Basic characterization and partial purification of β-glucosidase from cell-free extracts of Oenococcus oeni ST81

Aims: This study was designed to characterize a β‐glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. Methods and Results: The β‐glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²⁺ (10 mmol l^?1) and Cu²⁺ (5 mmol l^?1) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of β‐glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K_m and V_max values for 4‐nitrophenyl‐β‐d ‐glucopyranoside were 0·38 mmol l^?1 and 5·21 nmol min^?1, respectively. The enzyme was stable at pH 5·0 with a value of t_1/2 = 50 days for the crude extract. Conclusions: The β‐glucosidase of O. oeni ST81 is substantially different from those characterized from other wine‐related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a β‐glucosidase from O. oeni ATCC BAA‐1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine‐related compounds on the enzymatic activity and long‐term stability of the enzyme could be of interest for winemaking. Significance and Impact of the Study: Information regarding a β‐glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native β‐glucosidase purified from crude extracts of O. oeni ST81.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison of Fungal Activities on Wood and Leaf Litter in Unaltered and Nutrient-Enriched Headwater Streams

Fungi are the dominant organisms decomposing leaf litter in streams and mediating energy transfer to other trophic levels. However, less is known about their role in decomposing submerged wood. This study provides the first estimates of fungal production on wood and compares the importance of fungi in the decomposition of submerged wood versus that of leaves at the ecosystem scale. We determined fungal biomass (ergosterol) and activity associated with randomly collected small wood (<40 mm diameter) and leaves in two southern Appalachian streams (reference and nutrient enriched) over an annual cycle. Fungal production (from rates of radiolabeled acetate incorporation into ergosterol) and microbial respiration on wood (per gram of detrital C) were about an order of magnitude lower than those on leaves. Microbial activity (per gram of C) was significantly higher in the nutrient-enriched stream. Despite a standing crop of wood two to three times higher than that of leaves in both streams, fungal production on an areal basis was lower on wood than on leaves (4.3 and 15.8 g C m⁻² year⁻¹ in the reference stream; 5.5 and 33.1 g C m⁻² year⁻¹ in the enriched stream). However, since the annual input of wood was five times lower than that of leaves, the proportion of organic matter input directly assimilated by fungi was comparable for these substrates (15.4 [wood] and 11.3% [leaves] in the reference stream; 20.0 [wood] and 20.2% [leaves] in the enriched stream). Despite a significantly lower fungal activity on wood than on leaves (per gram of detrital C), fungi can be equally important in processing both leaves and wood in streams.     

Effects of nutrient enrichment on the decomposition of wood and associated microbial activity in streams

1. We determined the effects of nutrient enrichment on wood decomposition rates and microbial activity during a 3‐year study in two headwater streams at Coweeta Hydrologic Laboratory, NC, U.S.A. After a 1‐year pretreatment period, one of the streams was continuously enriched with inorganic nutrients (nitrogen and phosphorus) for 2 years while the other stream served as a reference. We determined the effects of enrichment on both wood veneers and sticks, which have similar carbon quality but differ in physical characteristics (e.g. surface area to volume ratios, presence of bark) that potentially affect microbial colonisation and activity. 2. Oak wood veneers (0.5 mm thick) were placed in streams monthly and allowed to decompose for approximately 90 days. Nutrient addition stimulated ash‐free dry mass loss and increased mean nitrogen content, fungal biomass and microbial respiration on veneers in the treatment stream compared with the reference. The magnitude of the response to enrichment was great, with mass loss 6.1 times, and per cent N, fungal biomass and microbial respiration approximately four times greater in the treatment versus reference stream. 3. Decomposition rate and nitrogen content of maple sticks (ca. 1–2 cm diameter) also increased; however, the effect was less pronounced than for veneers. Wood response overall was greater than that determined for leaves in a comparable study, supporting the hypothesis that response to enrichment may be greater for lower quality organic matter (high C : N) than for higher quality (low C : N) substrates. 4. Our results show that moderate nutrient enrichment can profoundly affect decomposition rate and microbial activity on wood in streams. Thus, the timing and availability of wood that provides retention, structure, attachment sites and food in stream ecosystems may be affected by nutrient concentrations raised by human activities.     

Does nitrogen become limiting under high‐P conditions in detritus‐based tropical streams?

