Soil ecosystems are home to a diverse range of microorganisms, but they are only partially understood because no single-cell sequencing or whole-community sequencing provides a complete picture of these complex communities. Using one of such metagenomics approaches, we succeeded in monitoring the microbial diversity and stress-response gene in the soil samples. This study aims to test whether known differences in taxonomic diversity and composition are reflected in functional gene profiles by implementing whole gene sequencing (WGS) metagenomic analysis of geographically dispersed soils from two distinct pristine forests. The study was commenced by sequencing three rainforest soil samples and three peat swamp soil samples. Soil richness effects were assessed by exploring the changes in specific functional gene abundances to elucidate physiological constraints acting on different soil systems and identify variance in functional pathways relevant to soil biogeochemical cycling. Proteobacteria shows abundances of microbial diversity for 52.15% in Royal Belum Reserved Forest and 48.28% in Raja Musa; 177 out of 1,391,841 and 449 out of 3,586,577 protein coding represent acidic stress-response genes for Royal Belum and Raja Musa, respectively. Raja Musa indicates pH 2.5, which is extremely acidic. The analysis of the taxonomic community showed that Royal Belum soils are dominated by bacteria (98% in Sungai Kooi (SK), 98% in Sungai Papan (SP), and 98% in Sungai Ruok (SR), Archaea (0.9% in SK, 0.9% in SP, and 1% in SR), and the remaining were classed under Eukaryota and viruses. Likewise, the soils of Raja Muda Musa are also dominated by bacteria (95% in Raja Musa 1 (RM1), 98% in Raja Musa 2 (RM2), and 96% in Raja Musa 3 (RM3)), followed by Archaea (4% in RM1, 1% in RM2, and 3% in RM3), and the remaining were classed under Eukaryota and viruses. This study revealed that RBFR (Royal Belum Foresr Reserve) and RMFR (Raja Musa Forest Reserve) metagenomes contained abundant stress-related genes assigned to various stress-response pathways, many of which did not show any difference among samples from both sites. Our findings indicate that the structure and functional potential of the microbial community will be altered by future environmental potential as the first glimpse of both the taxonomic and functional composition of soil microbial communities.
A novel wastewater treatment system consisting of an up-flow anaerobic sludge blanket (UASB) reactor and a down-flow hanging sponge (DHS) reactor with sulfur-redox reaction was developed for treatment of municipal sewage under low-temperature conditions. In the UASB reactor, a novel phenomenon of anaerobic sulfur oxidation occurred in the absence of oxygen, nitrite and nitrate as electron acceptors. The microorganisms involved in anaerobic sulfur oxidation have not been elucidated. Therefore, in this study, we studied the microbial communities existing in the UASB reactor that probably enhanced anaerobic sulfur oxidation. Sludge samples collected from the UASB reactor before and after sulfur oxidation were used for cloning and terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes of the bacterial and archaeal domains. The microbial community structures of bacteria and archaea indicated that the genus Smithella and uncultured bacteria within the phylum Caldiserica were the dominant bacteria groups. Methanosaeta spp. was the dominant group of the domain archaea. The T-RFLP analysis, which was consistent with the cloning results, also yielded characteristic fingerprints for bacterial communities, whereas the archaeal community structure yielded stable microbial community. From these results, it can be presumed that these major bacteria groups, genus Smithella and uncultured bacteria within the phylum Caldiserica, probably play an important role in sulfur oxidation in UASB reactors.
The activities of lipase produced by five lipases-producing thermophilic bacteria strains (SY1, SY5, SY6, SY7 and SY9) isolated from Selayang Hot Spring in the western part of Peninsular Malaysia were analyzed and compared. SY7 and SY9 had considerably higher lipolytic activities than those of SY1, SY5 and SY6. Thermostabilities of lipase produced by all strains were determined after heating at 80°C for 30 minutes. Strain SY7 retained the highest lipolytic activity of 77%, while others had infinitesimally low thermostability (retaining less than 34% of their original activity) at the same temperature and time. SY7 was chosen for further characterization because it showed exceptionally high lipase activity and thermostability. It was identified as belonging to Bacillus species by the conventional Gram-staining technique, Biochemical tests and Biolog Microstation system. By using 16S rRNA gene sequencing, strain SY7 generated the same expected PCR product with molecular weight of 1500 base pair. It displayed 98% sequence similarity to Bacillus cereus strain J-1 16S rRNA gene partial sequence with accession number: AY305275 and has been deposited in the database of Genbank.
