusions Our experimental results confirmed the mathematical model predictions as well as the ESEM images on the role of drying in soil aggregation as an agent for transport and deposition of binding agents.

Description: Background and aims Many plant growth-promoting endophytes (PGPE) possessing 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity can reduce the level of stress ethylene and assist their host plants cope with various biotic and abiotic stresses. However, information about the endophytic bacteria colonizing in the coastal halophytes is still very scarce. This study aims at isolating efficient ACC deaminase-producing plant growth-promoting (PGP) bacterial strains from the inner tissues of a traditional Chinese folk medicine Limonium sinense (Girard) Kuntze, a halophyte which has high economic and medicinal values grown in the coastal saline soils. Their PGP activity and effects on host seed germination and seedling growth under salinity stress were also evaluated. Methods A total of 126 isolates were obtained from the surface sterilized roots, stems and leaves of L. sinense (Girard) Kuntze. They were initially selected for their ability to produce ACC deaminase as well as other PGP properties such as production of indole-3-acetic acid (IAA), N 2 -fixation, and phosphate-solubilizing activities and subsequently identified by the 16S rRNA gene sequencing. For selected strains, seed germination, seedling growth, and flavonoids production in axenically growth L. sinense (Girard) Kuntze seedlings at different NaCl concentrations (0–500 mM) were quantified. Results Thirteen isolates possessing ACC deaminase activity were obtained. The 16S rRNA gene sequencing analysis showed them to belong to eight genera: Bacillus , Pseudomonas , Klebsiella , Serratia , Arthrobacter , Streptomyces , Isoptericola , and Microbacterium . Inoculation with four of the selected ACC deaminase-producing strains not only stimulated the growth of the host plant but also influenced the flavonoids accumulation. All four strains could colonize and can be re-isolated from the host plant interior tissues. Conclusions These results demonstrate that ACC deaminase-producing habitat-adapted symbiotic bacteria isolated from halophyte could enhance plant growth under saline stress conditions and the PGPE strains could be appropriate as bioinoculants to enhance soil fertility and protect the plants against salt stress.

Description: Bacillus amyloliquefaciens FZB42 has been shown to stimulate plant growth and to suppress the growth of plant pathogenic organisms including nematodes. However, the mechanism underlying its effect against nematodes remains unknown. In this study, we screened a random mutant library of B. amyloliquefaciens FZB42 generated by the mariner transposon Tn YLB-1 and identified a mutant strain F5 with attenuated nematicidal activity. Reversible polymerase chain reaction revealed that three candidate genes RAMB_007470 , yhdY , and prkA that were disrupted by the transposon in strain F5 potentially contributed to its decreased nematicidal activity. Bioassay of mutants impaired in the three candidate genes demonstrated that directed deletion of gene RBAM_007470 resulted in loss of nematicidal activity comparable with that of the F5 triple mutant. RBAM_007470 has been reported as being involved in biosynthesis of plantazolicin, a thiazole/oxazole-modified microcin with hitherto unknown function. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) analyses of surface extracts revealed that plantazolicin bearing a molecular weight of 1,354 Da was present in wild-type B. amyloliquefaciens FZB42, but absent in the Δ RABM_007470 mutant. Furthermore, bioassay of the organic extract containing plantazolicin also showed a moderate nematicidal activity. We conclude that a novel gene RBAM_007470 and its related metabolite are involved in the antagonistic effect exerted by B. amyloliquefaciens FZB42 against nematodes.

Description: Aims The objective of this study was to investigate the effects of future warming and drought on (1) the biochemical composition of above-ground biomass of forage plants ( Festuca arundinacea and Dactylis glomerata ), (2) the potential mineralization of this material in soil, and (3) its priming effect on native soil organic matter. Methods We sampled above-ground plant material from spring regrowth and summer regrowth of a climate change experiment. While in spring, the plants were well watered, the summer regrowth was exposed to drought and elevated temperature (+3 °C) by infrared heating of the canopy during 3 weeks. We assessed the elemental and isotopic composition, lignin and non-cellulosic carbohydrate content and composition of plant material grown under all three conditions. Its mineralization potential in soil and priming effects were evaluated during laboratory incubation. Results Warming had no significant effect on elemental and stable isotope composition of both plant materials. In contrast, it resulted in reduction of lignin content for both plant species and decrease of the lignin-to-N ratio for F. arundinacea and increased non-cellulosic carbohydrate content for D. glomerata . Summer regrowth was characterised by increase of δ 13 C values, which is consistent with variations in stomatal conductance due to water shortage. Moreover, summer drought induced an increase in N content leading to decrease of the C/N ratio and increase of lignin-to-N ratio of summer regrowth compared to spring regrowth. Differences in decomposition were small, while priming effects were more strongly altered by the different exposure to enviromental. Conclusion Our results provide direct experimental evidence that extreme climatic events (high temperature and precipitation deficit) have an influence on soil carbon storage particularly through their effect on priming of native soil organic matter induced by altered plant litter. These effects seem to be governed by alterations of stoichiometry and to a smaller extent by alterations of plant chemical composition.

Description: Syngas fermentation is a promising route for resource recovery. Acetate is an important industrial chemical product and also an attractive precursor for liquid biofuels production. This study demonstrated high fraction acetate production from syngas (H 2 and CO 2 ) in a hollow-fiber membrane biofilm reactor, in which the hydrogen utilizing efficiency reached 100 % during the operational period. The maximum concentration of acetate in batch mode was 12.5 g/L, while the acetate concentration in continuous mode with a hydraulic retention time of 9 days was 3.6 ± 0.1 g/L. Since butyrate concentration was rather low and below 0.1 g/L, the acetate fraction was higher than 99 % in both batch and continuous modes. Microbial community analysis showed that the biofilm was dominated by Clostridium spp., such as Clostridium ljungdahlii and Clostridium drakei , the percentage of which was 70.5 %. This study demonstrates a potential technology for the in situ utilization of syngas and valuable chemical production.

Description: Lignosulfonates(LSs), by-products from chemical pulping processes, are low-value products with limited dispersion properties. The ability of commercially available horseradish peroxidase (HRP) to polymerize LS macromolecules and improve the dispersion properties of LSs was investigated. The polymerization of LSs proceeded efficiently under mild reaction conditions in an aqueous solution with HRP/H 2 O 2 . Gel permeation chromatography showed a significant increase in weight-average molecular weight ( M w ) of sulfonated kraft lignin and sodium lignosulfonate (NaLS) by 8.5-fold and 4.7-fold, respectively. The mechanism of polymerization was investigated by elemental analysis, surface charge measurement, headspace gas chromatography, infrared spectroscopy (IR), and hydrogen nuclear magnetic resonance spectrometry ( 1 H-NMR). The functional group measurements indicated that HRP incubation did not reduce the sulfonic group content. However, it decreased the phenolic and methoxyl group contents. As the phenolic group content decreased, M w increased as a power function. The polymerization was proposed to involve the random coupling of phenoxy radical intermediates. The radicals coupled with each other to form different inter-unit linkages, most of which were the β-O-4’ type, as the 1 H-NMR spectra indicated. Moreover, the HRP/H 2 O 2 incubation induced a significant improvement in the adsorption and dispersion properties of LSs. Therefore, the HRP/H 2 O 2 incubation is a promising approach for industrial applications of LSs.

Description: Marine microalga Nannochloropsis oculata possesses nutrients valuable for human health. In this study, we added freeze-dried N. oculata powder to soybean oil and observed a remarkable inhibition in oil oxidation. The amount of microalgae powder added was positively correlated to the increase in oil stability. The addition of 5.0 % ( w / w ) microalgae powder increased the oil stability index (OSI) values of soybean oil more than twofold at the tested temperatures 120 and 130 °C. N . oculata contains high levels of both phenolic compounds and α-tocopherols that could be the contributors to such an increase of the OSI. Two methods were conducted to assay the active ingredients released from microalgae: one employed three solvent systems to extract the microalgae and the other was the soybean oil added with microalgae. Analyses of free radical scavenging and reducing power suggested that the phenolic compounds dominated the antioxidation activities in soybean oil when it was infused with the microalgae powder. Our results suggest that N. oculata could potentially be used as an additive in cooking oil to increase the shelf life and nutritional value of the oil and to reduce the production of free radicals from lipid oxidation when the oil is used at high-temperature cooking processes.

Description: The actinomycete Streptomyces platensis produces two compounds that display antibacterial activity: platensimycin and platencin. These compounds were discovered by the Merck Research Laboratories, and a complex insoluble production medium was reported. We have used this medium as our starting point in our studies. In a previous study, we developed a semi-defined production medium, i.e., PM5. In the present studies, by varying the concentration of the components of PM5, we were able to develop a superior semi-defined medium, i.e., PM6, which contains a higher concentration of lactose. Versions of PM6, containing lower concentrations of all components, were also found to be superior to PM5. The new semi-defined production media contain dextrin, lactose, MOPS buffer, and ammonium sulfate in different concentrations. We determined antibiotic production capabilities using agar diffusion assays and chemical assays via thin-layer silica chromatography and high-performance liquid chromatography. We reduced crude nutrient carryover from the seed medium by washing the cells with distilled water. Using these semi-defined media, we determined that addition of the semi-defined component soluble starch stimulated antibiotic production and that it and dextrin could both be replaced with glucose, resulting in the chemically defined medium, PM7.

