BACKGROUND: The global over-reliance on non-renewable fossil fuels has led to the emission of greenhouse gases, creating a critical global environmental challenge. There is an urgent need for alternative solutions like biofuels. Advanced biofuel is a renewable sustainable energy generated from lignocellulosic plant materials, which can significantly contribute to mitigating CO2 emissions. Microbial Carbohydrate Active Enzymes (CAZymes) are the most crucial enzymes for the generation of sustainable biofuel energy. The present study designed shotgun metagenomics approaches to assemble, predict, and annotate, aiming to gain an insight into the taxonomic diversity, annotate CAZymes, and identify carbohydrate hydrolyzing CAZymes from microbiomes in Menagesha suba forest soil for the first time.
RESULTS: The microbial diversity based on small subunit (SSU) rRNA analysis revealed the dominance of the bacterial domain representing 81.82% and 92.31% in the studied samples. Furthermore, the phylum composition result indicated the dominance of the phyla Proteobacteria (23.08%, 27.27%), Actinobacteria (11.36%, 20.51%), and Acidobacteria (10.26%, 15.91%). The study also identified unassigned bacteria which might have a unique potential for biopolymer hydrolysis. The metagenomic study revealed that 100,244 and 65,356 genes were predicted from the two distinct samples. A total number of 1806 CAZyme genes were identified, among annotated CAZymes, 758 had a known enzyme assigned to CAZymes. Glycoside hydrolases (GHs) CAZyme family contained most of the CAZyme genes with known enzymes such as β-glucosidase, endo-β-1,4-mannanase, exo-β-1,4-glucanase, α-L-arabinofuranosidase and oligoxyloglucan reducing end-specific cellobiohydrolase. On the other hand, 1048 of the identified CAZyme genes were putative CAZyme genes with unknown enzymatical activity and the majority of which belong to the GHs family.
CONCLUSIONS: In general, the identified putative CAZymes genes open up an opportunity for the discovery of new enzymes responsible for hydrolyzing biopolymers utilized for biofuel energy generation. This finding is used as a first-hand piece of evidence to serve as a benchmark for further and comprehensive studies to unveil novel classes of bio-economically valuable genes and their encoded products.

The environmental risks of migration of heavy metals (HMs) following applications of sewage sludge (SS) to forest soils are garnering increased attention. Plant litter at the forest floor may modify HM migration pathways through impacts on soil aggregates and water/soil erosion; however, HM migration responses to plant litter are poorly understood. The aim of this study was to determine the effects of plant litter cover on HMs migration, and water and soil erosion following the application of SS to subtropical forest soils. Effects of addition of SS along and SS plus plant litter at 0.75 or 1.5 kg m-2 on the migration of cadmium, chromium, copper, nickel, lead, and zinc in surface runoff, soil interflow, and sediments were quantified across nine simulated rainfall events in a laboratory experiment and following natural intense rain events in a field experiment. Addition of SS elevated HM concentrations in surface runoff by 38.7 to 98.5 %, in soil interflow by 48.3 to 312.5 %, and in sediment by 28.5 to 149.4 %, and increased the production of sediment aggregates <0.05 mm that led to greater cumulative migrations of HMs in surface runoff and sediment; sediment accounted for 89.5 % of HM migrations. Addition of plant litter reduced cumulative migration of HMs by 87.1-97.27 %; however, the higher rate of plant litter led to a decrease in surface runoff and sediment yield, and an increase in soil interflow. Addition of plant litter shifted the main pathway of HM migration from sediment to surface runoff and soil interflow. The potential ecological HM risk index was \"low\" for each treatment. We found consistency in HM concentrations and migrations via surface runoff between the field and laboratory experiments. Overall, the addition of plant litter with SS mitigated soil erosion and reduced total migration of HMs, resulting in a 88.7-97.3 % decrease in the ecological risk index of the six HMs. We conclude that the addition of plant litter may provide a management strategy for the mitigation of HM risks to environmental safety for the disposal of SS in subtropical forest systems.

