Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library

A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four open-reading frames (ORFs). The ORF, xyn10N18, encodes an endo-β-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical α8/ß8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0–12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of >40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cations Mn²⁺ and Co²⁺ and is dramatically reduced by Hg²⁺ and Cu²⁺. Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry.     

Effect of 1-MCP and low-temperature storage on postharvest conservation of camu–camu

Camu–camu, a native fruit from the Amazon region, is a rich source of bioactive compounds. However, its intense metabolic activity and high-water content limit the fruit’s postharvest storage and marketing. The aim of this study, conducted in two parts, was to evaluate the effects of 1-MCP and storage temperature on the physiology and postharvest preservation of camu–camu fruit. In part 1 of the study, fruit harvested at maturity stage 3 were divided into groups: control, 1-methylcyclopropene (1-MCP; 900 nL L^?1; 12 h) and ethylene (1000 µL L^?1; 24 h) and were stored at 22?±?1 °C and 85?±?5% RH for 9 days. In part 2, fruit harvested at maturity stage 3 were stored at 5, 10, 15, 20, or 25?±?1 °C and 85?±?5% RH for 9 days. During storage, fruit were evaluated daily for decay, mass loss, respiratory activity, and ethylene production, and every 3 days they were evaluated for peel color, pulp firmness, soluble solids content, total titratable acidity, ascorbic acid, total chlorophyll, and total anthocyanins. Fruit treated with 1-MCP exhibited delayed ripening due to lower metabolic activity, as evidenced by delay to softening, reduced mass loss and no decay. Storage at 5 °C prevented ethylene production, mass loss, color changes, and maintained pulp firmness, while did not affect soluble solids content. The results indicated that storage of camu–camu fruit at 5 °C or at 25 °C following application of 900 nL L^?1 1-MCP were effective strategies to delay ripening and maintain fruit quality up to 9 days.     

Purification and characterization of polygalacturonase produced by thermophilic <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Heterologous expression and characterization of a malathion-hydrolyzing carboxylesterase from a thermophilic bacterium, Alicyclobacillus tengchongensis

A carboxylesterase gene from thermophilic bacterium, Alicyclobacillus tengchongensis, was cloned and expressed in Escherichia coli BL21 (DE3). The gene coded for a 513 amino acid protein with a calculated molecular mass of 57.82 kDa. The deduced amino acid sequence had structural features highly conserved among serine hydrolases, including Ser204, Glu325, and His415 as a catalytic triad, as well as type-B carboxylesterase serine active site (FGGDPENITIGGQSAG) and type-B carboxylesterase signature 2 (EDCLYLNIWTP). The purified enzyme exhibited optimum activity with β-naphthyl acetate at 60 °C and pH 7 as well as stability at 25 °C and pH 7. One unit of the enzyme hydrolyzed 5 mg malathion l^?1 by 50 % within 25 min and 89 % within 100 min. The enzyme strongly degraded malathion and has a potential use for the detoxification of malathion residues.     

Temperature sensitivity of biomass‐specific microbial exo‐enzyme activities and CO2 efflux is resistant to change across short‐ and long‐term timescales

Accurate representation of temperature sensitivity (Q₁₀) of soil microbial activity across time is critical for projecting soil CO₂ efflux. As microorganisms mediate soil carbon (C) loss via exo‐enzyme activity and respiration, we explore temperature sensitivities of microbial exo‐enzyme activity and respiratory CO₂ loss across time and assess mechanisms associated with these potential changes in microbial temperature responses. We collected soils along a latitudinal boreal forest transect with different temperature regimes (long‐term timescale) and exposed these soils to laboratory temperature manipulations at 5, 15, and 25°C for 84 days (short‐term timescale). We quantified temperature sensitivity of microbial activity per g soil and per g microbial biomass at days 9, 34, 55, and 84, and determined bacterial and fungal community structure before the incubation and at days 9 and 84. All biomass‐specific rates exhibited temperature sensitivities resistant to change across short‐ and long‐term timescales (mean Q₁₀ = 2.77 ± 0.25, 2.63 ± 0.26, 1.78 ± 0.26, 2.27 ± 0.25, 3.28 ± 0.44, 2.89 ± 0.55 for β‐glucosidase, N‐acetyl‐β‐d ‐glucosaminidase, leucine amino peptidase, acid phosphatase, cellobiohydrolase, and CO₂ efflux, respectively). In contrast, temperature sensitivity of soil mass‐specific rates exhibited either resilience (the Q₁₀ value changed and returned to the original value over time) or resistance to change. Regardless of the microbial flux responses, bacterial and fungal community structure was susceptible to change with temperature, significantly differing with short‐ and long‐term exposure to different temperature regimes. Our results highlight that temperature responses of microbial resource allocation to exo‐enzyme production and associated respiratory CO₂ loss per unit biomass can remain invariant across time, and thus, that vulnerability of soil organic C stocks to rising temperatures may persist in the long term. Furthermore, resistant temperature sensitivities of biomass‐specific rates in spite of different community structures imply decoupling of community constituents and the temperature responses of soil microbial activities.     