Description: Pichia pastoris is widely used as a host system for heterologous protein expression in both academia and industry. Production is typically accomplished by a fed-batch induction process that is known to have negative impacts on cell physiology that impose limits on both protein yields and quality. We have analysed recombinant protein production in chemostat cultures to understand the physiological responses associated with methanol-induced production of two human lysozyme variants with different degrees of misfolding by P . pastoris . Confounding variables associated with nutrient stress or growth-rate are minimised during steady-state growth in chemostats. Comparison of transcriptome-level data obtained during the non-inducing and inducing steady states identified changes in expression of only about 1 % of the genome during production of either an amyloidogenic human lysozyme variant prone to intracellular aggregation (I56T) or a misfolded but secretable variant (T70N), indicating near-complete acclimation to their production. A marked, but temporary, stress response involving both the unfolded protein response (UPR) and ER-associated degradation pathway was observed during the transient between steady states, particularly following induction of the T70N variant synthesis, and was accompanied by changes in expression of around 50 antisense transcripts. The results suggest that optimal heterologous protein production could best be achieved by a continuous process that minimises the number of methanol-induced transients experienced by the cultures. The processing of HAC1 mRNA required for the UPR was found to be constitutive in the culture conditions used, even in the absence of recombinant protein induction.

Description: The Aminobacter sp. strain MSH1 has potential for pesticide bioremediation because it degrades the herbicide metabolite 2,6-dichlorobenzamide (BAM). Production of the BAM-degrading bacterium using aerobic bioreactor fermentation was investigated. A mineral salt medium limited for carbon and with an element composition similar to the strain was generated. The optimal pH and temperature for strain growth were determined using shaker flasks and verified in bioreactors. Glucose, fructose, and glycerol were suitable carbon sources for MSH1 ( μ  = 0.1 h −1 ); slower growth was observed on succinate and acetic acid ( μ  = 0.01 h −1 ). Standard conditions for growth of the MSH1 strain were defined at pH 7 and 25 °C, with glucose as the carbon source. In bioreactors (1 and 5 L), the specific growth rate of MSH1 increased from μ  = 0.1 h −1 on traditional mineral salt medium to μ  = 0.18 h −1 on the optimized mineral salt medium. The biomass yield under standard conditions was 0.47 g dry weight biomass/g glucose consumed. An investigation of the catabolic capacity of MSH1 cells harvested in exponential and stationary growth phases showed a degradation activity per cell of about 3 × 10 −9  μg BAM h −1 . Thus, fast, efficient, large-scale production of herbicide-degrading Aminobacter was possible, bringing the use of this bacterium in bioaugmentation field remediation closer to reality.

Description: Background and aim Saccharothrix algeriensis NRRL B-24137, isolated from a Saharan soil, has been described as a potential biocontrol agent against Botrytis cinerea and other phytopathogens. However, the plant protection mechanisms involved still need to be described. The aim of this study was to determine this protection phenomenon as well as parts of the mechanisms involved, using Arabidopsis thaliana seedlings and B. cinerea . Methods The bacterial colonization process was evaluated on A. thaliana seedlings using fluorescence in situ hybridization. Protection of A. thaliana seedlings inoculated with NRRL B-24137 against B. cinerea was then evaluated. Parts of the mechanisms involved in the systemic protection against B. cinerea were evaluated using known mutants of genes involved in jasmonate (JA)/ethylene (ET)/salicylic acid (SA) signaling. Other Arabidopsis mutants, AtrhbohD-3 , AtrhbohF-3 , and ups1-1 were also screened to determine other parts of the mechanisms involved. Results The results showed that the strain NRRL B-24137 colonized, epi- and endophytically, the roots of Arabidopsis seedlings but the strain was not a systemic colonizer during the time of the experiment. The strain NRRL B-24137 also reduced B. cinerea symptoms and the protection was linked to known mechanisms of induced systemic resistance (ISR; JA/ET signaling), as well as to functionality of AtrbohF oxidase and of UPS1. Crosstalk between ET/JA and SA signaling could also be involved. Conclusions The isolate NRRL B-24137, after colonizing the root systems of A. thaliana , induces an ISR against B. cinerea , which is JA/ET dependent, but could also require SA crosstalk and protection could also require NAPDH oxidases and UPS1 functionalities.

Description: Soy sauce is a traditional condiment manufactured by natural inoculation and mixed culture fermentation. As is well known, it is the microbial community that plays an important role in the formation of its flavors. However, to date, its dynamic changes during the long period of fermentation process are still unclear, intensively constraining the improvement and control of the soy sauce quality. In this work, we revealed the dynamic changes of the microbial community by combining a cultured dependent method and a cultured independent method of polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis. Results indicated that the two methods verified and complemented each other in profiling microbial community, and that significant dynamics of the microbial community existed during the fermentation process, especially the strong inhibition of the growth of most of the microbes when entering into the mash stage from the koji stage. In the analysis of bacterial community, Staphylococcus and Bacillus were found to be the dominant bacteria and detected in the whole fermentation process. Kurthia and Klebsiella began to appear in the koji stage and then fade away in the early stage of the mash fermentation. In the analysis of fungal community, Aspergillus sojae and Zygosaccharomyces rouxii were found to be the dominant fungi in the koji and mash fermentation, respectively. It was clearly shown that when A. sojae decreased and disappeared in the middle stage of the mash fermentation, Z. rouxii appeared and increased at the meantime. Aspergillus parasiticus , Trichosporon ovoides and Trichosporon asahii also appeared in the koji and the early period of the mash fermentation and disappeared thereafter. Similar to Z. rouxii , Millerozyma farinosa and Peronospora farinosa were also found spontaneously which appeared in the mid-late period of the mash fermentation. The principal component analysis suggested that the microbial community underwent significant changes in the early period of the fermentation and, thereafter, tended to the stabilization in the mid-late periods. This study gave us important clues to understand the fermentation process and can serve as a foundation for improving the quality of soy sauce in the future.

Description: Background and aims The effect of forest cover distribution and plant litter input on soil organic carbon were analyzed to better understand the dynamics of carbon cycling across ecosystems on the “Natural Oriented Reserve Bosco delle Pianelle”. Fluorescence spectroscopy represents a very useful tool to characterize soil organic matter properties, since it allows to directly monitor the molecular status of a fluorophore depending on its chemical environment, as well as on its structure, substituents of the aromatic moieties, and molecular weight. Here, fluorescence analysis was performed on humic acids isolated from four litters (HALs) and their underlying soils (HAs) at three depths. Methods All samples were collected from a protected forest area, Southern Italy, under different plant covering: Quercus ilex L. (Q), mixed Carpinus betulus L. and Carpinus orientalis Mill. (CC), Pinus halepensis L. (P), and mixed Quercus trojana Webb. and Quercus ilex L. (QQ). Results Data obtained showed a fast decomposition process for P and QQ litters, with HAs in the underlying soils characterized by the presence of simple, highly fluorescent structural components also in the deepest layers. On the contrary, a slow decomposition process was observed for Q and CC litters, whose underlying soil HAs were characterized by an increasing aromatic polycondensation and humification degree from the surface to the deepest layers, as supported by low values of fluorescence intensity and high wavelength maxima. Conclusions Results obtained indicate that P and QQ species promote C accumulation and stock in the underlying soils, thanks to a greater decomposition of their litter, and fluorescence spectroscopy is a very simple and suitable method to evaluate the influence of three species distribution on soil organic carbon pools.

Description: Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger . All enzymes were active toward p -nitrophenyl-β- d -galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N -acetylglucosamine or fucose, such as N -glycans derived from bovine fibrin and Caenorhabditis elegans . In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae , all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.

Description: A Lolium perenne ice-binding protein ( Lp IBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a “green” gas hydrate inhibitor. Recombinant production of Lp IBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized Lp IBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii . Both mixotrophic and photoautotrophic growth regimes supported Lp IBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant Lp IBP into a model gas hydrate offers the promise that algal production may eventually find application as a “green” hydrate inhibitor.

Description: The interest in biofertilizers is increasing and so is the potential for their use in sustainable agriculture. However, many of the products that are currently available worldwide are often of very poor quality, resulting in the loss of confidence from farmers. The formulation of an inoculant is a crucial multistep process that should result in one or several strains of microorganisms included in a suitable carrier, providing a safe environment to protect them from the often harsh conditions during storage and ensuring survival and establishment after introduction into soils. One of the key issues in formulation development and production is the quality control of the products, at each stage of the process. This review presents the different components and the major steps involved in the formulation of good quality biofertilizers, including the techniques used to assess the quality of the products following production. The quality of currently available inoculants is also reviewed, emphasizing the need for better quality control systems worldwide.