Previous studies on screening of lignin-degrading bacteria mainly focused on the ligninolytic ability of the isolated bacteria for the utilization of lignin monomers. In this study, we focused on the depolymerization of alkali lignin to prove the ability of the isolated thermophilic bacterial strains to utilize and depolymerize more than a monomer of alkali lignin within 7 days of incubation. Indigenous thermophilic bacterial isolates from the palm oil plantation were used to evaluate the depolymerization and utilization of alkali lignin. The confirmation of the bacterium-mediated depolymerization of oil palm empty fruit bunch was achieved through the removal of silica bodies, as observed with scanning electron microscopy. Stenotrophomonas sp. S2 and Bacillus subtilis S11Y were able to reduce approximately 50% and 20% of alkali lignin at 7 days of incubation without the requirement for additional carbon sources.
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein nuclease (Cas) is identified as an adaptive immune system in archaea and bacteria. Type II of this system, CRISPR-Cas9, is the most versatile form that has enabled facile and efficient targeted genome editing. Viral infections have serious impacts on global health and conventional antiviral therapies have not yielded a successful solution hitherto. The CRISPR-Cas9 system represents a promising tool for eliminating viral infections. In this review, we highlight 1) the recent progress of CRISPR-Cas technology in decoding and diagnosis of viral outbreaks, 2) its applications to eliminate viral infections in both pre-integration and provirus stages, and 3) various delivery systems that are employed to introduce the platform into target cells.
Clustered regulatory interspaced short palindromic repeats (CRISPR) in association with CRISPR-associated protein (Cas) is an adaptive immune system, playing a pivotal role in the defense of bacteria and archaea. Ease of handling and cost effectiveness make the CRISPR-Cas system an ideal programmable nuclease tool. Recent advances in understanding the CRISPR-Cas system have tremendously improved its efficiency. For instance, it is possible to recapitulate the chronicle CRISPR-Cas from its infancy and inaugurate a developed version by generating novel variants of Cas proteins, subduing off-target effects, and optimizing of innovative strategies. In summary, the CRISPR-Cas system could be employed in a number of applications, including providing model systems, rectification of detrimental mutations, and antiviral therapies.
Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ. Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of 13C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.
Malaysia has a great number of hot springs, especially along the flank of the Banjaran Titiwangsa mountain range. Biological studies of the Malaysian hot springs are rare because of the lack of comprehensive information on their microbial communities. In this study, we report a cultivation-independent census to describe microbial communities in six hot springs. The Ulu Slim (US), Sungai Klah (SK), Dusun Tua (DT), Sungai Serai (SS), Semenyih (SE), and Ayer Hangat (AH) hot springs exhibit circumneutral pH with temperatures ranging from 43°C to 90°C. Genomic DNA was extracted from environmental samples and the V3-V4 hypervariable regions of 16S rRNA genes were amplified, sequenced, and analyzed. High-throughput sequencing analysis showed that microbial richness was high in all samples as indicated by the detection of 6,334-26,244 operational taxonomy units. In total, 59, 61, 72, 73, 65, and 52 bacterial phyla were identified in the US, SK, DT, SS, SE, and AH hot springs, respectively. Generally, Firmicutes and Proteobacteria dominated the bacterial communities in all hot springs. Archaeal communities mainly consisted of Crenarchaeota, Euryarchaeota, and Parvarchaeota. In beta diversity analysis, the hot spring microbial memberships were clustered primarily on the basis of temperature and salinity. Canonical correlation analysis to assess the relationship between the microbial communities and physicochemical variables revealed that diversity patterns were best explained by a combination of physicochemical variables, rather than by individual abiotic variables such as temperature and salinity.
Microbial communities of two oil reservoirs from Malaysia, denoted as Platform Bo and Platform Pe were studied using
culture-independent approach. Environmental DNA was extracted and the universal amplified ribosomal region (UARR)
was target amplified for both prokaryotes and eukaryotes. The amplified products were purified and cloned into pTZ57R/T
vector to construct the 16S/18S rDNA library. Restriction endocucleases HhaI and MspI were used to screen the library.
