Extracellular Enzyme Assays Research Articles

Genetic and Biochemical Characterization of Monokaryotic Progeny Strains of Button Mushroom (Agaricus bisporus)

To promote the selection of promising monokaryotic strains of button mushroom (Agaricus bisporus) during breeding, 61 progeny strains derived from basidiospores of two different lines of dikaryotic parental strains, ASI1038 and ASI1346, were analyzed by nucleotide sequencing of the intergenic spacer I (IGS I) region in their rDNA and by extracellular enzyme assays. Nineteen different sizes of IGS I, which ranged from 1,301 to 1,348 bp, were present among twenty ASI1346-derived progeny strains, while 15 different sizes of IGS I, which ranged from 700 to 1,347 bp, were present among twenty ASI1038-derived progeny strains. Phylogenetic analysis of the IGS sequences revealed that different clades were present in both the ASI10388- and ASI1346-derived progeny strains. Plating assays of seven kinds of extracellular enzymes (β-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease) also revealed apparent variation in the ability to produce extracellular enzymes among the 40 tested progeny strains from both parental A. bisporus strains. Overall, this study demonstrates that characterization of IGS I regions and extracellular enzymes is useful for the assessment of the substrate-degrading ability and heterogenicity of A. bisporus monokaryotic strains.

Responses of soil fungi to logging and oil palm agriculture in Southeast Asian tropical forests.

Human land use alters soil microbial composition and function in a variety of systems, although few comparable studies have been done in tropical forests and tropical agricultural production areas. Logging and the expansion of oil palm agriculture are two of the most significant drivers of tropical deforestation, and the latter is most prevalent in Southeast Asia. The aim of this study was to compare soil fungal communities from three sites in Malaysia that represent three of the most dominant land-use types in the Southeast Asia tropics: a primary forest, a regenerating forest that had been selectively logged 50years previously, and a 25-year-old oil palm plantation. Soil cores were collected from three replicate plots at each site, and fungal communities were sequenced using the Illumina platform. Extracellular enzyme assays were assessed as a proxy for soil microbial function. We found that fungal communities were distinct across all sites, although fungal composition in the regenerating forest was more similar to the primary forest than either forest community was to the oil palm site. Ectomycorrhizal fungi, which are important associates of the dominant Dipterocarpaceae tree family in this region, were compositionally distinct across forests, but were nearly absent from oil palm soils. Extracellular enzyme assays indicated that the soil ecosystem in oil palm plantations experienced altered nutrient cycling dynamics, but there were few differences between regenerating and primary forest soils. Together, these results show that logging and the replacement of primary forest with oil palm plantations alter fungal community and function, although forests regenerating from logging had more similarities with primary forests in terms of fungal composition and nutrient cycling potential. Since oil palm agriculture is currently the mostly rapidly expanding equatorial crop and logging is pervasive across tropical ecosystems, these findings may have broad applicability.

Seasonal dynamics of fungal communities in a temperate oak forest soil

Fungi are the agents primarily responsible for the transformation of plant-derived carbon in terrestrial ecosystems. However, little is known of their responses to the seasonal changes in resource availability in deciduous forests, including photosynthate allocation below ground and seasonal inputs of fresh litter. Vertical stratification of and seasonal changes in fungal abundance, activity and community composition were investigated in the litter, organic and upper mineral soils of a temperate Quercus petraea forest using ergosterol and extracellular enzyme assays and amplicon 454-pyrosequencing of the rDNA-ITS region. Fungal activity, biomass and diversity decreased substantially with soil depth. The highest enzyme activities were detected in winter, especially in litter, where these activities were followed by a peak in fungal biomass during spring. The litter community exhibited more profound seasonal changes than did the community in the deeper horizons. In the litter, saprotrophic genera reached their seasonal maxima in autumn, but summer typically saw the highest abundance of ectomycorrhizal taxa. Although the composition of the litter community changes over the course of the year, the mineral soil shows changes in biomass. The fungal community is affected by season. Litter decomposition and phytosynthate allocation represent important factors contributing to the observed variations.