Description: Aims The objective of our study was to confirm if hydraulic lift (HL) promotes nutrient uptake in field-grown plants in the same way as demonstrated previously in pot-grown plants. Methods We conducted a field experiment in an agroforestry system, over an entire growing season that included a dry period and a wet period. We used a shallow-root crop plant, mung bean ( Vigna radiata L.), intercropped with walnut ( Juglans regia L.) and jujube ( Zizyphus jujube Mill.), as an indicator for the presence of HL and its effects on nutrient uptake. To monitor HL, we artificially applied deuterium isotope to the deep roots of trees. Results We demonstrated the presence of a natural nitrogen, phosphorus, and potassium gradient along the soil depth, and the occurrence of HL, evidenced by deuterium signature in the shallow soil layers and V. radiata stem, only during the dry season. J. regia and Z. jujube both had deep root systems, but the former species exhibited stronger HL to the shallow soil than the latter. Meanwhile, the upper soil layers of J. regia had significantly higher moisture content, and the intercropped V. radiata had higher nutrient content. Conclusion HL can facilitate water uptake by V. radiata from the upper soil layers in the field condition during the dry season, which relates to nutrient acquisition by the crop.

Description: A large number of molds serve as producer strains for the industrial production of pharmaceuticals, foods, or organic chemicals. To optimize strains for production processes, conventional strain development programs use random mutagenesis and, more recently, recombinant technologies to generate microbial strains with novel and advantageous properties. The recent detection of mating type genes in fungal production strains and the discovery of cryptic sexuality in presumably asexual fungi open up novel strategies for generating progeny with new, as yet unobserved properties. Mating type genes, which can be considered as “sex genes,” not only direct sexual development but also regulate a broad range of fungal secondary metabolites. In addition, they control hyphal morphology, which has a direct impact on production processes that are often conducted in huge fermenter tanks. Here, we survey the occurrence and function of mating type genes that have been discovered in a wide range of industrial fungal producer strains. The possibility to obtain progeny from industrial producers by sexual mating provides an exciting alternative to conventional strain improvement programs aiming to generate optimized recombinant production strains.

Description: Endo-1,4-β-xylanases (EC 3.2.1.8) hydrolyze the 1,4-β-D-xylosidic linkages in xylans, the most abundant hemicellulose in plant cell walls. Xylanase enzymes have numerous industrial applications, including the manufacturing of animal feed, bread, juice and wine, pulp and paper, and biofuels. In this study, two glycosyl hydrolase family 10 members designated Gt Xyn10A and Gt Xyn10B and two glycosyl hydrolase family 11 members, Op Xyn11A and Cc Xyn11C, were functionally expressed and subjected to biochemical characterization. The K M , V max , and k cat values of the four xylanases, determined using birchwood xylan, ranged from 0.27 to 1.1 mg/mL, 130 to 980 μmol/min/mg, and 109 to 344 s −1 , respectively, where Op Xyn11A gave the highest and Gt Xyn10B the lowest values for all three parameters. Substrate specificity studies and analysis of the products released during the degradation of xylo-oligosaccharides and three types of xylan revealed significant differences in catalytic properties, particularly between Op Xyn11A and the other xylanases and between the family 10 and the family 11 xylanases. Molecular modeling suggests that the unique substrate specificity of Op Xyn11A can be attributed to the presence of a serine rather that an asparagine or aspartate residue at the +1 substrate binding site. Additionally, all four xylanases exhibited biochemical characteristics of interest for various commercial applications.

Description: Hand, foot, and mouth disease (HFMD) has caused significant morbidity and mortality in the Asia-Pacific regions, particularly in infants and young children. Coxsackievirus A16 (CA16) represents one of the major causative agents for HFMD, and the development of a safe and effective vaccine preventing CA16 infections has become a public health priority. In this study, we have developed a yeast system for the production of virus-like particles (VLPs) for CA16 by co-expressing P1 and 3CD of CA16 in Saccharomyces cerevisiae . These VLPs exhibit similarity in both protein composition and morphology as empty particles from CA16-infected cells. Immunization with CA16 VLPs in mice potently induced CA16-specific IgG and neutralization antibodies in a dose-dependent manner. IgG subclass isotyping revealed that IgG1 and lgG2b were dominantly induced by VLPs. Meanwhile, cytokine profiling demonstrated that immunization with VLPs significantly induced the secretion of IFN-γ, indicating potent cellular immune response. Furthermore, in vivo challenge experiments showed that passive immunization with anti-VLPs sera conferred full protection against lethal CA16 challenge in neonate mice. Taken together, our data demonstrated that VLPs produced in yeast might have the potential to be further developed as a vaccine candidate against HFMD.

Description: Studies were carried out to assess the utility of the cellular and extracellular constituents of Bacillus megaterium for the flotation of sphalerite and galena minerals. Based on the flotation results on the individual minerals, it was observed that sphalerite was preferentially floated compared to galena. A maximum selectivity index (SI) value of 11.7 was achieved in the presence of the soluble fraction of the thermolysed cells, which was higher than that obtained with the intact cells (SI of 6.5) and the insoluble fraction of the thermolysed cells (SI of 9.6). The results of the various enzymatic treatment tests revealed that extracellular DNA played a vital role in the selective flotation of sphalerite. A noteworthy finding was that the single-stranded DNA (ssDNA) had a higher biocollector capacity vis-à-vis the double-stranded DNA (dsDNA), leading to better flotation efficiency. About 95 % recovery of sphalerite could be achieved from the mineral mixture by the combined addition of the ssDNA with the non-DNA components of the bacterial cells, resulting in a maximum SI of 19.1. Calcium and phosphate components of the nutrient media were found to be essential for better selectivity of separation of sphalerite. The mechanisms of microbe–mineral interaction are discussed.

Description: While the use of anaerobic digestion to generate methane as a source of bioenergy is increasing worldwide, our knowledge of the microbial communities that perform biomethanation is very limited. Using next-generation sequencing, bacterial population profiles were determined in three full-scale mesophilic anaerobic digesters operated on dairy farms in the state of Vermont (USA). To our knowledge, this is the first report of a metagenomic analysis on the bacterial population of anaerobic digesters using dairy manure as their main substrate. A total of 20,366 non-chimeric sequence reads, covering the V1-V2 hypervariable regions of the bacterial 16S rRNA gene, were assigned to 2,176 operational taxonomic units (OTUs) at a genetic distance cutoff value of 5 %. Based on their limited sequence identity to validly characterized species, the majority of OTUs identified in our study likely represented novel bacterial species. Using a naïve Bayesian classifier, 1,624 anaerobic digester OTUs could be assigned to 16 bacterial phyla, while 552 OTUs could not be classified and may belong to novel bacterial taxonomic groups that have yet to be described. Firmicutes , Bacteroidetes , and Chloroflexi were the most highly represented bacteria overall, with Bacteroidetes and Chloroflexi showing the least and the most variation in abundance between digesters, respectively. All digesters shared 132 OTUs, which as a “core” group represented 65.4 to 70.6 % of sequences in individual digesters. Our results show that bacterial populations from microbial communities of anaerobic manure digesters can display high levels of diversity despite sharing a common core substrate.

Description: The discovery of Dehalococcoides mccartyi reducing perchloroethene and trichloroethene (TCE) to ethene was a key landmark for bioremediation applications at contaminated sites. D. mccartyi -containing cultures are typically grown in batch-fed reactors. On the other hand, continuous cultivation of these microorganisms has been described only at long hydraulic retention times (HRTs). We report the cultivation of a representative D. mccartyi -containing culture in continuous stirred-tank reactors (CSTRs) at a short, 3-d HRT, using TCE as the electron acceptor. We successfully operated 3-d HRT CSTRs for up to 120 days and observed sustained dechlorination of TCE at influent concentrations of 1 and 2 mM TCE to ≥97 % ethene, coupled to the production of 10 12 D. mccartyi cells L culture −1 . These outcomes were possible in part by using a medium with low bicarbonate concentrations (5 mM) to minimize the excessive proliferation of microorganisms that use bicarbonate as an electron acceptor and compete with D. mccartyi for H 2 . The maximum conversion rates for the CSTR-produced culture were 0.13 ± 0.016, 0.06 ± 0.018, and 0.02 ± 0.007 mmol Cl − L culture −1 h −1 , respectively, for TCE, cis -dichloroethene, and vinyl chloride. The CSTR operation described here provides the fastest laboratory cultivation rate of high-cell density Dehalococcoides cultures reported in the literature to date. This cultivation method provides a fundamental scientific platform for potential future operations of such a system at larger scales.