From that, 125 and 253 recombinant plasmid representative clones from Platform Bo and Platform Pe, respectively, were
sent for DNA sequencing. Twenty-six operational taxonomic units (OTUs) consist of 20 genera detected at Platform Bo
and 17 OTUs consist of 13 genera detected at Platform Pe. Marinobacter and Acinetobacter species co-occurred in both
platforms whereas the rest are site-specific. Gammaproteobacteria accounted for 86.0% of the microbial community in
Platform Bo, where OTUs affiliated to Marinobacter, Pseudomonas and Marinobacterium that were the most abundant. The
major OTUs in the Platform Pe were with affinities to Achromobacter, followed by Stenotrophomonas and Serratia. The
only archaeal isolates were detected in Platform Pe, which affiliated to Thermocladium. The singletons and doubletons
accounted for about 50.0% of the OTU abundance in both platforms, which considerably significant despite their rare
occurrence.
Singapore, an equatorial island in South East Asia, is influenced by a bi-annual reversal of wind directions which defines two monsoon seasons. We characterized the dynamics of the microbial communities of Singapore coastal waters by collecting monthly samples between February 2017 and July 2018 at four sites located across two straits with different trophic status, and sequencing the V6-V8 region of the small sub-unit ribosomal RNA gene (rRNA gene) of Bacteria, Archaea, and Eukaryota. Johor Strait, which is subjected to wider environmental fluctuations from anthropogenic activities, presented a higher abundance of copiotrophic microbes, including Cellvibrionales and Rhodobacterales. The mesotrophic Singapore Strait, where the seasonal variability is caused by changes in the oceanographic conditions, harboured a higher proportion of typically marine microbe groups such as Synechococcales, Nitrosupumilales, SAR11, SAR86, Marine Group II Archaea and Radiolaria. In addition, we observed seasonal variability of the microbial communities in the Singapore Strait, which was possibly influenced by the alternating monsoon regime, while no seasonal pattern was detected in the Johor Strait.
The unique cellular enzymatic machinery of halophilic microbes allows them to thrive in extreme saline environments. That these microorganisms can prosper in hypersaline environments has been correlated with the elevated acidic amino acid content in their proteins, which increase the negative protein surface potential. Because these microorganisms effectively use hydrocarbons as their sole carbon and energy sources, they may prove to be valuable bioremediation agents for the treatment of saline effluents and hypersaline waters contaminated with toxic compounds that are resistant to degradation. This review highlights the various strategies adopted by halophiles to compensate for their saline surroundings and includes descriptions of recent studies that have used these microorganisms for bioremediation of environments contaminated by petroleum hydrocarbons. The known halotolerant dehalogenase-producing microbes, their dehalogenation mechanisms, and how their proteins are stabilized is also reviewed. In view of their robustness in saline environments, efforts to document their full potential regarding remediation of contaminated hypersaline ecosystems merits further exploration.
Several recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor.
Tropical peat swamp forests are important and endangered ecosystems, although little is known of their microbial diversity and ecology. We used molecular and enzymatic techniques to examine patterns in prokaryotic community structure and overall microbial activity at 0-, 10-, 20-, and 50-cm depths in sediments in a peat swamp forest in Malaysia. Denaturing gradient gel electrophoresis profiles of amplified 16S ribosomal ribonucleic acid (rRNA) gene fragments showed that different depths harbored different bacterial assemblages and that Archaea appeared to be limited to the deeper samples. Cloning and sequencing of longer 16S rRNA gene fragments suggested reduced microbial diversity in the deeper samples compared to the surface. Bacterial clone libraries were largely dominated by ribotypes affiliated with the Acidobacteria, which accounted for at least 27-54% of the sequences obtained. All of the sequenced representatives from the archaeal clone libraries were Crenarchaeota. Activities of microbial extracellular enzymes involved in carbon, nitrogen, and phosphorus cycling declined appreciably with depth, the only exception being peroxidase. These results show that tropical peat swamp forests are unusual systems with microbial assemblages dominated by members of the Acidobacteria and Crenarchaeota. Microbial communities show clear changes with depth, and most microbial activity is likely confined to populations in the upper few centimeters, the site of new leaf litter fall, rather than the deeper, older, peat layers.