1. We examined effects of nutrients on leaf breakdown in interior forest streams at La Selva Biological Station, Costa Rica. We tested the hypothesis that dissolved inorganic nitrogen (DIN) becomes limiting when ambient phosphorus (P) concentration is high. We also compared the breakdown of relatively ‘low quality’ leaves (lower C : N, Trema integerrima) with that of ‘higher quality’ leaves (higher C : N, Ficus insipida) in a high‐P stream. 2. Litterbags were incubated in two streams: one enriched experimentally with P [target concentration 200 μg soluble reactive phosphorus (SRP) L^?1] and one control (naturally low P concentration approximately 10 μg SRP L^?1). Ammonium enrichment was achieved by adding fertiliser upstream of half of the litterbags in each stream. 3. Phosphorus addition stimulated leaf breakdown, microbial respiration, ergosterol and leaf %P. Leaf breakdown rate was consistent with those in La Selva streams with naturally high P concentration. 4. Nitrogen (N) addition had no effect on leaf breakdown, microbial respiration, ergosterol or leaf chemistry in either the P‐enriched or the reference stream, in spite of low N : P ratios. We conclude that N is probably not limiting in streams at La Selva that are naturally high in P. This may be due to moderately high ambient N concentration (>200 μg DIN L^?1) prevailing throughout the year. 5. The species with a lower C : N decomposed more rapidly and supported higher microbial activity than that with a higher C : N. Subtle differences in leaf N content, as well as dissolved P concentration, may be important in determining microbial colonisation and subsequent leaf breakdown.     

Enzyme‐triggered fluorescence turn‐off/turn‐on of carbon dots for monitoring β‐glucosidase and its inhibitor in living cells

Energy transfer engineering based on fluorescent probes for directly sensing enzyme activities are in great demand as enzyme‐mediated transformations, which are central to all biological processes. Here, a fluorescence carbon dot (CD)‐based assay exhibiting selective responses to the quantitation of β‐glucosidase and the effect of its inhibitor was developed. The most common substrate, para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPG) was hydrolyzed by β‐glucosidase to release p‐nitrophenol (pNP), which can efficiently quench fluorescence of CDs via an inner filter effect and electron transfer. However, in the presence of inhibitors of β‐glucosidase, the fluorescence intensity gradually recovered as the concentration of inhibitors increased. Therefore, the enzyme‐triggered fluorescence turn‐off/turn‐on of specific CDs successfully achieved sensitive detection of β‐glucosidase and monitored the effect of its inhibitors. This new strategy was applied to detect β‐glucosidase and monitor β‐glucosidase inhibitor in hepatoma cells using cell imaging. All results suggest that the new method is sensitive and promising for use in cancer diagnosis and treatment.     

Warmer night‐time temperature promotes microbial heterotrophic activity and modifies stream sediment community

Diel temperature patterns are changing because of global warming, with higher temperatures being predicted to be more pronounced at night. Biological reactions are temperature dependent, with some occurring only during the daylight hours (e.g., light photosynthesis) and other during the entire day (e.g., respiration). Consequently, we expect the modification of daily temperature cycles to alter microbial biological reactions in stream sediments. Here, we aimed to study the effect of warming and changes of the diel temperature patterns on stream sediment biofilm functions tied to organic carbon decomposition, as well as on biofilm meiofaunal community structure. We performed an eight‐week experiment with 12 artificial streams subjected to three different diel temperature patterns: warming, warmer nights and control. Significant effects of warming on biofilm function and structure were mainly detected in the long term. Our results showed that warming altered biofilm function, especially in the warmer nights’ treatment, which enhanced β‐glucosidase enzyme activity. Interestingly, clear opposite diel patterns were observed for dissolved organic carbon and β‐glucosidase activity, suggesting that, at night, sediment bacteria quickly consume the input of photosynthetic dissolved organic carbon labile compounds created during light‐time. The biofilm structure was also altered by warming, as both warming and warmer night treatments enhanced copepod abundance and diminished abundances of turbellaria and nematodes, which, in turn, controlled bacterial, algal and ciliate communities. Overall, we conclude that warming has strong effect on sediment biofilm structure and enhanced microbial organic matter degradation which might, consequently, affect higher trophic levels and river carbon cycling.     