Description: Aims and background Despite increasing knowledge of the role of allelochemicals in the productivity decline of replanted Chinese fir plantations, relatively little is known about the levels and sources of allelochemicals in relation to autoinhibition. Methods Allelopathic potential of litter, root exudates, and soils in successive rotations of Chinese fir plantations were detected. An allelochemical cyclic dipeptide (6-hydroxy-1,3-dimethyl-8-nonadecyl-[1,4]-diazocane-2,5-dione) from litter, root exudates, and soils in successive rotations was quantified. Results Extracts of leaf litter, fine root, and root exudates significantly inhibited the growth of Chinese fir germinants, and inhibition increased with successive rotations. Similar results were observed in the rhizosphere soil, basal soil, and bulk soil. The largest observed inhibition occurred in the rhizosphere soil. Furthermore, cyclic dipeptide was found in litter, root exudates, and soils, and the concentrations increased with successive rotations. The rhizosphere soil had the highest cyclic dipeptide level, followed by basal soil, while bulk soil contained the lowest concentration. There was a significant positive relationship between the inhibition of radicle growth of Chinese fir germinants and the concentration of cyclic dipeptide. Annual release of cyclic dipeptide through root exudation was 2.08–9.78 mol ha −1 annum, but the annual release of cyclic dipeptide through leaf litter decomposition was lowered to 0.32–1.41 mol ha −1 annum. Conclusions Cyclic dipeptide which caused autoinhibition of Chinese fir may be released into the soil through litter decomposition and root exudation. Root exudates provided more contributions to soil cyclic dipeptide levels than litter in Chinese fir plantations.

Description: Background and aims In the Central Negev hills (Israel) many ancient terraced wadis exist, which captured run-off and caused gradual soil aggradation, which enabled agricultural practices. In these terraces, dark colored soil horizons were observed, containing charcoal, as can be found in Terra Preta soils, suggesting higher fertility compared to natural soils. The aim of our investigation was to investigate these anthropogenic soils and to study the effects of charcoal and ash addition on soil properties and crop growth. Methods We investigated 12 soil profiles, focusing on possible differences between light and dark colored soil horizons. We also investigated the effects of amendment of charcoal and ash on the growth of wheat ( Triticum Aestivum L. ) in a 40-day pot experiment involving two water regimes. Results Results show that charcoal content in light and dark horizons were both low (〈0.2 %), but significantly lower bulk densities were found in dark colored horizons. In the crop experiment, charcoal addition resulted in decreased crop growth, while, in the water deficit regime, ash addition resulted in increased crop growth. Conclusions Considering the observed charcoal and the results from the crop experiment, we hypothesize that, in ancient run-off capturing agricultural systems, ash was purposefully added as fertilizer.

Description: κ-Carrageenases exhibit apparent distinctions in gene sequence, molecular weight, enzyme properties, and posttranslational processes. In this study, a new κ-carrageenase gene named cgk Z was cloned from the marine bacterium Zobellia sp. ZM-2. The gene comprised an open reading frame of 1,638 bp and encoded 545 amino acids. The natural signal peptide of κ-carrageenase was used successfully for the secretory production of the recombinant enzyme in Escherichia coli . A posttranslational process that removes an amino acid sequence of about 20 kDa from the C-terminal end of κ-carrageenase was first discovered in E. coli . An increase in enzyme activity by 167.3 % in the presence of 5 mM DTT was discovered, and Na + at a certain concentration range was positively correlated with enzyme activity. The κ-carrageenase production of E. coli was 9.0 times higher than that of ZM-2. These results indicate the potential use of the enzyme in the biotechnological industry.

Description: Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei , and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter , Agrobacterium , Burkholderia , Cronobacter , Enterobacter , Mesorhizobium , Novosphingobium , Pantoea , Pseudomonas , Rahnella , Rhizobium , Serratia , and Variovorax . Seven isolates were Gram-positive bacteria belonging to the genera Bacillus , Paenibacillus , Planomicrobium , and Rhodococcus . Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA , glnII , rpoB , and 16S rRNA gene sequences, Rhizobium , Mesorhizobium , and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter , Rhizobium sp., Rhizobium phaseoli , Mesorhizobium sp., and putative new Rhizobium species. The strains from R . phaseoli , Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium , Burkholderia , Paenibacillus , Pantoea , Pseudomonas , Rhizobium , and Serratia , were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.

Description: Introduction In a recent paper, Warren et al. ( 2013 ) illustrated the potential of neutron radiography to visualize water dynamics in soil and plants. Methods After injection of deuterated water (D 2 O) in soil, the authors could monitor the changes of D 2 O concentration in roots. Results Based on the radiographs, the authors concluded that D 2 O was transported from roots growing in a wet soil region to roots in a dry region, proving hydraulic redistribution between roots. However, this interpretation depends on the correct estimation of D 2 O concentration in soil. Conclusions The experiments of Warren et al. ( 2013 ) could also be explained by diffusion of D 2 O from soil to roots, without hydraulic redistribution within the root system.

Description: Aims The mechanisms of belowground competition are not well understood. Addressing literature reports on competition-induced changes in tree fine root morphology, we conducted a growth experiment with tree saplings to investigate competition effects on important root morphological and functional traits in a root order-focused analysis. Methods European beech and European ash saplings were grown for 34 months in containers under greenhouse conditions in monoculture (2 conspecific plants), in mixture (1 beech and 1 ash) or as single plants. The root system was fractionated according to root orders and eight morphological and functional properties were determined. Results Root order was the most influential factor affecting the fine root traits (except for root diameter and δ 13 C); a significant species identity effect was found for root diameter, tissue density, N concentration and δ 13 C. Ash fine roots were thicker, but had lower tissue densities, contained more N and had systematically higher δ 13 C values than beech roots. The competition treatments had no significant effect on morphological root traits but altered δ 13 C in the 2nd root order. Conclusion Neither intra- nor interspecific root competition affected fine root morphology significantly suggesting that competition-induced root modification may not be a universal phenomenon in temperate trees.

Description: Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD + . To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E . coli BA016 was investigated. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD + ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l −1 glucose and produced 14.08 g l −1 succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased.

Description: 1,2-Benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl (flavipin) was found to be antagonistic against nematodes and fungi. Here we demonstrated that flavipin is a potent antioxidant in vitro and in vivo, which has great potential in the therapy for free radical-associated diseases. Therefore, flavipin-producing bio-source was screened from 80 endophytes in Ginkgo biloba . Seven endophytic fungi were able to synthesize antioxidant substances and identified by ITS rDNA sequences. Among them, Chaetomium globosum CDW7 was a remarkable producer of flavipin. The fermentation parameters of CDW7 were then optimized for high flavipin production. Cultured under the optimal condition (25 °C, 100/250 mL flask, 12 discs/flask, 150 rpm, pH 6.5) for 14 days, CDW7 was able to synthesize flavipin at a production of 315.5 mg/L. In addition, flavipin output was positively correlated to antioxidant activities of crude extracts with a correlation coefficient of 0.8235, indicating that flavipin was the major antioxidant component of CDW7's metabolites. These data demonstrated that CDW7 was a highly yielded bio-source of antioxidant flavipin.

Description: Background and aims Distinct metal distribution patterns within leaves of metal hyperaccumulating plants are repeatedly observed however, the presumable role of key structural biochemical molecules in determining and regulating their allocation remains largely unknown. We aimed to characterise in a spatially resolved manner the distribution of the main biochemical components in leaves of field-collected Cd/Zn-hyperaccumulating Noccaea praecox in order to relate them to metal distribution patterns at tissue level. Methods The biomolecular composition of the leaves was spatially analysed using synchrotron radiation Fourier Transform Infrared (FTIR) and the distribution of Zn with synchrotron radiation Low-Energy X-Ray Fluorescence (LEXRF) microspectroscopy was determined on the same tissues of interest (epidermis, sub-epidermis, mesophyll). Results In epidermal cells high proportion of free-carboxyl, nitro and phosphate groups standing for pectin, nitroaromatics, phytic and other organic acids were found. Adjacent mesophyll cells had higher proportions of proteins, carbohydrates and cellulosic compounds. Conclusions Pectin compounds were indicated as important components of Zn enriched epidermal cell walls. In addition, intense lignification of epidermal cell walls might limit leakage of the trapped metals back to the metabolically active and thus more sensitive mesophyll. Distribution of metal-binding compounds in particular cell types/tissues may therefore predispose metal distribution patterns and tolerance in leaves.

Description: Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis , ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC 50 ) at concentrations of 〉8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC = 1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus , Enterococcus faecalis , Staphylococcus epidermidis , Bacillus subtilis 168, and Clostridium acetobutylicum . The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.

Description: The compartmentalized anaerobic reactor (CAR) is a patent novel high-rate reactor, which consists of three compartments. The reactor has a great potential for application due to its many advantages. In this work, the microbial consortium, spatial distribution, and their relationship with performance of CAR were investigated by means of polymerase chain reaction, denaturing gradient gel electrophoresis, and fluorescence in situ hybridization. The results showed that the predominant archaea were Methanobacterium , Methanosaeta , and Methanospirillum , and the predominant bacteria were Firmicutes , Deltaproteobacteria , Spirochaetes , Actinobacteria , and Gammaproteobacteria in the microbial consortium. The methanogenic archaea (MA), the hydrogen-producing acetogenic bacteria (HAB), and the hydrolytic fermentative bacteria (HFB) were found to be predominant in the upper, middle, and bottom compartments, respectively. The results revealed that the granular sludge took on a stratified microbial structure. The HFB, HAB, and MA were located in the outer shell, middle layer, and core, respectively. The microbial populations from the bottom compartment were relatively homogeneous in the granular sludge, and from the middle and upper compartments, they were relatively heterogeneous in the granular sludge. The microbial consortia and their spatial distribution were in accordance with the organic loading rate and chemical components in the three compartments.