Afforestation with Norway spruce on a subalpine pasture alters carbon dynamics but only moderately affects soil carbon storage

There is a strong trend toward reforestation of abandoned grasslands in alpine regions which may impact the carbon balance of alpine ecosystems. Here, we studied the effects of afforestation with Norway spruce (Picea abies L.) on an extensively grazed subalpine pasture in Switzerland on soil organic carbon (SOC) cycling and storage. Along a 120-year long chronosequence with spruce stands of 25, 30, 40, 45, and >120 years and adjacent pastures, we measured tree biomass, SOC stocks down to the bedrock, natural ¹³C abundances, and litter quality. To unravel controls on SOC cycling, we have monitored microclimatic conditions and quantified SOC decomposability under standardized conditions as well as soil respiration in situ. Stocks of SOC were only moderately affected by the afforestation: in the mineral soil, SOC stocks transiently decreased after tree establishment, reaching a minimum 40–45 years after afforestation (?25 %) and increased thereafter. Soils of the mature spruce forest stored the largest amount of SOC, 13 % more than the pasture soils, mainly due to the accumulation of an organic layer (23 t C ha^?1). By comparison, C accumulated in the tree biomass exceeded the SOC pool by a factor of three in the old forest. In contrast to the small impact on C storage, afforestation strongly influenced the composition and quality of the soil organic matter (SOM). With increasing stand age, δ¹³C values of the SOM became consistently more positive, which can be interpreted as a gradual replacement of grass- by spruce-derived C. Fine roots of spruce were enriched in ¹³C, in lignin and had a higher C/N ratio in comparison to grass roots. As a consequence, SOM quality as indicated by the lower fraction of readily decomposable (labile) SOM and higher C:N ratios declined after the land-use change. Furthermore, spruce plantation induced a less favorable microclimate for microbial activity with the average soil temperature during the growing season being 5 °C lower in the spruce stands than in the pasture. In situ soil respiration was approximately 50 % lower after the land use conversion, which we primarily attribute to the colder conditions and the lower SOM quality, but also to drier soils (?25 %) and to a decreased fine root biomass (?40 %). In summary, afforestation on subalpine pastures only moderately affected SOC storage as compared to the large C sink in tree biomass. In contrast, SOC cycling rates strongly decreased as a result of a less favorable microclimate for decomposition of SOM, a lower C input by roots, and a lower litter quality.     

The importance of amino sugar turnover to C and N cycling in organic horizons of old-growth Douglas-fir forest soils colonized by ectomycorrhizal mats