Beta-xylosidase activity of a GH3 glucosidase/xylosidase from yak rumen metagenome promotes the enzymatic degradation of hemicellulosic xylans

Aims: To characterize the duel activities of a glycosyl hydrolase family 3 β‐glucosidase/xylosidase from rumen bacterial metagenome and to investigate the capabilities of its β‐d ‐xylosidase activities for saccharification of hemicellulosic xylans. Methods and Results: A β‐glucosidase/xylosidase gene RuBGX1 was cloned from yak (Bos grunniens) rumen using the metagenomic technology. Recombinant RuBGX1, expressed in Escherichia coli, demonstrated high hydrolytic activities on both p‐nitrophenyl‐β‐d ‐glucopyranoside (pNP‐Glc) and p‐nitrophenyl‐β‐d ‐xylopyranoside (pNP‐Xyl) substrates. Analysis of the kinetic properties indicated that RuBGX1 had a lower affinity for pNP‐Glc substrate as the K_m was 0·164 mmol l^?1 for pNP‐Glc and 0·03 mmol l^?1 for pNP‐Xyl at pH 6·0 and 50°C, respectively. The capabilities of RuBGX1 β‐xylosidase for hydrolysis of xylooligosaccharide substrates were further investigated using an endoxylanase‐coupled assay. Hydrolysis time courses illustrated that a significant increase (about 50%) in the reducing sugars, including xylobiose, xylotriose and xylotetraose, was achieved by supplementing endoxylanase with RuBGX1. Enzymatic product analysis using high‐performance anion‐exchange chromatography‐pulsed amperometric detection showed that RuBGX1 could release xyloses from intermediate xylooligosaccharides produced by endoxylanase. Conclusions: The RuBGX1 shows β‐glucosidase activity in hydrolysis of cello‐oligosaccharides; meanwhile, it has β‐xylosidase activity and functions synergistically with endoxylanase to promote the degradation of hemicellulosic xylans. Significance and Impact of the study: This was the first to report the β‐xylosidase activity of family 3 β‐glucosidase/xylosidase functioned in the degradation of hemicellulosic xylans. The bifunctional β‐glucosidase/xylosidase property of RuBGX1 can be used in simultaneous saccharification of cellulose and xylan into fermentable glucose and xylose.     

Isolation and basic characterization of a β‐glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture

Aims: To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular β‐glucosidase from this strain. Methods and Results: Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for β‐glycosidase activities. A strain of Lact. brevis showing high intracellular β‐d ‐glucosidase, β‐d ‐xylosidase and α‐l ‐arabinosidase activities was selected for purification and characterization of its β‐glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3·5. The K_m for p‐nitrophenyl‐β‐d ‐glucopyranoside and p‐nitrophenyl‐β‐d ‐xylopyranoside is 0·22 and 1·14 mmol l^?1, respectively. The β‐glucosidase activity was strongly inhibited by gluconic acid δ‐lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7·0 (t_1/2 = 50 day) but not at pH 4·0 (t_1/2 = 4 days). Conclusions: The β‐glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei ( Coulon et al. 1998 ) and Lactobacillus plantarum ( Sestelo et al. 2004 ). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long‐term stability could be of great interest for the release of aroma compounds during winemaking. Significance and Impact of the study: Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular β‐glucosidase isolated from a wine‐related strain of Lact. brevis.     

ATP and Ergosterol as Indicators of Fungal Biomass during Leaf Decomposition in Streams: a Comparative Study

Ergosterol and ATP concentrations, microbial respiration and sporulation rates of aquatic hyphomycetes associated with leaves of Castanea sativa decomposing in a 5^th order stream were determined periodically over a period of 102 days in order to compare ergosterol and ATP as indicators of fungal biomass. ATP and ergosterol concentrations exhibited a significant positive correlation (F = 4.459, DF = 28, P < 0.001) during the first stages of leaf breakdown (until day 39), i.e., during periods of increasing fungal biomass. No correlation was found between ATP and ergosterol concentrations during later stages of decomposition (days 39 to 102). Respiration rates increased rapidly up to 0.525 mg O₂ h^–1 g^–1 AFDM during the first month and remained high until the end of the experiment. Sporulation rates peaked at day 9 (1069 conidia day^–1 mg^–1 AFDM) and decreased during later stages of decomposition. ATP‐to‐biomass conversion factors were determined for both fungi (0.59 μmol ATP g^–1 dry mass) and bacteria (1.30 μmol ATP g^–1 dry mass) collected from the stream and grown in the laboratory. Estimates of fungal biomass based on ATP concentrations were similar to those calculated from ergosterol concentrations during the first 39 days of breakdown. The results here presented suggest that ATP is a reliable method to quantify microbial biomass in streams and that the relative importance of bacteria increases at later stages of decomposition. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Organic matter quantity and source affects microbial community structure and function following volcanic eruption on Kasatochi Island,Alaska