Description: We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum , and direct l -lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans , was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of l -lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l -lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum .

Description: Aims Tree species affect herb layer species through their effects on soil quality and light regime but their relative importance and interactions are insufficiently known. Methods Pot experiment with soil taken from stands planted with tree species with contrasting effects on soil acidification, two light regimes and six forest perennials. Results The survival or growth of Mercurialis perennis, Lamium galeobdolon , Anemone nemorosa and Primula elatior was lower in the acid Alnus soils than in the less acid Fraxinus soils. By contrast, the acid tolerant Convallaria majalis and Dryopteris dilatata were barely affected by tree species. Light conditions had less impact than soil chemistry and did not compensate for unfavourable soil conditions. Ca and P concentrations increased in plants grown in Fraxinus soils. The Mg and Al shoot/root ratios of respectively one and two of the acid tolerant species was elevated in the most acid soil. Conclusions Tree species effects on forest perennials are mainly explained by increased Al concentrations under acidifying species. Changed plant concentrations and allocation are likely associated to Al antagonism. We found no light compensation for the soil effect on the studied species. However, light alters the plant nutrient concentrations and allocation which may suggest an indirect effect.

Description: The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose–response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

Description: Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.

Description: Glucosamine (GlcN), an amino sugar, is a compound derived from substitution of a hydroxyl group of a glucose molecule with an amino group. GlcN and its acetylated derivative, N -acetylglucosamine (GlcNAc), have been widely used in food, cosmetics, and pharmaceutical industries and are currently produced by acid hydrolysis of chitin (a linear polymer of GlcNAc) extracted from crab and shrimp shells. Microbial fermentation by filamentous fungi or recombinant Escherichia coli , as an alternative method for the production of GlcN and GlcNAc, is attracting increasing attention because it is an environmentally friendly process. Although the microbial production of GlcN and GlcNAc is hampered by low yield and high production cost, considerable advances have been made in recent years. Here we review the applications, commercial market, and production of GlcN and GlcNAc, with emphasis on the metabolic and process engineering strategies used to improve GlcN and GlcNAc production by recombinant microbes.

Description: Aims The aim of this study was to determine whether goat grazing in the understory of a pine forest at Doñana Natural Park could accelerate the decomposition of the pine needles accumulated on the soil surface and, if so, through which mechanisms. Specifically, the roles of trampling (mechanical fragmentation) and nutrient enrichment through defecation (fertilization) were evaluated in terms of their effect on pine needle decomposition rates. Methods An experiment was conducted featuring the following 4 treatments: 1) intact needles (control), 2) trampled needles, 3) intact needles fertilized with liquid manure, and 4) trampled needles fertilized with liquid manure. Litter decomposition was determined as a function of mass loss over time, using the litter-bag method. Bags were recovered 4, 8, 16, 24 and 36 months after burial in soil, dried and weighed. Needle length, leaf mass per area and C and N concentration were also measured in the buried litter-bags. Results Four months after burial, mass loss was greater in the trampled (23–27 %) than non-trampled (14–16 %) treatments. However, from 8 months onwards, decomposition rates in the fertilized treatments were significantly higher than those in the non-fertilized treatments (between 5 % and 15 % less mass loss). Meanwhile, fertilized treatments presented higher N content (2.1 %) than the non-fertilized ones (1.2 %), with a significantly lower C:N ratio also found in the in the fertilized treatment. Conclusions Trampling and fertilization during grazing accelerates litter decomposition and thus promotes the incorporation of N into the system. Acceleration of decomposition reduces the accumulation of pine needles on the soil surface, reducing the risk of fire.

Description: In this work, the successful coupling of enzymatic oxidation and aldol addition reactions for the synthesis of a Cbz-aminopolyol from a Cbz-amino alcohol was achieved for the first time in a multienzymatic one-pot system. The two-step cascade reaction consisted of the oxidation of Cbz-ethanolamine to Cbz-glycinal catalyzed by chloroperoxidase from the fungus Caldariomyces fumago and aldol addition of dihydroxyacetone phosphate to Cbz-glycinal catalyzed by rhamnulose-1-phosphate aldolase expressed as a recombinant enzyme in Escherichia coli , yielding (3 R ,4 S )-5-{[(benzyloxy)carbonyl]amino}-5-deoxy-1- O -phosphonopent-2-ulose. Tools of enzymatic immobilization, reactor configurations, and modification of the reaction medium were applied to highly increase the production of the target compound. While the use of soluble enzymes yielded only 23.6 % of Cbz-aminopolyol due to rapid enzyme inactivation, the use of immobilized ones permitted an almost complete consumption of Cbz-ethanolamine, reaching Cbz-aminopolyol yields of 69.1 and 71.9 % in the stirred-tank and packed-bed reactor, respectively. Furthermore, the reaction production was 18-fold improved when it was catalyzed by immobilized enzymes in the presence of 5 % ( v / v ) dioxane, reaching a value of 86.6 mM of Cbz-aminopoliol (31 g/L).

Description: The complete genome of Acinetobacter oleivorans DR1 contains AqsR and AqsI genes, which are LuxR and LuxI homolog, respectively. In a previous study, we demonstrated that quorum sensing (QS) signals play an important role in biofilm formation and hexadecane biodegradation. However, the regulation of genes controlled by the QS system in DR1 remains unexplored. We constructed an aqsR mutant and performed RNA sequencing analysis to understand the QS system. A total of 353 genes were differentially expressed during the stationary phase of wild-type cells compared to that of the aqsR mutant. AqsR appears to be an exceptionally important regulator because knockout of aqsR affected global gene expression. Genes involved in posttranslational modification, chaperones, cell wall structure, secondary metabolites biosynthesis, and stress defense were highly upregulated only in the wild type. Among upregulated genes, both the AOLE_03905 (putative surface adhesion protein) and the AOLE_11355 (L-asparaginase) genes have putative LuxR binding sites at their promoter regions. Soluble AqsR proteins were successfully purified in Escherichia coli harboring both aqsR and aqsI . Comparison of QS signals in an AqsI–AqsR co-overexpression strain with N -acyl homoserine lactone standards showed that the cognate N -acyl homoserine lactone binding to AqsR might be 3OH C12HSL. Our electrophoretic mobility shift assays with purified AqsR revealed direct binding of AqsR to those promoter regions. Our data showed that AqsR functions as an important regulator and is associated with several phenotypes, such as hexadecane utilization, biofilm formation, and sensitivity to cumene hydroperoxide.

Description: A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558 T . Although F . aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117 U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80 % amino acid (AA) identity to AdhA and 68 % AA identity to AdhB of Ga . europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.

Description: Enantioselective oxidation of racemic phenyl-1,2-ethanediol into ( R )-(−)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1 was demonstrated. It was found that optically active ( R )-(−)-mandelic acid ( e.e.p  〉 99.9 %) is produced leaving the other enantiomer ( S )-(+)-phenyl-1,2-ethanediol intact. Using fed-batch method, a total of 172.9 mM ( R )-(−)-mandelic acid accumulated in the reaction mixture after the seventh feed. Moreover, oxidation of phenyl-1,2-ethanediol using calcium alginate-entrapped resting cells was carried out in the aqueous system, and efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 27.94 g ( R )-(−)-mandelic acid g −1 dry cell weight cell after 16 cycles of repeated use.

Description: The bacterium Bacillus amyloliquefaciens anti-CA isolated from mangrove system was found to be able to actively kill Candida albicans isolated from clinic. The bacterial strain anti-CA could produce high level of bioactive substance, amylase and protease in the cheap medium containing 2.0 % soybean meal, 2.0 % wheat flour, pH 6.5 within 26 h. After purification, the main bioactive substance was confirmed to be a cyclic lipopeptide containing a heptapeptide, L-Asp→L-Leu→L-Leu→L-Val→L-Val→L-Glu→L-Leu and a 3-OH fatty acid (15 carbons). In addition to C. albicans , the purified lipopeptide can also kill many yeast strains including Metschnikowia bicuspidata , Candida tropicalis , Yarrowia lipolytica and Saccharomyces cerevisiae. After treated by the purified lipopeptide, both the whole cells and protoplasts of C. albicans were destroyed.

Description: Background and aims Cadmium (Cd) could activate activity of mitogen-activated protein kinase MPK6 in plants. In this study, we investigated the role of MPK6 in mediating Cd toxicity in plants. Methods The wild type Arabidopsis plants (WT) and the mpk6 - 2 mutants were subjected either 0 (Control) or 10 μM Cd treatment. Kinase activity of MPK6, nitric oxide (NO) level, Cd concentration, and oxidative stress were measured. Results In WT plants, Cd exposure rapidly stimulated kinase activity of MPK6. However, upon Cd exposure, mpk6 - 2 showed better growth than the WT. Although Cd-induced production of NO in roots was greater in WT than in mpk6 - 2 , there was no difference in Cd concentration between the two plants. Nevertheless, the Cd-induced hydroperoxide burst, lipid peroxidation and loss of membrane integrity, were all more severe in the WT than in mpk6 - 2 . Foliar applications of antioxidant ascorbic acid, vigorously improved the growth of both the WT and mpk6 - 2 under Cd exposure. Thereby the growth difference between these two plants was minimized. Conclusions Mutation of mpk6 enhances Cd tolerance in plants by alleviating oxidative stress, but did not affect cadmium accumulation in plants.