Subsoil (below 20 cm), storing over 50 % of soil organics carbon (SOC) within the 1 m depth, plays a critical role in regulating climate and ecosystem function. However, little was known on the changes in SOC decomposition induced by exogenous C input (i.e., priming effect) across the whole soil profile under nitrogen (N) enrichment and climate warming. We designed an incubation system of soil columns with minor physical disturbance, which allows the manual additions of exogenous C and N and incubation under ambient or elevated temperature. A negative priming effect by glucose was observed in all layers of ambient soil, while the negative priming effect was enhanced by soil depth but inhibited by warming. Nitrogen addition shifted the priming effect from negative to positive under ambient temperature, and decreased the magnitude of negative priming effect under elevated temperature. Nitrogen uplift effect on priming effect was more pronounced in subsoil compared to topsoil, while this effect diminished with rising temperature. Soil microbial activity (e.g., the CO2 production within 3 days) and acid phosphatase activity had important roles in regulating the variations in priming effect across the soil profile. Our results indicated that increase in labile substrate (e.g., exogenous C input) input would not lead to native SOC destabilization in subsoil, N addition shifted the priming effect from negative to positive, increasing the SOC decomposition under ambient temperature, while labile C input together with N addition benefited SOC sequestration by inducing negative priming effects in forest soil under warming climate.

Lignin, a heterogeneous aromatic polymer present in plant biomass, is intertwined with cellulose and hemicellulose fibrils, posing challenges to its effective utilization due to its phenolic nature and recalcitrance to degradation. In this study, three lignin utilizing bacteria, Klebsiella sp. LEA1, Pseudomonas sp. LEA2, and Burkholderia sp. LEA3, were isolated from deciduous forest soil samples in Nan province, Thailand. These isolates were capable of growing on alkali lignin and various lignin-associated monomers at 40 °C under microaerobic conditions. The presence of Cu2+ significantly enhanced guaiacol oxidation in Klebsiella sp. LEA1 and Pseudomonas sp. LEA2. Lignin-related monomers and intermediates such as 2,6-dimethoxyphenol, 4-vinyl guaiacol, 4-hydroxybenzoic acid, benzoic acid, catechol, and succinic acid were detected mostly during the late stage of incubation of Klebsiella sp. LEA1 and Pseudomonas sp. LEA2 in lignin minimal salt media via GC-MS analysis. The intermediates identified from Klebsiella sp. LEA1 degradation suggested that conversion and utilization occurred through the β-ketoadipate (ortho-cleavage) pathway under limited oxygen conditions. The ability of these bacteria to thrive on alkaline lignin and produce various lignin-related intermediates under limited oxygen conditions suggests their potential utility in oxygen-limited processes and the production of renewable chemicals from plant biomass.