Methodologies for Extracellular Enzyme Assays from Wetland Soils

Measurement of extracellular enzymic activity in wetland soils can give an indication of the ecosystems biogeochemical processes, and rates of nutrient and carbon cycling. Analysis of these have allowed researchers to gain an understanding of the ecosystems’ microbial ecology and how it can be affected by environmental factors. Here we give a detailed description of the assays necessary to determine the activity of a suite of key hydrolase enzymes and phenol oxidases. These enzymes control the rates of decomposition and consequently the production of biogenic greenhouse gases. Knowing the processes responsible for the breakdown of organic matter is therefore essential if it becomes necessary to curb these emissions. Our protocols allow for cost effective analysis of a large number of samples and provide sufficient accuracy to determine differences between soil types. When coupled with contemporary microbial techniques these enzyme assays permit entire biochemical pathways to be determined, giving unparalleled knowledge on the processes involved in wetland ecosystems.

Fine scale variability in soil extracellular enzyme activity is insensitive to rain events and temperature in a mesic system

Abstract While soil extracellular enzyme assays (EEAs) are frequently used to infer soil microbial function, the data typically reflect a small number of sampling points across a season, and it is unclear to what extent soil EEA may vary on the time scale of days to weeks. Rain events, in particular, may cause rapid shifts in EEA, and fine scale temporal data are needed to properly assess the generality of EEA data collected at coarser time scales. We examined soil EEA 2–3 times per week in the field from June to November in the context of natural rain events and temperature fluctuations, and explored how long-term water addition altered EEA responses. We also tested the short-term effects of water addition on the distribution of EEA in intact soil mesocoms and leachate. There was little temporal variation in EEA for the hydrolases phosphatase, N-acetyl-glucosaminidase and β-glucosidase, despite the occurrence of multiple large rain events and large soil temperature fluctuations. Phenol oxidase activity correlated significantly with seasonal trends in temperature and soil moisture, but was highly variable at short time scales, and the latter did not correlate significantly with short-term changes in soil microclimate. EEA generally increased in response to long-term water addition, and in soil mesocosms water addition did not significantly redistribute EEA among the upper and lower soil layers, and leachate EEA was three orders of magnitude lower than soil EEA. Overall, our results reveal relatively minor short-term variation in EEA for hydrolase enzymes, and no discernable response to temperature fluctuations or precipitation over the short term. However, high short-term variation in phenol oxidase activity suggests that it may be difficult to infer temporal trends in EEA for this enzyme from a limited number of sampling points.

Cellulolytic Enzymes Production from Submerged Fermentation of Different Substrates by Newly Isolated Bacillus spp. FME

Newly isolated strains Bacillus sp. FME 1 and FME 2 were evaluated for the cellulolytic enzymes production during submerged fermentation (SmF) of different substrates including rice husk, Whatman filter paper and cellulose powder CF 11. Extracellular enzyme assays for CMCase, FPase and β-glucosidase were examined up to 8 days of submerged fermentation. Among the three substrates, rice husk was the most suitable substrate for higher production of cellulolytic enzymes. Maximum titers of 100, 45, and 3.5 U/mL in respect of CMCase, FPase and β-glucosidase in Bacillus sp. FME 2 were recovered as against 45, 12, and 0.39 U/mL in Bacillus sp. FME 1 respectively, at their respective peak time intervals. Bacillus sp. FME 2 was found to produce higher cellulolytic enzyme activities than Bacillus sp. FME 1.