Description: Background and Aims The role and linkage of endophytic bacteria to resistance of peanut seeds to biotic stress is poorly understood. The aims of the present study were to survey the experimental (axenic) and control (conventional) peanut plants for the predominant endophytic bacteria, and to characterize isolates with activity against selected A. flavus strains. Methods Young axenic plants were grown from presumably bacteria-free embryos in the lab, and then they were grown in a field. Endophytic bacterial species were identified by the analysis of DNA sequences of their 16S-ribosomal RNA gene. DNA extracted from soil was also analyzed for predominant bacteria. Results Mature seeds from the experimental and control plants contained several species of nonpathogenic endophytic bacteria. Among the eight bacterial species isolated from seeds, and DNA sequences detected in soil, Bacillus thuringiensis was dominant. All B. amyloliquefaciens isolates, the second abundant species in seeds demonstrated activity against A. flavus . This effect was not observed with any other bacterial isolates. There was no significant difference in number and relative occurrence of the two major bacterial species between the experimental and conventionally grown control seeds. Conclusion Endophytic bacterial colonization derives from local soil and not from the seed source, and the peanut plant accommodates only selected species of bacteria from diverse soil populations. Some bacterial isolates showed antibiosis against A. flavus.

Description: Background and Aims Climate warming and increased atmospheric nitrogen (N) deposition both have the potential to increase plant productivity over the next century, yet they can also increase decomposition and respiration. Our aim was to examine the extent to which warming and N addition can, on balance, alter net ecosystem CO 2 exchange (NEE) in a grass-dominated system. Methods We measured NEE responses to warming and N addition over two growing seasons in a temperate old field using steady-state flow-through chambers, which allowed for the integrated measurement of respiration and photoassimilation effects on net CO 2 flux over diel periods. We also assessed the relationship between NEE and plant biomass responses to the warming and N treatments. Results In both years, our study system was a net source of carbon (C) during the snow-free season. N addition did not significantly affect diel NEE or dark respiration in either year, despite a doubling in aboveground plant biomass in response to N addition in the second year, and a corresponding increase in peak daily net CO 2 photoassimilation in N addition plots. The warming treatment also had no significant effect on NEE, although the flow-through chambers required warming to be temporarily halted during NEE measurements. Conclusions Overall, our results both highlight the potential divergence of plant and soil responses to N addition and demonstrate the capacity for a grass-dominated system to function as a net source of C in consecutive years.

Description: Background and aim Intuitively, access to water from the soil at key phenological stages is important for adaptation to drought. This study aimed to assess the temporal pattern of water extraction under terminal drought stress. Methods Pearl millet genotypes with varying levels of terminal drought tolerance were grown in a lysimetric system with a soil volume and plant spacing similar to field conditions. Water extraction was monitored until maturity under differing water regimes. Results The yield did not differ among genotypes under well-watered (WW) conditions, and the water extraction profile of WW plants was similar across all genotypes. In contrast, the yield of sensitive genotypes was 30–100 % lower than that of tolerant lines under water stress (WS). The total volumes of water extracted by tolerant and sensitive genotypes were similar under WS; however, tolerant genotypes extracted less water prior to anthesis, and more water after anthesis. Grain yield was positively related to the amount of water extracted during week three after panicle emergence. Increased water extraction after anthesis benefitted the tillers more than the main culm and was correlated with higher staygreen scores. Conclusion Increased water uptake after anthesis, which results from earlier water conservation during pre-anthesis, increases yield under terminal drought in pearl millet.

Description: Background and aims Efficient accumulation of arsenic (As) in rice ( Oryza sativa L.) poses a potential health risk to rice consumers. The aim of this study was to investigate the mechanisms of uptake, transport and distribution of inorganic arsenic (As i ) and dimethylarsinic acid (DMA) in rice plants. Methods Rice was exposed to As i (As(V)) and DMA in hydroponics. High-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and synchrotron X-ray fluorescence (SXRF) microprobe were used to determine As concentration and the in situ As distribution. Results DMA induced abnormal florets before flowering and caused a sharp decline in the seed setting rate after flowering compared to As i . Rice grains accumulated 2-fold higher DMA than As i . The distribution of As i concentration (root 〉 leaf 〉 husk 〉 caryopsis) in As(V) treatments was different from that of the DMA concentration (caryopsis 〉 husk 〉 root ≥ leaf) in DMA treatments. SXRF showed that As i mainly accumulated in the vascular trace of caryopsis with limited distribution to the endosperm, whereas DMA was observed in both tissues. Conclusions DMA tended to accumulate in caryopsis and induced higher toxicity to the reproductive tissues resulting in markedly reduced grain yield, whereas As i mainly remained in the vegetative tissues and had no significant effect on yield. DMA is more toxic than As i to the reproductive tissues when both of them are at similar concentrations in nutrient solution.

Description: Background and aims Variations in responses to soil N between a non-N-fixing shrub, Baccharis halimifolia L., and a N-fixing shrub, Morella cerifera (L.) Small, were tested over 12 weeks to determine whether N availability is the sole cause of persistent dominance of M. cerifera on barrier islands. Methods Plants were supplied increasing levels of soil N up to 200 mg kg −1 . Measurements included gas exchange and chlorophyll fluorescence parameters across treatments, species, and time. Tissues were analyzed for differences in biomass and nutrients. Results Baccharis halimifolia had reduced physiological responses across all treatment levels, but M. cerifera had comparatively few variations. Across all treatments B. halimifolia photosynthesis and stomatal conductance were reduced by 62 and 76 %, respectively,by week 12. Increasing foliar δ 15 N values across treatments for M. cerifera indicated a shift from utilizing fixed N to available soil N. Biomass was highest at 200 mg kg −1  N for both species. Baccharis halimifolia showed indications of stress response and resource limitation based on physiological responses, nutrient contents, and isotope effects. Conclusions Baccharis halimifolia showed signs of co-limitation of both N and P whereas M. cerifera was limited by neither, suggesting that dominance of M. cerifera is only partially explained by actinorhizal symbiosis and N availability.

Description: Background and Aims Soil mineralization, nitrification, and dynamic changes in abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were studied to validate our hypothesis that soil mineralization and nitrification decreased along the chronosequence of rice cultivation. Methods Paddy soils with a 300, 700 and 2000-year cultivation history (P300, P700 and P2000) were selected to study net mineralization and nitrification processes. Dynamic abundance of AOB and AOA was estimated by quantifying their respective amoA gene copies. Results The net mineralization rate was higher for P300 than P700 and P2000. Potential nitrification ( N p ) and average nitrification rates ( V a ) were similar for P300 and P700 soils, but the simulated potential nitrification rate ( V p ) and nitrification rate (k 1 ) was 72 % and 88 % higher for P300 than P700, respectively. V a was about 70 % lower than for P2000 than P300 and P700. AOB amoA gene copies were higher for P300 than P700 and P2000, whereas AOA abundance did not show significant differences. AOB abundance showed a positive response to NH 4 supply but AOA did not. Conclusions Both N mineralization and nitrification were depressed with increased cultivation time. Archaea responded to mineralization positively rather than nitrification, which suggested that readily mineralized organic matter may play an important role in AOA.

Description: Aims The current study aimed to assess the potential of peanut ( Arachis hypogaea L.) for bioenergy production via phytoextraction in cadmium (Cd) -contaminated soils and screen appropriate cultivars for this approach. Methods A life-cycle pot experiment was conducted to determine the biomass, seed yield, oil content and Cd accumulation of seven peanut cultivars under Cd concentration gradients of 0, 2, and 4 mg kg −1 . Results Peanut exhibits genotypic variations in Cd tolerance, seed production, oil content, and Cd accumulation. Exposure of plants to 2 and 4 mg kg −1 Cd did not inhibit shoot biomass, seed yield, and oil content for most of the cultivars tested. There are large amounts of Cd accumulated in the shoots. Although the seed Cd concentration of peanut was relatively high, the Cd concentration in seed oils was very low (0.04-0.08 mg kg −1 ). Among the cultivars, Qishan 208 showed significant Cd tolerance, high shoot biomass, high pod and seed yield, high seed oil content, considerable shoot Cd concentration, and the largest translocation factor and total Cd in shoots. Conclusions The cultivation of peanut in Cd-contaminated farmland was confirmed to be feasible for bioenergy production via phytoextraction, and Qishan 208 is a good candidate for this approach.