Amino sugar dynamics represent an important but under-investigated component of the carbon (C) and nitrogen (N) cycles in old-growth Douglas-fir forest soils. Because fungal biomass is high in these soils, particularly in areas colonized by rhizomorphic ectomycorrhizal fungal mats, organic matter derived from chitinous cell wall material (or the monomeric building block of chitin, N-acetylglucosamine (NAG)) could be a significant source of C or N to the soil microbiota, and thus an important driver of microbial C and N processing. This paper reports the results of incubation experiments initiated to measure chitin degradation, NAG utilization, and the contribution of these substrates to soil respiration and N mineralization rates in mat-colonized and non-mat soil organic horizons. Amendments of chitin and NAG stimulated respiration, N mineralization, and biomass accumulation in mat and non-mat soils, and responses to NAG amendment were stronger than to chitin amendment. NAG-induced respiration was consistently two-fold higher in mat soils than non-mat soils, but induced N mineralization was similar between the two soil patch types. Assimilation of both C and N into microbial biomass was apparent, biomass C:N ratio decreased in all treatments, and microbial N use efficiency (treatment means 0.25 ± 0.06–0.50 ± 0.05) was greater than C use efficiency (treatment means 0.12 ± 0.04–0.32 ± 0.02). NAGase enzyme response was non-linear and showed the same pattern in chitin and NAG amendments. Responses to NAG and chitin amendment differed between mat and non-mat soils, indicating different mechanisms driving NAG and chitin utilization or differences in saprotrophic community composition between the two soil patch types. Net chitin and NAG processing rates were 0.08–3.4 times the basal respiration rates and 0.07–14 times the ambient net N mineralization rates, high enough for the turnover of total soil amino sugars to potentially occur in days to weeks. The results support the hypotheses that amino sugars are important microbial C and N sources and drivers of C and N cycling in these soils.     

Will CO<Subscript>2</Subscript> Emissions from Drained Tropical Peatlands Decline Over Time? Links Between Soil Organic Matter Quality,Nutrients, and C Mineralization Rates

Conversion, drainage, and cultivation of tropical peatlands can change soil conditions, shifting the C balance of these systems, which is important for the global C cycle. We examined the effect of soil organic matter (SOM) quality and nutrients on CO₂ production from peat decomposition using laboratory incubations of Indonesian peat soils from undrained forest in Kalimantan and drained oil palm plantations in Kalimantan and Sumatra. We found that oil palm soils had higher C/N and lower SOM quality than forest soils. Higher substrate quality and nutrient availability, particularly lower ratios of aromatic/aliphatic carbon and C/N, rather than total SOM or carbon, explained the higher rate of CO₂ production by forest soils (10.80 ± 0.23 µg CO₂–C g C h^?1) compared to oil palm soils (5.34 ± 0.26 µg CO₂–C g C h^?1) from Kalimantan. These factors also explained lower rates in Sumatran oil palm (3.90 ± 0.25 µg CO₂–C g C h^?1). We amended peat with nitrogen (N), phosphorus (P), and glucose to further investigate observed substrate and nutrient constraints across the range of observed peat quality. Available N limited CO₂ production, in unamended and amended soils. P addition raised CO₂ production when substrate quality was high and initial P state was low. Glucose addition raised CO₂ production in the presence of added N and P. Our results suggest that decline in SOM quality and nutrients associated with conversion may decrease substrate-driven rates of CO₂ production from peat decomposition over time.     

Decomposition and stabilization of root litter in top- and subsoil horizons: what is the difference?

Mechanisms leading to high mean residence times of organic matter in subsoil horizons are poorly understood. In lower parts of the soil profile root material contributes greatly to soil organic matter (SOM). The objective of this study was to elucidate the decomposition dynamics of root-derived C and N in different soil depths during a 3 year field experiment and to examine the importance of different protection mechanisms as well as abiotic factors for the decomposition dynamics. Additionally, we assessed the effect of root litter addition on native SOM. Our conceptual approach included the exposure of litterbags with ¹³C and ¹⁵N labeled wheat root material mixed to loamy agricultural soil at three different soil depths (30, 60 and 90 cm). During the incubation period, we monitored soil temperature, humidity and the incorporation of root derived C and N into the soil microbial biomass and physical SOM fractions. Our results showed that abiotic decay conditions were better in subsurface horizons compared to the topsoil. Root litter addition significantly increased the size of microbial biomass in all three soil horizons. In the topsoil, root-derived C decomposition was significantly higher in the first 6 months of incubation compared to subsoil horizons. In 60 and 90 cm depths, a lag phase with development of soil microbial biomass seemed to be prevailing before decomposition was activated. For root-derived N, similar decomposition kinetics could be observed in top- and subsoil horizons. Despite of higher SOM contents, better soil structure and higher microbial activity in the topsoil horizon compared to subsoil horizons, the amounts of root-derived C and N remaining after 3 years were similar for all three depths. Most of the root-derived C and N was present as organo-mineral complexes or occluded in soil aggregates (oPOM), illustrating similar importance of these two protection mechanisms in all three soil depths. Addition of fresh root litter caused small losses of native soil C whereas native N was retained. We conclude that despite of similar SOM protection mechanisms, there are distinct differences in decomposition dynamics of root litter between top- and subsoil horizons. In the long run, the better abiotic decay conditions prevailing in subsoil horizons may compensate for their poorer physico-chemical characteristics.     