Trees contribute to bedrock weathering in a variety of ways. However, evaluating their full impact is complicated by a lack of direct observation of unexposed root systems of individual trees, especially when the scale of the analysis goes down to the level of microbiomes. In the present study, we investigated the contribution of tree root systems to bioweathering and soil production at the macro- and microscale. Soil profiles developed under trees on granite bedrock were investigated in two parts of the Sudety Mountains, SW Poland: the Rudawy Janowickie Mountains, and the Stołowe Mountains. Soil profiles were gradually excavated and soil samples collected from pre-defined positions of the root zone: 1) bulk soil, 2) rhizosphere, 3) cracks, 4) topsoil, and 5) control positions. In total, we analyzed 103 samples for soil chemistry and microbiological activity. In addition, we analyzed 19 samples using XRF (X-ray Fluorescence). Four parent rock samples, in the form of thin-sections, were the subject of mineralogical evaluation. Soil analyses included: total organic carbon (C) and nitrogen (N) content, soil pHH2O, soluble iron (Fed), and aluminum (Ald), non-crystalline (amorphous) iron (Feox), and aluminum (Alox). For microbiological analyses, we used a Biolog (EcoPlate) system to determine the functional diversity of soil microorganisms. We evaluated the results on soil chemistry and microbiological activity statistically by principal component analysis (PCA) and redundancy analysis (RDA). Differences between soil sampling positions were assessed using a non-parametric Kruskal-Wallis (K-W) rank sum test and a post-hoc pairwise Dunn test. Trees developed different root architectures, likely shaped by the depth to bedrock and its pre-existing net of fractures and fissures. Tree roots were able to enter bedrock cracks at one study site (at Pstrążna, Stołowe Mountains). The soil profile was too deep for root system penetration at the second study site (Mt Jańska, Rudawy Janowickie Mountains, RJM). The rhizospheric soil along the roots had significantly different chemical properties compared to non-rhizospheric soil types. At Mt. Jańska, soil differed from the crack soil in terms of Alox (pHolm-adj. < 0.0006) and Feox (pHolm-adj. < 0.004), and from the bulk soil (pHolm-adj. < 0.02) and topsoil (pHolm-adj. < 0.007). In addition, at Pstrążna, the soil differed from the control soil in terms of C (pHolm-adj. < 0.009) and soil pHH2O (pHolm-adj. < 0.0008) and from the topsoil in terms of soil pHH2O. The highest metabolic activity was in cracks at Mt. Jańska and in control samples from Pstrążna. In general, the spatial distribution of soil microbial activity, and the weathering that results from that portion of the soil biome, is spatially heterogeneous and appears to be partially determined by the interaction of root growth and bedrock fracture patterns.

The central carbon (C) metabolic network is responsible for most of the production of energy and biosynthesis in microorganisms and is therefore key to a mechanistic understanding of microbial life in soil communities. Many upland soil communities have shown a relatively high C flux through the pentose phosphate (PP) or the Entner-Doudoroff (ED) pathway, thought to be related to oxidative damage control. We tested the hypothesis that the metabolic organization of the central C metabolic network differed between two ecosystems, an anoxic marsh soil and oxic upland soil, and would be affected by altering oxygen concentrations. We expected there to be high PP/ED pathway activity under high oxygen concentrations and in oxic soils and low PP/ED activity in reduced oxygen concentrations and in marsh soil. Although we found high PP/ED activity in the upland soil and low activity in the marsh soil, lowering the oxygen concentration for the upland soil did not reduce the relative PP/ED pathway activity as hypothesized, nor did increasing the oxygen concentration in the marsh soil increase the PP/ED pathway activity. We speculate that the high PP/ED activity in the upland soil, even when exposed to low oxygen concentrations, was related to a high demand for NADPH for biosynthesis, thus reflecting higher microbial growth rates in C-rich soils than in C-poor sediments. Further studies are needed to explain the observed metabolic diversity among soil ecosystems and determine whether it is related to microbial growth rates.IMPORTANCEWe observed that the organization of the central carbon (C) metabolic processes differed between oxic and anoxic soil. However, we also found that the pentose phosphate pathway/Entner-Doudoroff (PP/ED) pathway activity remained high after reducing the oxygen concentration for the upland soil and did not increase in response to an increase in oxygen concentration in the marsh soil. These observations contradicted the hypothesis that oxidative stress is a main driver for high PP/ED activity in soil communities. We suggest that the high PP/ED activity and NADPH production reflect higher anabolic activities and growth rates in the upland soil compared to the anaerobic marsh soil. A greater understanding of the molecular and biochemical processes in soil communities is needed to develop a mechanistic perspective on microbial activities and their relationship to soil C and nutrient cycling. Such an increased mechanistic perspective is ecologically relevant, given that the central carbon metabolic network is intimately tied to the energy metabolism of microbes, the efficiency of new microbial biomass production, and soil organic matter formation.