Description: Background and aims Sufficient soil phosphorus (P) is important for achieving optimal crop production, but excessive soil P levels may create a risk of P losses and associated eutrophication of surface waters. The aim of this study was to determine critical soil P levels for achieving optimal crop yields and minimal P losses in common soil types and dominant cropping systems in China. Methods Four long-term experiment sites were selected in China. The critical level of soil Olsen-P for crop yield was determined using the linear-plateau model. The relationships between the soil total P, Olsen-P and CaCl 2 -P were evaluated using two-segment linear model to determine the soil P fertility rate and leaching change-point. Results The critical levels of soil Olsen-P for optimal crop yield ranged from 10.9 mg kg −1 to 21.4 mg kg −1 , above which crop yield response less to the increasing of soil Olsen-P. The P leaching change-points of Olsen-P ranged from 39.9 mg kg −1 to 90.2 mg kg −1 , above which soil CaCl 2 -P greatly increasing with increasing soil Olsen-P. Similar change-point was found between soil total P and Olsen-P. Overall, the change-point ranged from 4.6 mg kg −1 to 71.8 mg kg −1 among all the four sites. These change-points were highly affected by crop specie, soil type, pH and soil organic matter content. Conclusions The three response curves could be used to access the soil Olsen-P status for crop yield, soil P fertility rate and soil P leaching risk for a sustainable soil P management in field.

Description: The epothilones, compounds with anticancer mechanisms similar to that of paclitaxel (Taxol), are produced by strains of the myxobacterium Sorangium cellulosum , and the gene cluster responsible for epothilone biosynthesis is organised as a large operon. In this work, we showed that the 440-bp promoter regions of the operons from eight S. cellulosum strains have 94.27 % DNA sequence identity and 50 % variability in promoter activity in Escherichia coli . A primer extension analysis revealed two transcriptional start sites (TSSs) at 246 (TSS1) and 193 bp (TSS2) upstream of the translation start site (TLS), respectively. Promoter truncation determined that the basal promoter from the So0157-2 strain is located within a 264-bp region containing weak promoter activity; whereas in the 38-bp region upstream, the 264-bp promoter was required for the strong promoter activity, which was dramatically increased by 11-fold in average. There was a conserved stem–loop structure between TSS2 and the TLS, which was identified in E. coli as a negative regulatory element. In addition, the upstream non-conserved 357-bp non-coding region contributes to the promoter activity, increasing it by 1.5-fold. In conclusion, the expression of the epothilone operon non-coding region in E. coli is regulated by a double promoter (with −35 and −10 regions and two distinct TSSs), a stem–loop structure, and a distal non-coding region.

Description: Bacterial community compositions were characterized using denaturing gradient gel electrophoresis analysis of bacterial 16S rRNA gene in the sediments of the Pearl River estuary. Sequencing analyses of the excised bands indicated that Gram-negative bacteria, especially Gammaproteobacteria , were dominant in the Pearl River estuary. The diversity of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) gene in this estuary was then assessed by clone library analysis. The phylogenetic analyses showed that all PAH-RHD gene sequences of Gram-negative bacteria (PAH-RHD [GN] ) were closely related to the nagAc gene described for Ralstonia sp. U2 or nahAc gene for Pseudomonas sp. 9816–4, while the PAH-RHD gene sequences of Gram-positive bacteria (PAH-RHD [GP] ) at sampling site A1 showed high sequence similarity to the nidA gene from Mycobacterium species. Meanwhile, molecular diversity of the two functional genes was higher at the upstream of this region, while lower at the downstream. Redundancy analysis indicated that environmental factors, such as NH 4 -N, ∑PAHs, pH, SiO 3 -Si, and water depth, affected the distribution of the PAH-RHD [GN] gene in the Pearl River estuary.

Description: In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture–PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log 10 increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n  = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.

Description: Background and aims Gaseous losses of ammonia (NH 3 ) have been observed in citrus orchards when urea is surface-applied to the soils, and this loss might significantly limit the effectiveness of the nitrogen (N) fertilizer. However, a portion of the volatilized NH 3 might be absorbed by the plants through the leaves. To quantify the contribution of the leaf absorption of 15 NH 3 , a study with sweet oranges was conducted in two field areas where trees were grown at standard (480 trees ha −1 ) and high densities (617 trees ha −1 ). Methods Plastic trays were filled with soil, covered with mown grass to simulate field management conditions, fertilized with 15 N labeled urea (12 atom % excess) and placed under each of three trees in the orchards. This experimental procedure prevented the uptake of N from the labeled urea by the roots. Two weeks after 15 N fertilization, the trays were removed from the field, and the soil was homogenized and sampled for chemical analyses. The citrus trees under which the trays were placed were destructively harvested, and the total N concentrations and 15 N/ 14 N ratios were determined. Results After urea application, the NH 3 losses peaked within three days and subsequently decreased to negligible amounts after 10 days. The total NH 3 losses accounted for 55–82 % of the applied N. Although the NH 3 absorption by the citrus leaves was proportional to the tree density in the field, only 3–7 % of the 15 NH 3 volatilized from the soil was recovered by the citrus trees, and the NH 3 absorption was also influenced by the proximity of citrus trees to the site of urea application and the leaf areas of the trees. Conclusions The citrus trees can absorb the NH 3 volatilized from urea, even though, the amount recovered by the trees is small and does not represent a significant proportion of total gaseous N losses, what demonstrates the importance of enhanced N use efficiency practices in field to reduce losses of NH 3 when urea is applied to soil surfaces.

Description: Background Rice can accumulate arsenic (As) to relatively high concentrations due to the general flooding practices in rice cultivation, and organic matter in the soil strongly affected As bioavailability to rice plants. The influence of organic matter input on the As transformation in paddy soil and As uptake into rice plants is an area that is rarely investigated. Methods Biogas slurry (BGS), a commonly used organic fertilizer, was applied to an As contaminated paddy soil, in order to investigate the influence of organic matter on As transformation in the paddy soil and As accumulation in rice plants. Results Application of BGS significantly increased the As accumulation in rice plants, especially for methylated As species. Results showed that the concentrations of dissolved organic carbon (DOC) and dissolved Fe(II) in the soil solution were significantly increased by the BGS addition into the paddy soil, and were significantly correlated to the As concentration in the soil solution ( P  〈 0.01). The increase of soil pH and the decrease of the soil redox potential (Eh) were observed as well. These alteration of soil characteristics elevated the As release from soil particles to the soil solution under the addition of BGS. The increased concentrations of dimethylarsinic acid (DMAs(V)) and monomethylarsonic acid (MMAs(V)) in the soil solution, and the volatilized As of trimethylarsine (TMAs) from the paddy soil, suggested that As methylation and volatilization in the soil were also enhanced by BGS addition. The concentrations of methylated As species in rice husks and grains were increased by 105.8–105.9 % and 99.7–112.2 %, respectively. Conclusion These results suggested that the use of organic fertilizer, such as BGS in As-contaminated paddy soil, can significantly alter the behavior of As in soil-rice system and enhance As accumulation in rice plants and should therefore be avoided.

Description: Proteins with a Zn(II) 2 Cys 6 domain, Cys-X 2 -Cys-X 6 -Cys-X 5-12 -Cys-X 2 -Cys-X 6-9 -Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overview of this family of genes. Annotation of this gene family in most fungal genomes is still far from perfect and refined bioinformatic algorithms are urgently needed. Aspergillus flavus is a saprophytic soil fungus that can produce the carcinogenic aflatoxin. It is the second leading causative agent of invasive aspergillosis. The 37-Mb genome of A . flavus is predicted to encode 12,000 proteins. Two and a half percent of the total proteins are estimated to contain the C6 domain, more than twofold greater than those estimated for yeast, which is about 1 %. The variability in the spacing between cysteines, C 3 -C 4 and C 5 -C 6 , in the zinc cluster enables classification of the domains into distinct subgroups, which are also well conserved in Aspergillus nidulans . Sixty-six percent (202/306) of the A . flavus C6 proteins contain a specific transcription factor domain, and 7 % contain a domain of unknown function, DUF3468. Two A . nidulans C6 proteins containing the DUF3468 are involved in asexual conidiation and another two in sexual differentiation. In the anamorphic A . flavus , a homolog of the latter lacks the C6 domain. A . flavus being heterothallic and reproducing mainly through conidiation appears to have lost some components involved in homothallic sexual development. Of the 55 predicted gene clusters thought to be involved in production of secondary metabolites, only about half have a C6-encoding gene in or near the gene clusters. The features revealed by the A . flavus C6 proteins likely are common for other ascomycete fungi.

Description: A range of isolates of Pseudomonas aeruginosa from widely different environmental sources were examined for their ability to synthesise rhamnolipid biosurfactants. No significant differences in the quantity or composition of the rhamnolipid congeners could be produced by manipulating the growth conditions. Sequences for the rhamnolipid genes indicated low levels of strain variation, and the majority of polymorphisms did lead to amino acid sequence changes that had no evident phenotypic effect. Expression of the rhlB and rhlC rhamnosyltransferase genes showed a fixed sequential expression pattern during growth, and no significant up-regulation could be induced by varying producer strains or growth media. The results indicated that rhamnolipids are highly conserved molecules and that their gene expression has a rather stringent control. This leaves little opportunity to manipulate and greatly increase the yield of rhamnolipids from strains of P. aeruginosa for biotechnological applications.