The attractive recombinant phytase from Bacillus licheniformis: biochemical and molecular characterization

The phyL gene encoding phytase from the industrial strain Bacillus licheniformis ATCC 14580 (PhyL) was cloned, sequenced, and overexpressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant enzyme has an apparent molecular weight of nearly 42 kDa. Interestingly, this enzyme was optimally active at 70–75 °C and pH 6.5–7.0. This enzyme is distinguishable by the fact that it preserved more than 40 % of its activity at wide range of temperatures from 4 to 85 °C. This new phytase displayed also a high specific activity of 316 U/mg. For its maximal activity and thermostability, this biocatalyst required only 0.6 mM of Ca²⁺ ion and exhibited high catalytic efficiency of 8.3 s^?1 μM^?1 towards phytic acid.     

How interactions between microbial resource demands,soil organic matter stoichiometry,and substrate reactivity determine the direction and magnitude of soil respiratory responses to warming

Recent empirical and theoretical advances inform us about multiple drivers of soil organic matter (SOM) decomposition and microbial responses to warming. Absent from our conceptual framework of how soil respiration will respond to warming are adequate links between microbial resource demands, kinetic theory, and substrate stoichiometry. Here, we describe two important concepts either insufficiently explored in current investigations of SOM responses to temperature, or not yet addressed. First, we describe the complete range of responses for how warming may change microbial resource demands, physiology, community structure, and total biomass. Second, we describe how any relationship between SOM activation energy of decay and carbon (C) and nitrogen (N) stoichiometry can alter the relative availability of C and N as temperature changes. Changing availabilities of C and N liberated from their organic precursors can feedback to microbial resource demands, which in turn influence the aggregated respiratory response to temperature we observe. An unsuspecting biogeochemist focused primarily on temperature sensitivity of substrate decay thus cannot make accurate projections of heterotrophic CO₂ losses from diverse organic matter reservoirs in a warming world. We establish the linkages between enzyme kinetics, SOM characteristics, and potential for microbial adaptation critical for making such projections. By examining how changing microbial needs interact with inherent SOM structure and composition, and thus reactivity, we demonstrate the means by which increasing temperature could result in increasing, unchanging, or even decreasing respiration rates observed in soils. We use this exercise to highlight ideas for future research that will develop our abilities to predict SOM feedbacks to climate.     

Endogenous arabitol and mannitol improve shelf life of encapsulated <Emphasis Type="Italic">Metarhizium brunneum</Emphasis>

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6?±?0.2 and 2.1?±?0.2 µg/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1?±?4.4 to 55.0?±?6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5?±?3.3% at 5 °C followed by 54.5?±?1.6% at 18 °C and 19.4?±?1.3% at 25 °C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3?±?7.6 to 98.0?±?1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.     

Continuous biohydrogen production from fruit wastewater at low pH conditions

Biohydrogen production from a simulated fruit wastewater (soluble COD = 3.17 ± 0.10 g L^?1) was carried out in a continuous stirred tank reactor (CSTR) of 2 L operational volume without biomass inoculation, heat pre-treatment or pH adjustment, resulting in a low operational pH (3.75 ± 0.09). The hydraulic retention time (HRT) varied from 15 to 5 h. A strong negative correlation (p < 0.01) between the biogas production rate and the HRT was observed. Biogas production rates were higher at 30 °C than at 25 °C (p < 0.01), when the CSTR was operated under the same HRT. The biogas hydrogen content was estimated as high as 55.8 ± 2.3 % and 55.4 ± 2.5 % at 25 and 30 °C, respectively. The main fermentation end products were acetic and butyric acids, followed by ethanol. Significant differences (p < 0.01) during the operation of the CSTR at 25 or 30 °C were identified for butyric acid at almost all HRTs examined. Simulation of the acidogenesis process in the CSTR (based on COD and carbon balances) indicated the possible metabolic compounds produced at 25 and 30 °C reactions and provided an adequate fit of the experimental data.     