In August 2008, Kasatochi volcano erupted and buried a small island in pyroclastic deposits and fine ash; since then, microbes, plants and birds have begun to re‐colonize the initially sterile surface. Five years post‐eruption, bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) copy numbers and extracellular enzyme activity (EEA) potentials were one to two orders of magnitude greater in pyroclastic materials with organic matter (OM) inputs relative to those without, despite minimal accumulation of OM (< 0.2%C). When normalized by OM levels, post‐eruptive surfaces with OM inputs had the highest β‐glucosidase, phosphatase, NAGase and cellobiohydrolase activities, and had microbial population sizes approaching those in reference soils. In contrast, the strongest factor determining bacterial community composition was the dominance of plants versus birds as OM input vectors. Although soil pH ranged from 3.9 to 7.0, and %C ranged 100×, differentiation between plant‐ and bird‐associated microbial communities suggested that cell dispersal or nutrient availability are more likely drivers of assembly than pH or OM content. This study exemplifies the complex relationship between microbial cell dispersal, soil geochemistry, and microbial structure and function; and illustrates the potential for soil microbiota to be resilient to disturbance.     

Senescence‐induced loss in photosynthesis enhances cell wall β‐glucosidase activity

A link between senescence‐induced decline in photosynthesis and activity of β‐glucosidase is examined in the leaves of Arabidopsis. The enzyme is purified and characterized. The molecular weight of the enzyme is 58 kDa. It shows maximum activity at pH 5.5 and at temperature of 50°C. Photosynthetic measurements and activity of the enzyme are conducted at different developmental stages including senescence of leaves. Senescence causes a significant loss in total chlorophyll, stomatal conductance, rate of evaporation and in the ability of the leaves for carbon dioxide fixation. The process also brings about a decline in oxygen evolution, quantum yield of photosystem II (PS II) and quantum efficiency of PS II photochemistry of thylakoid membrane. The loss in photosynthesis is accompanied by a significant increase in the activity of the cell wall‐bound β‐glucosidase that breaks down polysaccharides to soluble sugars. The loss in photosynthesis as a signal for the enhancement in the activity of the enzyme is confirmed from the observation that incubation of excised mature leaves in continuous dark or in light with a photosynthesis inhibitor 3‐(3,4‐dichlorophenyl)‐1, 1‐dimethylurea (DCMU) that leads to sugar starvation enhances the activity of the enzyme. The work suggests that in the background of photosynthetic decline, the polysaccharides bound to cell wall that remains intact even during late phase of senescence may be the last target of senescing leaves for a possible source of sugar for remobilization and completion of the energy‐dependent senescence program.     

Fungal biomass associated with decaying leaf litter in a stream

Summary Fungal biomass, measured as ergosterol content, was determined on alder leaf litter incubated during autumn in a softwater Pyrenean stream. The ergosterol content of the leaf litter increased rapidly to a maximum of 462 μg/g detrital dry mass. Ergosterol contents of aquatic Hyphomycetes grown in shake culture were typically ≤5 mg/g mycelial dry mass. Using the corresponding ergosterol-to-biomass conversion factor of 200, peak fungal mass accounted for 9.2% of total system mass, or 10.2% of leaf dry mass. For the period of highest activity (incubation days 7–28), net fungal production on leaf litter was estimated as 2.3 mg d⁻¹ g⁻¹ leaf mass. A conservative estimate of the growth efficiency for the same period was 105 mg mycelial mass per gram leaf mass degraded, assuming that non-leaf organic matter did not constitute an important carbon source supporting fungal production.     

EXTRACELLULAR ENZYMES OF THE MICROCYSTIS AERUGINOSA PCC 7813 STRAIN ARE INHIBITED IN THE PRESENCE OF HYDROQUINONE AND PYROGALLOL,ALLELOCHEMICALS PRODUCED BY AQUATIC PLANTS1

Several cyanobacterial species have a high potential to dominate in marine environments and freshwater reservoirs, and the ecological and physiological reasons for this phenomenon are not understood comprehensively. In this study, the ability of a Microcystis aeruginosa Kütz. strain to produce free dissolved enzymes was documented. We have observed that this highly toxic strain releases alkaline phosphatase, leucine aminopeptidase, and β‐glucosidase into the ambient environment. Additionally, the inhibitory activity of selected phenols produced by aquatic plants on the activity of these enzymes was analyzed. The investigated compounds, pyrogallol and, to a lesser degree, hydroquinone, decreased the activity of extracellular enzymes produced by M. aeruginosa, with leucine aminopeptidase being the most sensitive to the inhibitors. The noncompetitive character of enzymatic inhibition suggests that the polyphenols produced by aquatic plants are able to influence the activity of different extracellular or membrane‐bound enzymes.     

