Legume Overseeding and P Fertilization Increases Microbial Activity and Decreases the Relative Abundance of AM Fungi in Pampas Natural Pastures.

Natural grasslands provide a valuable resource for livestock grazing. In many parts of South America, legume overseeding and P fertilization are commonly used to enhance primary productivity. The effect of this practice on the plant community is well established. However, how this management regime affects the soil microbiome is less known. Here, to contribute to filling this knowledge gap, we analyzed the effect of Lotus subbiflorus overseeding, together with P fertilization, on soil microbial community diversity and activity in the Uruguayan Pampa region. The results showed that plant communities in the natural grassland paddocks significantly differed from those of the managed paddocks. In contrast, neither microbial biomass and respiration nor microbial diversity was significantly affected by management, although the structure of the bacterial and fungal communities were correlated with those of the plant communities. AM Fungi relative abundance, as well as several enzyme activities, were significantly affected by management. This could have consequences for the C, N, and P content of SOM in these soils, which in turn might affect SOM degradation.

Potential impacts of seasonal and altitudinal changes on enzymatic peat decomposition in the High Andean Paramo region of Ecuador.

The Andean Paramo is a vast ecosystem, characterized by distinct vegetational zones at several altitudinal levels with huge water storage and carbon fixation capacity within its peat-like andosols, due to a slow decomposition rate of organic matter. These characteristics become mutually related as enzymatic activities increase with temperature and are associated with oxygen penetration, restricting the activity of many hydrolytic enzymes according to the enzyme Latch Theory. This study describes the changing activity of sulfatase (Sulf), phosphatase (Phos), n-acetyl-glucosaminidase (N-Ac), cellobiohydrolase (Cellobio), ß-glucosidase (ß-Glu), and peroxidase (POX) on an altitudinal scale from 3600 to 4200 m, in rainy and dry seasons at 10 and 30 cm sampling depth, related to physical and chemical soil characteristics, like metals and organic elements. Linear fixed-effect models were established to analyze these environmental factors to determine distinct decomposition patterns. The data suggests a strong tendency towards decreasing enzyme activities at higher altitudes and in the dry season up to two-fold stronger activation for Sulf, Phos, Cellobio, and ß-Glu. Especially the lowest altitude showed considerably stronger activity of N-Ac, ß-Glu, and POX. Although sampling depth revealed significant differences for all hydrolases but Cellobio, it had minor effects on model outcomes. Further organic rather than physical or metal components of the soil explain the enzyme activity variations. Although the levels of phenols coincided mostly with the soil organic carbon content, there was no direct relation between hydrolases, POX activity, and phenolic substances. The outcome suggests that slight environmental changes with global warming might cause important changes in enzyme activities leading to increased organic matter decomposition at the borderline between the paramo region and downslope ecosystems. Expected extremer dry seasons could cause critical changes as aeration increases peat decomposition leading to a constant liberation of carbon stocks, which puts the paramo region and its ecosystem services in great danger.

Extracellular enzymatic activity of two hydrolases in wastewater treatment for biological nutrient removal.

Due to the complex nature of the wastewater (both domestic and non-domestic) composition, biological processes are widely used to remove nutrients, such as carbon (C), nitrogen (N), and phosphorous (P), which cause instability and hence contribute to the damage of water bodies. Systems with different configurations have been developed (including anaerobic, anoxic, and aerobic conditions) for the joint removal of carbon, nitrogen, and phosphorus. The goal of this research is to evaluate the extracellular activity of ß-glucosidase and phosphatase enzymes in a University of Cape Town (UCT) system fed with two synthetic wastewaters of different molecular complexity. Both types of waters have medium strength characteristics similar to those of domestic wastewater with a mean C/N/P ratio of 100:13:1. The operation parameters were hydraulic retention time (HRT) of 10 h, solid retention time (SRT) of 12 days, mean concentration of the influent in terms of chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorus (TP) of 600, 80, and 6 mg/L, respectively. According to the results obtained, statistically significant differences have been found in the extracellular enzyme activities with the evaluated wastewaters and in the units comprising the treatment system in some of the cases. An analysis of principal components showed that the extracellular enzymatic activity has been correlated to nutrient concentration in wastewater, biomass concentration in the system, and metabolic conditions of treatment phases. Additionally, this research has allowed determining an inverse relationship between wastewater biodegradability and the extracellular enzyme activity of ß-glucosidase and phosphatase. These results highlight the importance of including the analysis of biomass biochemical characteristics as control methods in wastewater treatment systems for the nutrient removal.

Escherichia coli expressing endoglucanase gene from Thai higher termite bacteria for enzymatic and microbial hydrolysis of cellulosic materials

Background: Endoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme, but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, the endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli. Then, the crude recombinant endoglucanase (EglS) along with a commercial cellulase (Cel) was used for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells. Results: E. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50°C). EglS was highly stable (over 80% activity retained) at 40­50°C after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose, and corncob about 5.2-, 1.7-, and 4.0-folds, respectively, compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37°C after 48 h. Conclusions: The recombinant endoglucanase activity improved by 17 times compared with that of the native strain and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase.

Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli

Background Bacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12 ± 0.01 μmol/min/mL. However, for economically viable industrial applications, the enzyme needs to be expressed in a heterologous host to overcome the low enzyme production from the wild-type strain. Results The endoglucanase gene from B. subtilis UMC7 was successfully cloned and expressed. A higher enzyme activity was observed in the intracellular fraction of the recombinant clone (0.51 ± 0.02 μmol/min/mL) compared with the cell-bound fraction (0.37 ± 0.02 μmol/min/mL) and the extracellular fraction (0.33 ± 0.01 μmol/min/mL). The recombinant endoglucanase was approximately 56 kDa, with optimal enzyme activity at 60°C and pH 6.0. The activity of the enzyme was enhanced by the addition of Ca2 +. However, the enzyme was inhibited by other metal ions in the following order: Fe3 + > Ni2 + > Cu2 + > Mn2 + = Zn2 + > Mg2 + > Cd2 + > Cr2 +. The enzyme was able to hydrolyze both low- and high-viscosity carboxymethyl-cellulose (CMC), avicel, cotton linter, filter paper and avicel but not starch, xylan, chitin, pectin and p-nitrophenyl α-d-glucopyranoside. Conclusions The recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose.

Preliminary data on growth and enzymatic abilities of soil fungus Humicolopsis cephalosporioides at different incubation temperatures.

BACKGROUND: Nothofagus pumilio (Poepp & Endl.) Krasser, known as "lenga" is the most important timber wood species in southernmost Patagonia (Argentina). Humicolopsis cephalosporioides Cabral & Marchand is a soil fungus associated with Nothofagus pumilio forests, which has outstanding cellulolytic activity. However, there is no information about the ability of this fungus to use organic substrates other than cellulose, and its ability to produce different enzyme systems, as well as its response to temperature. AIMS: The aim of this study was to examine the role of H. cephalosporioides in degradation processes in N. pumilio forests in detail by evaluating the in vitro ability of four isolates of this fungus to grow and produce different lytic enzyme systems, and their response to incubation temperature. METHODS: The ability of the fungi to grow and produce enzyme systems was estimated by inoculating them on agar media with specific substrates, and the cultures were incubated at three temperatures. RESULTS: A differential behavior of each strain in levels of growth and enzyme activity was found according to the medium type and/or incubation temperature. CONCLUSIONS: A intra-specific variability was found in H. cephalosporioides. Likewise a possible link between the saprotrophic role of this fungus in N. pumilio forests and the degradation of organic matter under stress conditions, such as those from frosty environments, was also discussed.