Impact of coleopteran targeting toxin (Cry3Bb1) of Bt corn on microbially mediated decomposition

Genetically engineered corn expressing crystalline proteins for insect control and encoded by genes derived from soil bacterium Bacillus thuringiensis ( Bt) are widely adopted in the United States. Among the seven different events of Bt corn available commercially, YieldGard ® Rootworm (MON863) expresses a variant of the cry3Bb1 protein in the root tissue to control corn rootworm larvae. Although numerous laboratory and field studies show no unexpected ecological risks at the insect community-level above-ground, few studies have addressed the possible impact of cry proteins released from living or decaying roots of Bt corn on soil microbial communities. Here, we test the hypothesis that coleopteran-active Bt corn does not affect nontarget ecological processes, such as decomposition or the function of the associated saprophytic microbial community. Experimental treatments were: (1) a Bt hybrid; (2) a non- Bt, isogenic hybrid treated with a conventional soil insecticide; and (3) a non- Bt, isogenic hybrid without insecticide. Soil and root samples were collected at various times throughout 2 years from experimental plots to estimate microbial community function by quantifying activity of extracellular enzymes on 10 substrates. Decomposition was measured as mass loss by root decay in litter bags. Bt corn (MON863) exuding the cry3Bb1 toxin does not appear to have adverse effects on saprophytic microbial communities of soil and decaying roots or on decomposition. The addition of the soil insecticide had greater effects on microbial function in soil and decaying roots than Bt corn. Our results are similar to those found previously for the cry3Bb1 protein that showed no adverse effects on microbial community composition in controlled and natural environments. This field study is one of the first to report the use of extracellular enzyme assays to examine the effect of transgenic crops on the functional activity of microbes in soil and decaying roots.

High levels of microbial biomass and activity in unvegetated tropical and temperate alpine soils

Barren high-altitude soils are among the most extreme terrestrial environments on Earth. The present study was undertaken to quantify broad-scale patterns of total microbial biomass in unvegetated soils in the subnival zone of the Colorado Front Range and the high Andes of Perú. In order to better understand the limiting factors and substrates used by this community, we measured microbial biomass C, dissolved organic carbon (DOC), total organic carbon (TOC), soil gravimetric water content, and extracellular enzyme activity. To further investigate substrate limitation in these alpine soils, respiration after substrate addition was measured for samples from three sites in Colorado. In general, the abundance of microbes in these soils is positively correlated with soil water content. However, Perú talus soils had higher average microbial biomass than Colorado soils despite the Perú soils being higher in altitude and drier than the Colorado sites. Furthermore, the activity of the heterotrophic portion of the microbial community appears to be limited first by carbon and then by phosphorus as indicated both by results from extracellular enzyme assays and substrate addition experiments.

Soil microbial community responses to altered lignin biosynthesis in Populus tremuloides vary among three distinct soils

The development and use of transgenic plants has steadily increased, but there are still little data about the responses of soil microorganisms to these genetic modifications. We utilized a greenhouse trial approach to evaluate the effects of altered stem lignin in trembling aspen (Populus tremuloides) on soil microbial communities in three soils which differed in their chemical and physical properties; they included a sandy loam (CO-Colorado), a silt loam (KS-Kansas), and a clay loam (TX-Texas). Three transgenic aspen lines were developed from a natural clone common to the Great Lakes region of North America. The concentrations of stem lignin concentrations were reduced by 35% (Line 23), 40% (Line 141) and 50% (Line 72). Line 72 and Line 141 also had a 40 and 20% increase in syringyl-type stem lignin, respectively. Indirectly, these modifications resulted in increased (5–13%) and decreased (−5 to −57%) levels of root production across the lines and soil types. Responses of the soil microbial communities were investigated using: phospholipid fatty acids (PLFA), neutral lipid fatty acids (NLFA), and 3) extracellular enzyme assays. PLFA analyses indicated that there were large differences in microbial community composition between the three soils. Similarly, there were large differences in total NLFA between soils, with the KS soils having the highest amount and CO the lowest. Enzyme activities did not differ between soils, except for cellubiohydrolase, which was highest in CO soil. Across all three soils, responses to the four genetic lines were not consistent. Interactions between soil type and genetic line make it difficult to assess the potential ecological impacts of transgenic aspen on soil microbial communities and their associated functions. Given these interactions, field trials with transgenic aspen should encompass the wide range of soils targeted for commercial planting in order to determine their effect(s) on the resident soil microbial community.