Description: Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. It was previously demonstrated that polysaccharides from fermented A . membranaceus by S . alactolyticus had some similar properties to those from A . membranaceus in terms of its ability to help heal hepatic fibrosis in rats and modulate immunopotentiation of broiler chicken. However, methods to increase the yield of the polysaccharides during fermentation of A . membranaceus are not well understood. In this paper, we investigated the involvement of uridine diphosphate (UDP)-glucose 4-epimerase ( galE ) and glucan-1,6-α-glucosidase ( dexB ) during A . membranaceus fermentation through real-time reverse transcription quantitative PCR. The galE and dexB genes of S . alactolyticus were cloned by homology-based cloning and the genome walking method for the first time, and the 3D structure of dexB was analyzed by Swiss-PdbViewer 4.0.1 software. The expression of both the galE and dexB genes in A . membranaceus fermentation was studied using the determined ideal reference gene ldh for transcript normalization. The results showed that these two genes were both highly induced and peaked after 12 h of fermentation. The expression level of galE was stepwise increased from 48 to 72 h, while dexB transcripts were markedly increased at 60 h and decreased by 72 h. These data suggested that dexB and galE of S . alactolyticus strain FGM were involved in the regulation of A . membranaceus fermentation and they might play some roles in the increase of polysaccharides.

Description: Rhodococcus erythropolis WZ010 was capable of producing optically pure (2 S ,3 S )-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli , and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % ( v / v ) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2 S ,3 S )-2,3-butanediol via ( S )-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2 S ,3 S )-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2 S ,3 S )-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2 S ,3 S )-2,3-butanediol and NAD + , suggesting its physiological role in favor of (2 S ,3 S )-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for ( S )-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2 S ,3 S )-2,3-butanediol and ( R )-1-phenylethanol in great conversions and enantiomeric excess values.

Description: Regioselective glycosylation of flavonoids cannot be easily achieved due to the presence of several hydroxyl groups in flavonoids. This hurdle could be overcome by employing uridine diphosphate-dependent glycosyltransferases (UGTs), which use nucleotide sugars as sugar donors and diverse compounds including flavonoids as sugar acceptors. Quercetin rhamnosides contain antiviral activity. Two quercetin diglycosides, quercetin 3- O -glucoside-7- O -rhamnoside and quercetin 3,7- O -bisrhamnoside, were synthesized using Escherichia coli expressing two UGTs. For the synthesis of quercetin 3- O -glucoside-7- O -rhamnoside, AtUGT78D2, which transfers glucose from UDP-glucose to the 3-hydroxyl group of quercetin, and AtUGT89C1, which transfers rhamnose from UDP-rhamnose to the 7-hydroxyl group of quercetin 3- O -glucoside, were transformed into E. coli . Using this approach, 67 mg/L of quercetin 3- O -glucoside-7- O -rhamnoside was synthesized. For the synthesis of quercetin 3,7- O -bisrhamnoside, AtUGT78D1, which transfers rhamnose to the 3-hydroxy group of quercetin, and AtUGT89C1 were used. The RHM2 gene from Arabidopsis thaliana was coexpressed to supply the sugar donor, UDP-rhamnose. E. coli expressing AtUGT78D1 , AtUGT89C1 , and RHM2 was used to obtain 67.4 mg/L of quercetin 3,7- O -bisrhamnoside.

Description: Biological soil disinfestations (BSDs) were developed separately in Japan and in The Netherlands as an alternative to chemical fumigations. In Japan, it was developed based on the knowledge of irrigated paddy rice and upland crop rotation system that was rather tolerant of soil-borne disease development. The methods consist of application of easily decomposable organic matter, irrigation, and covering the soil surface with plastic film, thereby inducing anaerobic (reductive) soil conditions and suppressing many soil-borne pests including fungi, bacteria, nematodes, and weeds. The methods are widely used by organic farmers in the area where residences and agricultural fields are intermingled. To note one advantage of these methods, maintenance of soil suppressiveness to Fusarium wilt of tomato was suggested, while soil treated with chloropicrin became conducive to the disease. Suppression of soil-borne fungal pathogens by BSDs might be attributed to anaerobicity and high temperature, organic acids generated, and metal ions released into soil water. Contributions of respective factors to suppression of respective pathogens might be diverse. Presumably, these factors might vary on the fungal community structure in BSD-treated soil. These factors also work in paddy fields. Therefore, the BSDs developed in Japan are probably a method to raise the efficacy of paddy–upland rotation through intensive organic matter application and through maintenance of a strongly anaerobic (reductive) soil condition.

Description: The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0–7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.

Description: Microbial enhanced oil recovery (MEOR) refers to the process of using bacterial activities for more oil recovery from oil reservoirs mainly by interfacial tension reduction and wettability alteration mechanisms. Investigating the impact of these two mechanisms on enhanced oil recovery during MEOR process is the main objective of this work. Different analytical methods such as oil spreading and surface activity measurements were utilized to screen the biosurfactant-producing bacteria isolated from the brine of a specific oil reservoir located in the southwest of Iran. The isolates identified by 16S rDNA and biochemical analysis as Enterobacter cloacae (Persian Type Culture Collection (PTCC) 1798) and Enterobacter hormaechei (PTCC 1799) produce 1.53 g/l of biosurfactant. The produced biosurfactant caused substantial surface tension reduction of the growth medium and interfacial tension reduction between oil and brine to 31 and 3.2 mN/m from the original value of 72 and 29 mN/m, respectively. A novel set of core flooding tests, including in situ and ex situ scenarios, was designed to explore the potential of the isolated consortium as an agent for MEOR process. Besides, the individual effects of wettability alteration and IFT reduction on oil recovery efficiency by this process were investigated. The results show that the wettability alteration of the reservoir rock toward neutrally wet condition in the course of the adsorption of bacteria cells and biofilm formation are the dominant mechanisms on the improvement of oil recovery efficiency.

Description: Bioaugmentation with degrading bacteria is an effective method to improve the treatment of refractory industrial wastewater; nevertheless there were controversial opinions about the fate of inoculated bacteria and microbial community dynamics. In this study, two lab-scale sequencing batch reactors filled with modified zeolite were used to treat a coking wastewater with pyridine and quinoline shock load, and a bacterial consortium containing three degrading strains was added in one reactor for bioaugmentation. During 120-day operation, the bioaugmented reactor removed over 99 % pyridine, 99 % quinoline, 85 % TOC, 65 % COD, and 95 % NO 3 − -N with higher resistance to the shock load than the non-bioaugmented reactor. Based on the terminal restriction fragment length polymorphism of 16S rDNA, bacterial community diversity increased in the bioaugmented reactor. Principal component analysis revealed that, to cope with the shock load, the indigenous bacterial community recovered to the initial structure by acclimatizing itself constantly to the inhospitable environment; but bioaugmentation accelerated the shift of whole bacterial community, resulting in a far different structure from the initial one. Canonical correspondence analysis indicated that the environmental parameters of pyridine, quinoline, TOC, and NO 3 − -N had close negative correlations with bioaugmentation; and NH 3 -N and COD were the main parameters to impact on the bacterial community changes and treatment efficiency.

Description: Pseudomonads are metabolically versatile microbes that employ complex regulatory networks to control gene expression, particularly with respect to carbon and nitrogen metabolism. The aim of this study was to characterise the regulatory networks that control pyrimidine metabolism (hydantoin-hydrolysing activity) in Pseudomonas putida strain RU-KM3 S , focussing on transcriptional activation of dihydropyrimidinase (Dhp) and β-ureidopropionase (Bup), encoding dhp and bup , respectively. The two genes are arranged divergently on the chromosome and are separated by ORF1, encoding a putative transporter, which lies upstream of and in the same orientation as bup . The results from this study reveal that pyrimidine metabolism, as a function of Bup and Dhp activity in P . putida RU-KM3 S , is controlled by a complex regulatory network including several global pathways in addition to induction by the substrate. Three major control pathways act at the level of transcriptional and include: (1) induction of transcriptional activation in the presence of hydantoin, (2) carbon catabolite repression mediated via a pathway independent of Crc and (3) quorum sensing that does not require a putative lux box located upstream of the dhp transcriptional start. Finally, the data suggest a minor role for the global regulators Anr, Vfr and Crc, likely through regulation of the activity of transcription factors interacting directly with the bup /ORF1– dhp promoter.

Description: Background & aims Plants may have dissimilar effects on ecosystem processes because they possess different attributes. Given increasing biodiversity losses, it is important to understand which plant traits are key drivers of ecosystem functions. To address this question, we studied the response of two ecosystem functions that remove nitrogen (N) from wetland soils, the accumulation of N in plant biomass and denitrification potential (DNP), to variation in plant trait composition. Methods Our experiment manipulated plant composition in a riparian wetland. We determined relative importance of plant traits and environmental variables as predictors of each ecosystem function. Results We demonstrate that Water Use Efficiency (WUE) had a strong negative effect on biomass N. Root porosity and belowground biomass were negatively correlated with DNP. Trait ordination indicated that WUE was largely orthogonal to traits that maximized DNP. Conclusions These results indicate that plant species with different trait values are required to maintain multiple ecosystem functions, and provide a more mechanistic, trait-based link between the recent findings that higher biodiversity is necessary for multi-functionality. While we selected plant traits based on ecological theory, several of the plant traits were not good predictors of each ecosystem function suggesting the ecological theory linking traits to function is incomplete and requires strengthening.