Physiological responses of soil microorganisms to global warming are important for soil ecosystem function and the terrestrial carbon cycle. Here, we investigate the effects of weeks, years, and decades of soil warming across seasons and time on the microbial protein biosynthesis machineries (i.e. ribosomes), the most abundant cellular macromolecular complexes, using RNA:DNA and RNA:MBC (microbial biomass carbon) ratios as proxies for cellular ribosome contents. We compared warmed soils and non-warmed controls of 15 replicated subarctic grassland and forest soil temperature gradients subject to natural geothermal warming. RNA:DNA ratios tended to be lower in the warmed soils during summer and autumn, independent of warming duration (6 weeks, 8-14 years, and > 50 years), warming intensity (+3°C, +6°C, and +9°C), and ecosystem type. With increasing temperatures, RNA:MBC ratios were also decreasing. Additionally, seasonal RNA:DNA ratios of the consecutively sampled forest showed the same temperature-driven pattern. This suggests that subarctic soil microorganisms are depleted of ribosomes under warm conditions and the lack of consistent relationships with other physicochemical parameters besides temperature further suggests temperature as key driver. Furthermore, in incubation experiments, we measured significantly higher CO2 emission rates per unit of RNA from short- and long-term warmed soils compared to non-warmed controls. In conclusion, ribosome reduction may represent a widespread microbial physiological response to warming that offers a selective advantage at higher temperatures, as energy and matter can be reallocated from ribosome synthesis to other processes including substrate uptake and turnover. This way, ribosome reduction could have a substantial effect on soil carbon dynamics.

Although the importance of the soil bacterial community for ecosystem functions has long been recognized, there is still a limited understanding of the associations between its community composition, structure, co-occurrence patterns, and soil physicochemical properties. The objectives of the present study were to explore the association between soil physicochemical properties and the composition, diversity, co-occurrence network topological features, and assembly mechanisms of the soil bacterial community. Four typical forest types from Liziping Nature Reserve, representing evergreen coniferous forest, deciduous coniferous forest, mixed conifer-broadleaf forest, and its secondary forest, were selected for this study. The soil bacterial community was analyzed using Illumina MiSeq sequencing of 16S rRNA genes. Nonmetric multidimensional scaling was used to illustrate the clustering of different samples based on Bray-Curtis distances. The associations between soil physicochemical properties and bacterial community structure were analyzed using the Mantel test. The interactions among bacterial taxa were visualized with a co-occurrence network, and the community assembly processes were quantified using the Beta Nearest Taxon Index (Beta-NTI). The dominant bacterial phyla across all forest soils were Proteobacteria (45.17%), Acidobacteria (21.73%), Actinobacteria (8.75%), and Chloroflexi (5.06%). Chao1 estimator of richness, observed ASVs, faith-phylogenetic diversity (faith-PD) index, and community composition were distinguishing features of the examined four forest types. The first two principal components of redundancy analysis explained 41.33% of the variation in the soil bacterial community, with total soil organic carbon, soil moisture, pH, total nitrogen, carbon/nitrogen (C/N), carbon/phosphorous (C/P), and nitrogen/phosphorous (N/P) being the main soil physicochemical properties shaping soil bacterial communities. The co-occurrence network structure in the mixed forest was more complex compared to that in pure forests. The Beta-NTI indicated that the bacterial community assembly of the four examined forest types was collaboratively influenced by deterministic and stochastic ecological processes.