Pulp and paper mill sludge (PPMS) was found to be poorly colonised with thermophilic microorganisms. However,
evidence to support the need for inoculation to facilitate PPMS composting has only been demonstrated in one instance.
In this study, we aimed to: screen and identify PPMS digesting thermophilic bacterial strains; investigate effects of the
mixture of selected thermophilic bacterial strains on PPMS digestion; and utilize this mixture as start inoculum in PPMS
composting and assess the quality of compost product. The results showed that eleven thermophilic bacterial strains were
isolated from Bai Bang PPMS by the enrichment culture method. Among these, three strains which reflected high growth
rates on the plates of Minimal Media Agar supplemented with Bai Bang PPMS and showed hydrolytic and ligninolytic
activities on the agar plates containing appropriate inductive substrates were selected. Based on the morphological,
biochemical characteristics and 16S rRNA gene sequencing, they were identified as Bacillus subtilis. The inoculation
with the mixture of selected strains enhanced remarkably Bai Bang PPMS digestion. The dry weight decrease, volatile
suspended solids removal, dehydrogenase and protease activities in the inoculated sludge were 2.1-, 1.5-, 1.3- and 1.2-
fold higher, respectively, compared to the non-inoculated sludge. The assessment of compost quality based on stability
using the alkaline trap method and maturity using the germination and root elongation test showed that the inoculated
compost was stable and mature while the non-inoculated compost was unstable and immature. These thermophilic
bacterial strains therefore have great potential for Bai Bang PPMS composting.
Microbial diversity within formation water and oil from two compartments in Bokor oil reservoir from a Malaysian petroleum oil field was examined. A total of 1,056 16S rRNA gene clones were screened from each location by amplified ribosomal DNA restriction analysis. All samples were dominated by clones affiliated with Marinobacter, some novel Deferribacteraceae genera and various clones allied to the Methanococci. In addition, either Marinobacterium- or Pseudomonas-like operational taxonomic units were detected from either compartment. A systematic comparison with the existing pertinent studies was undertaken by analysing the microbial amplicons detected and the PCR primers used. The analyses demonstrated that bacterial communities were site specific, while Archaea co-occurred more frequently. Amplicons related to Marinobacter, Marinobacterium and Pseudomonas were detected in a number of the studies examined, suggesting they may be ubiquitous members in oil reservoirs. Further analysis of primers used in those studies suggested that most primer pairs had fairly broad but low matches across the bacterial and archaeal domains, while a minority had selective matches to certain taxa or low matches to all the microbial taxa tested. Thus, it indicated that primers may play an important role in determining which taxa would be detected.
Pretreatment of waste-activated sludge (WAS) is an effective way to destabilize sludge floc structure and release organic matter for improving sludge digestion efficiency. Nonetheless, information on the impact of WAS pretreatment on digestion sludge microbiomes, as well as mechanistic insights into how sludge pretreatment improves digestion performance, remains elusive. In this study, a genome-centric metagenomic approach was employed to investigate the digestion sludge microbiome in four sludge digesters with different types of feeding sludge: WAS pretreated with 0.25 mol/liter alkaline/acid (APAD), WAS pretreated with 0.8 mol/liter alkaline/acid (HS-APAD), thermally pretreated WAS (thermal-AD), and fresh WAS (control-AD). We retrieved 254 metagenome-assembled genomes (MAGs) to identify the key functional populations involved in the methanogenic digestion process. These MAGs span 28 phyla, including 69 yet-to-be-cultivated lineages, and 30 novel lineages were characterized with metabolic potential associated with hydrolysis and fermentation. Interestingly, functional populations involving carbohydrate digestion were enriched in APAD and HS-APAD, while lineages related to protein and lipid fermentation were enriched in thermal-AD, corroborating the idea that different substrates are released from alkaline/acid and thermal pretreatments. Among the major functional populations (i.e., fermenters, syntrophic acetogens, and methanogens), significant correlations between genome sizes and abundance of the fermenters were observed, particularly in APAD and HS-APAD, which had improved digestion performance.IMPORTANCE Wastewater treatment generates large amounts of waste-activated sludge (WAS), which consists mainly of recalcitrant microbial cells and particulate organic matter. Though WAS pretreatment is an effective way to release sludge organic matter for subsequent digestion, detailed information on the impact of the sludge pretreatment on the digestion sludge microbiome remains scarce. Our study provides unprecedented genome-centric metagenomic insights into how WAS pretreatments change the digestion sludge microbiomes, as well as their metabolic networks. Moreover, digestion sludge microbiomes could be a unique source for exploring microbial dark matter. These results may inform future optimization of methanogenic sludge digestion and resource recovery.