The relation between glucovanillin, β-d-glucosidase activity and cellular compartmentation during the senescence, freezing and traditional curing of vanilla beans

The aim of this research was to improve our understanding of the mechanism of glucovanillin hydrolysis by β‐d ‐glucosidase activity in vanilla beans by studying their senescence, freezing and traditional curing. A batch of green pods from Madagascar was ripened at 30°C until fruits turned black; another batch was frozen for few days at ?18°C and defrosted at 35°C for 24 h and a third batch was cured using traditional methods. During treatments, samples were analysed for the yield of glucovanillin hydrolysis, and β‐glucosidase activity was measured. Cellular structures were also examined by light and transmission electron microscopy. Green fruits had a low yield of glucovanillin hydrolysis (<5%), a high level of β‐glucosidase activity (～1000 nkatal g^?1 fresh weight) and a perfect cellular integrity. Senescent fruits had a high yield of glucovanillin hydrolysis (>95%), no measurable β‐glucosidase activity and complete cellular degradation. Similar results were observed in beans after defrosting. During curing, beans had a medium yield of glucovanillin hydrolysis (<50%), no measurable β‐glucosidase activity and partial cellular degradation compared with senescent or defrosted beans. Results show that the mechanism of glucovanillin hydrolysis in vanilla beans is regulated by cellular compartmentation and that the β‐glucosidase activity level is not the limiting factor for complete hydrolysis. If total decompartmentation is obtained, then complete glucovanillin hydrolysis is observed even if most of the β‐glucosidase activity is lost. The β‐glucosidase activity level only has an effect on glucovanillin hydrolysis kinetics.     

Microbial enzyme activity at the watershed scale: response to chronic nitrogen deposition and acute phosphorus enrichment

Microbial enzymes play a critical role in organic matter decomposition and enzyme activity can dynamically respond to shifts in inorganic nutrient and substrate availability, reflecting the nutrient and energy limitation of the microbial community. We characterized microbial enzyme response to shifting nitrogen (N) and phosphorus (P) availability across terrestrial and aquatic environments at the Bear Brook Watershed in Maine, the site of a whole-watershed N enrichment experiment. We compared activity of β-1,4-glucosidase (BG); β-1,4-N-acetylglucosaminidase (NAG); acid phosphatase (AP) in soil, leaf litter in terrestrial and stream habitats and stream biofilms in a reference and N enriched watershed, representing whole-ecosystem response to chronic N enrichment. In addition, we used shorter, experimental P enrichments to address potential P limitation under ambient and elevated N availability. We found that BG and NAG activity were not affected by the long-term N enrichment in either habitat. Enhanced P limitation due to N enrichment was evident only in the aquatic habitats with 5- and 8-fold higher treated watershed AP activity in stream biofilms and stream litter, respectively. Acute P additions reduced AP activity and increased BG activity and these effects were also most pronounced in the streams. The stoichiometry of enzyme activity was constrained across ecosystem compartments with regression slopes for lnBG:lnNAG, lnBG:lnAP, and lnNAG:lnAP close to 1, ranging 1.142–1.241. We found that microbial enzyme response to shifting N and P availability varied among watershed compartments, typically with stronger effects in aquatic habitats. This suggests that understanding the response of ecosystem function to disturbance at the watershed scale requires simultaneous consideration of all compartments.     

Salinity affects microbial activity and soil organic matter content in tidal wetlands