To scrutinize the spatial distribution attributes of soil heavy metal content and discern its pollution status within the expanse of Wulongdong National Forest Park, a meticulous investigation is imperative. Three altitude gradients of 900, 1000, and 1069 m were selected on the shady and sunny slopes of Wulongdong National Forest Park, and a total of 300 soil sample points were collected. Soil samples were collected in layers, and the contents of seven soil heavy metal elements, Cr, Cd, Hg, Ni, Se, As, and Pb, were measured. With regard to the national soil element background values, the single factor index method, Nemerow index method, and pollution load index were employed to undertake a thorough assessment of soil heavy metal pollution. (1) The contents of heavy metal elements Cr, Se, As, and Pb in the 0-20 cm soil layer of Wulongdong National Forest Park are lower than the national soil element background value and the Henan soil element background value; the Cd and Hg contents exceed the national soil element background value. The value and Henan soil element background value are 2.2 times and 2.92 times the national soil element background value, and 2.75 times and 9.5 times the Henan soil element background value respectively; Ni content is lower than the Henan soil element background value, but higher than the national soil element background value. The background value is 1.03 times its content. The coefficients of variation of the contents of seven heavy metal elements are all greater than 50%, among which Hg shows extreme variation, and the remaining six are highly variable. (2) In the same soil layer, the Cr and As contents are lower on sunny slopes than on shady slopes, and the contents of Pb, Ni, and Hg are generally higher on sunny slopes than on shady slopes. On sunny slopes, the contents of As, Cd, and Hg decrease with increasing altitude, and the Se content increases with increasing altitude; while on shady slopes, the contents of Cr, Se, and As decrease with increasing altitude, and Pb and Hg content increase with the increase of altitude; the content of heavy metal element As increases with the deepening of the soil layer on shady slopes, and the Hg content decreases with the deepening of the soil layer on sunny slopes. The contents of other heavy metal elements have no obvious regularity among different slope directions, altitudes and soil layers. (3) The single factor index evaluation results show that in the 0 ~ 20c soil layer and on the sunny slope, Hg is heavily polluted, Cd is moderately polluted, Ni is lightly polluted, and Cr, Se, As, and Pb are all non-polluted; On the shady slope, Cd and Hg are moderately polluted, and the other five heavy metal elements are in a non-polluting state. (4) The Nemerow index method evaluation results show that in the 0 ~ 20 cm soil layer, the soil on sunny slopes is significantly more polluted by heavy metals than on shady slopes, and the main pollutants are Ni, Cd and Hg. (5) In the 0 ~ 20 cm soil layer of Wulongdong National Forest Park, the three heavy metal elements Ni, Cd and Hg have reached pollution levels, of which Ni is slightly polluted, Cd and Hg are moderately or above polluted; the sunny slope soil is slightly polluted. Heavy metal pollution, no heavy metal pollution on shady slopes.

In dry deciduous tropical forests, both seasons (winter and summer) offer habitats that are essential ecologically. How these seasonal changes affect soil properties and microbial communities is not yet fully understood. This study aimed to investigate the influence of seasonal fluctuations on soil characteristics and microbial populations. The soil moisture content dramatically increases in the summer. However, the soil pH only gradually shifts from acidic to slightly neutral. During the summer, electrical conductivity (EC) values range from 0.62 to 1.03 ds m-1, in contrast to their decline in the winter. The levels of soil macronutrients and micronutrients increase during the summer, as does the quantity of soil organic carbon (SOC). A two-way ANOVA analysis reveals limited impacts of seasonal fluctuations and specific geographic locations on the amounts of accessible nitrogen (N) and phosphorus (P). Moreover, dehydrogenase, nitrate reductase, and urease activities rise in the summer, while chitinase, protease, and acid phosphatase activities are more pronounced in the winter. The soil microbes were identified in both seasons through 16S rRNA and ITS (Internal Transcribed Spacer) gene sequencing. Results revealed Proteobacteria and Ascomycota as predominant bacterial and fungal phyla. However, Bacillus, Pseudomonas, and Burkholderia are dominant bacterial genera, and Aspergillus, Alternaria, and Trichoderma are dominant fungal genera in the forest soil samples. Dominant bacterial and fungal genera may play a role in essential ecosystem services such as soil health management and nutrient cycling. In both seasons, clear relationships exist between soil properties, including pH, moisture, iron (Fe), zinc (Zn), and microbial diversity. Enzymatic activities and microbial shift relate positively with soil parameters. This study highlights robust soil-microbial interactions that persist mainly in the top layers of tropical dry deciduous forests in the summer and winter seasons. It provides insights into the responses of soil-microbial communities to seasonal changes, advancing our understanding of ecosystem dynamics and biodiversity preservation.