Acute Toxicity of Arsenic under Different Temperatures and Salinity Conditions on the White Shrimp Litopenaeus vannamei

The aim of this study was to determine acute toxicity in the post larvae of the white shrimp Litopenaeus vannamei after 96 h of exposure to dissolved arsenic under three different temperatures and salinity conditions. Recent reports have shown an increase in the presence of this metalloid in coastal waters, estuaries, and lagoons along the Mexican coast. The white shrimp stands out for its adaptability to temperature and salinity changes and for being the main product for many commercial fisheries; it has the highest volume of oceanic capture and production in Mexican shrimp farms. Lethal concentrations (LC₅₀–96 h) were obtained at nine different combinations (3?×?3 combinations in total) of temperature (20, 25, and 30 °C) and salinity (17, 25, and 33) showing mean LC₅₀–96 h values (±standard error) of 9.13?±?0.76, 9.17?±?0.56, and 6.23?±?0.57 mgAs?L^?1(at 20 °C and 17, 25, and 33 salinity); 12.29?±?2.09, 8.70?±?0.82, and 8.03?±?0.59 mgAs?L^?1 (at 25 °C and 17, 25, and 33 salinity); and 7.84?±?1.30, 8.49?±?1.40, and 7.54?±?0.51 mgAs?L^?1 (at 30 °C and 17, 25, and 33 salinity), respectively. No significant differences were observed for the optimal temperature and isosmotic point of maintenance (25 °C–S 25) for the species, with respect to the other experimental conditions tested, except for at 20 °C–S 33, which was the most toxic. Toxicity under 20 °C–S 33 conditions was also higher than 25 °C–S 17 and 20 °C (S 17 or 25). The least toxic condition was 25 °C–S 17. All this suggests that the toxic effect of arsenic is not affected by temperature changes; it depends on the osmoregulatory pattern developed by the shrimp, either hyperosmotic at low salinity or hiposmotic at high salinity, as observed at least on the extreme salinity conditions here tested (17 and 33). However, further studies testing salinities near the isosmotic point (between 20 and 30 salinities) are needed to clarify these mechanisms.     

Temperature-dependent age-specific demography of grapevine moth (Lobesia botrana) (Lepidoptera: Tortricidae): jackknife vs. bootstrap techniques

Grapevine moth, Lobesia botrana (Lep. Tortricidae) is a key pest of grape in Iran and other vineyards of the world. In this study, eight constant rearing temperatures (5, 10, 15, 20, 25, 30, 32 and 35 ± 1 °C) along with 60 ± 10% RH and a 16:8 (L:D) h photoperiod were chosen for demographic studies of the grapevine moth. Immature stages were unable to develop when reared at 5 and 35 °C, and the progeny moths were unable to successfully mate at 10, 15 and 32 °C. The overall developmental time of juveniles decreased at 30 °C (from 320.7 ± 3.4 d at 10 °C to 34.2 ± 0.2 d) followed by an increase to 42.5 ± 0.6 d at 32 °C. Based on values of the stable population growth parameters, the temperature of 25 °C was found to be optimal for propagation of grapevine moth. The highest values of the intrinsic rate of increase, gross and net reproductive rates were 0.0719 d?^?1, 55.5 and 23 females per generation, respectively, at 25 °C. Since jackknife and bootstrap estimates of mean and standard error were mainly similar, both methods may equally be used for uncertainty estimates. Our data suggest that cold storage of grapes will help to control grapevine moth infestations and damage. In many grape growing regions of Iran, the first generation is expected to cause damage. It is expected since our reproductive life table analysis suggests that the hot summer temperatures may restrict pest development during subsequent generations.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.     