Climate change‐associated sea level rise is expected to cause saltwater intrusion into many historically freshwater ecosystems. Of particular concern are tidal freshwater wetlands, which perform several important ecological functions including carbon sequestration. To predict the impact of saltwater intrusion in these environments, we must first gain a better understanding of how salinity regulates decomposition in natural systems. This study sampled eight tidal wetlands ranging from freshwater to oligohaline (0–2 ppt) in four rivers near the Chesapeake Bay (Virginia). To help isolate salinity effects, sites were selected to be highly similar in terms of plant community composition and tidal influence. Overall, salinity was found to be strongly negatively correlated with soil organic matter content (OM%) and C : N, but unrelated to the other studied environmental parameters (pH, redox, and above‐ and below‐ground plant biomass). Partial correlation analysis, controlling for these environmental covariates, supported direct effects of salinity on the activity of carbon‐degrading extracellular enzymes (β‐1, 4‐glucosidase, 1, 4‐β‐cellobiosidase, β‐D‐xylosidase, and phenol oxidase) as well as alkaline phosphatase, using a per unit OM basis. As enzyme activity is the putative rate‐limiting step in decomposition, enhanced activity due to salinity increases could dramatically affect soil OM accumulation. Salinity was also found to be positively related to bacterial abundance (qPCR of the 16S rRNA gene) and tightly linked with community composition (T‐RFLP). Furthermore, strong relationships were found between bacterial abundance and/or composition with the activity of specific enzymes (1, 4‐β‐cellobiosidase, arylsulfatase, alkaline phosphatase, and phenol oxidase) suggesting salinity's impact on decomposition could be due, at least in part, to its effect on the bacterial community. Together, these results indicate that salinity increases microbial decomposition rates in low salinity wetlands, and suggests that these ecosystems may experience decreased soil OM accumulation, accretion, and carbon sequestration rates even with modest levels of saltwater intrusion.     

A time for every season: soil aggregate turnover stimulates decomposition and reduces carbon loss in grasslands managed for bioenergy

A primary goal of many next‐generation bioenergy systems is to increase ecosystem services such as soil carbon (C) storage and nutrient retention. Evaluating whether bioenergy management systems are achieving these goals is challenging in part because these processes occur over long periods of time at varying spatial scales. Investigation of microbially mediated soil processes at the microbe scale may provide early insights into the mechanisms driving these long‐term ecosystem services. Furthermore, seasonal fluctuations in microbial activity are rarely considered when estimating whole ecosystem functioning, but are central to decomposition, soil structure, and realized C storage. Some studies have characterized extracellular enzyme activity within soil structures (aggregates); however, seasonal variation in decomposition at the microscale remains virtually unknown, particularly in managed ecosystems. As such, we hypothesize that temporal variation in aggregate turnover is a strong regulator of microbial activity, with important implications for decomposition and C and nitrogen (N) storage in bioenergy systems. We address variation in soil microbial extracellular enzyme activity spatially across soil aggregates and temporally across two growing seasons in three ecosystems managed for bioenergy feedstock production: Zea mays L. (corn) agroecosystem, fertilized and unfertilized reconstructed tallgrass prairie. We measured potential N‐acetyl‐glucosaminidase (NAG), β‐glucosidase (BG), β‐xylosidase (BX), and cellobiohydrolase (CB) enzyme activity. Aggregate turnover in prairie systems was driven by precipitation events and seasonal spikes in enzyme activity corresponded with aggregate turnover events. In corn monocultures, soil aggregates turned over early in the growing season, followed by increasing, albeit low, enzyme activity throughout the growing season. Independent of management system or sampling date, NAG activity was greatest in large macroaggregates (>2000 μm) and CB activity was greatest in microaggregates (<250 μm). High microbial activity coupled with greater aggregation in prairie bioenergy systems may reduce loss of soil organic matter through decomposition and increase soil C storage.     

Enzyme Production of the Edible Mushroom Pleorotus ostreatus in Shaken Cultures Completed with Agro‐Industrial Wastes

The enzyme production of the white‐rot fungus, the edible mushroom Pleurotus ostreatus, was determined in shaken culture media containing extracts of agro‐industrial wastes (tomato, potato and pepper residues) as an unique carbon source. The activity of β‐glucosidase, xylanase, laccase as well as manganese‐dependent and independent peroxidases was measured at 0, 3.5, 7.0, 10.5, 14.0, 17.5, 21.0, 24.5, 28.0 and 31.5 days of cultivation. A spectral mapping technique and non‐linear mapping were employed for the calculation of the relationships among the fermentation parameters, such as fermentation time, enzyme activity and selectivity of enzyme production. It was established that P. ostreatus produced β‐glucosidase, xylanase, laccase, manganese‐dependent and independent peroxidases in each culture medium and that the enzyme activities were higher in cultures containing agro‐industrial wastes than in the control containing glucose as a carbon source. The spectral mapping technique allowed demonstrating that the enzyme activities were the highest in the culture completed with pepper extract followed by cultures containing potato and tomato extracts. The differences among the selectivity of the enzyme activities were negligible up to 21.0 days of fermentation and reached the maximum at the end of the fermentation process. The production of laccase as well as manganese‐dependent and independent peroxidases showed similar patterns while the selectivity patterns of xylanase and β‐galactoside production were different. In addition, it became evident that the agro‐industrial wastes influenced the enzyme production in a distinct way.     

Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.