Kinetic comparisons of mesophilic and thermophilic aerobic biomass

Kinetic parameters describing growth and decay of mesophilic (30°C) and thermophilic (55°C) aerobic biomass were determined in continuous and batch experiments by using oxygen uptake rate measurements. Biomass was cultivated on a single soluble substrate (acetate) in a mineral medium. The intrinsic maximum growth rate (μ _max) at 55°C was 0.71±0.09 h⁻¹, which is 1.5 times higher than the μ _max at 30°C (0.48±0.11 h⁻¹). The biomass decay rates increased from 0.004 h⁻¹ at 30°C to 0.017 h⁻¹ at 55°C. Monod constants were very low for both types of biomass: 9±2 mg chemical oxygen demand (COD) l⁻¹at 30°C and 3±2 mg COD l⁻¹at 55°C. Theoretical biomass yields were similar at 30 and 55°C: 0.5 g biomass COD (g acetate COD)⁻¹. The observed biomass yields decreased under both temperature conditions as a function of the cell residence time. Under thermophilic conditions, this effect was more pronounced due to the higher decay rates, resulting in lower biomass production at 55°C compared to 30°C. Electronic Publication     

Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry

Objectives

To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing.

Results

A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T ₅₀ value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k _cat value were 42,734 U mg^?1 and 189 s^?1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml^?1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively.

Conclusions

The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

     

Root exudate components change litter decomposition in a simulated rhizosphere depending on temperature

The release of root exudates into the rhizosphere is known to enhance soil biological activity and alter microbial community structure. To assess whether root exudates also stimulated litter decomposition, in a rhizosphere model system we continuously injected solutions of glucose, malate or glutamate through porous Rhizon^® soil solution samplers into the soil at rhizosphere concentrations. The effect of these substances on the decomposition of ¹⁴C-labelled Lolium perenne shoot residues present in the soil was evaluated by monitoring ¹⁴CO₂ evolution at either 15°C or 25°C. The incorporation of the ¹⁴C into the microbial biomass and appearance in the dissolved organic matter (DOM) pool was estimated after 32 d incubation. The presence of malate and glutamate increased the mineralization of L. perenne residues by approximately 20% relative to the soil without their addition at 15°C, however, no significant effects on residue decomposition were observed at 25°C. The incorporation of the ¹⁴C-label into the microbial biomass and DOM pool was not affected by the addition of either glucose, malate or glutamate. Although nearly the same amount of L. perenne residues were mineralized at either temperature after 32 d, less ¹⁴C was recovered in the microbial biomass and DOM pools at 25°C compared to 15°C. Alongside other results, this suggests that the rate of microbial turnover is greater at 25°C compared to 15°C. We conclude that the addition of labile root exudate components to the rhizosphere induced a small but significant increase on litter decomposition but that the magnitude of this effect was regulated by temperature.     

Root carbon inputs to the rhizosphere stimulate extracellular enzyme activity and increase nitrogen availability in temperate forest soils

The exudation of carbon (C) by tree roots stimulates microbial activity and the production of extracellular enzymes in the rhizosphere. Here, we investigated whether the strength of rhizosphere processes differed between temperate forest trees that vary in soil organic matter (SOM) chemistry and associate with either ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi. We measured rates of root exudation, microbial and extracellular enzyme activity, and nitrogen (N) availability in samples of rhizosphere and bulk soil influenced by four temperate forest tree species (i.e., to estimate a rhizosphere effect). Although not significantly different between species, root exudation ranged from 0.36 to 1.10 g C m^?2 day^?1, representing a small but important transfer of C to rhizosphere microbes. The magnitude of the rhizosphere effects could not be easily characterized by mycorrhizal associations or SOM chemistry. Ash had the lowest rhizosphere effects and beech had the highest rhizosphere effects, representing one AM and one ECM species, respectively. Hemlock and sugar maple had equivalent rhizosphere effects on enzyme activity. However, the form of N produced in the rhizosphere varied with mycorrhizal association. Enhanced enzyme activity primarily increased amino acid availability in ECM rhizospheres and increased inorganic N availability in AM rhizospheres. These results show that the exudation of C by roots can enhance extracellular enzyme activity and soil-N cycling. This work suggests that global changes that alter belowground C allocation have the potential to impact the form and amount of N to support primary production in ECM and AM stands.