The oil palm industry, especially in Indonesia and Malaysia is being threatened by Basal Stem Rot (BSR) disease caused by Ganoderma boninense. There is no conclusive remedy in handling this disease effectively. In this study, metagenomics analysis of soil were analyzed for a better understanding of the microbial diversity in relation to BSR disease. Study was conducted in three plantation sites of Sabah, Malaysia which incorporated different disease management and agronomic practices. The estates are located at Sandakan (Kam Cheong Plantation), Lahad Datu (FGV Ladang Sahabat) and Tawau (Warisan Gagah). Soil samples were collected from disease free, high and low BSR incidence plots. Illumina MiSeq metagenomic analysis using V3-V4 region of 16S rRNA gene was employed to study the microbial diversity. Bacteria (97.4%) and Archaea (0.2%) were found majority in kingdom taxonomy level. The most abundant phyla were Proteobacteria, Acidobacteria, Actinobacteria, and Verrucomicrobia. Higher alpha diversity of all species was observed among all tested soil from each estates. Beta analysis was analyzed using non phylogenetic UnifRac matrix and visualized using Principal Coordinates Analysis (PCoA). The tested soil samples in Kam Cheong Plantation were found to have similar bacterial communities. The data provided is useful as an indicator in developing biology controls against Ganoderma boninense.
Despite efforts to address the composition of the microbial community during the anaerobic treatment of palm oil mill effluent (POME), its composition in relation to biodegradation in the full-scale treatment system has not yet been extensively examined. Therefore, a thorough analysis of bacterial and archaeal communities was performed in the present study using MiSeq sequencing at the different stages of the POME treatment, which comprised anaerobic as well as facultative anaerobic and aerobic processes, including the mixed raw effluent (MRE), mixing pond, holding tank, and final discharge phases. Based on the results obtained, the following biodegradation processes were suggested to occur at the different treatment stages: (1) Lactobacillaceae (35.9%) dominated the first stage, which contributed to high lactic acid production; (2) the higher population of Clostridiaceae in the mixing pond (47.7%) and Prevotellaceae in the holding tank (49.7%) promoted acetic acid production; (3) the aceticlastic methanogen Methanosaetaceae (0.6-0.8%) played a role in acetic acid degradation in the open digester and closed reactor for methane generation; (4) Syntrophomonas (21.5-29.2%) appeared to be involved in the degradation of fatty acids and acetic acid by syntrophic cooperation with the hydrogenotrophic methanogen, Methanobacteriaceae (0.6-1.3%); and (5) the phenols and alcohols detected in the early phases, but not in the final discharge phase, indicated the successful degradation of lignocellulosic materials. The present results contribute to a better understanding of the biodegradation mechanisms involved in the different stages of the full-scale treatment of POME.
In this study, productivity and physicochemical and microbiological (454 sequencing) parameters, as well as environmental criteria, were investigated in anaerobic reactors to contribute to the ongoing debate about the optimal temperature range for treating animal manure, and expand the general knowledge on the relation between microbiological and physicochemical process indicators. For this purpose, two reactor sizes were used (10 m(3) and 16 l), in which two temperature conditions (35°C and 50°C) were tested. In addition, the effect of the hydraulic retention time was evaluated (16 versus 20 days). Thermophilic anaerobic digestion showed higher organic matter degradation (especially fiber), higher pH and higher methane (CH₄) yield, as well as better percentage of ultimate CH₄ yield retrieved and lower residual CH₄ emission, when compared with mesophilic conditions. In addition, lower microbial diversity was found in the thermophilic reactors, especially for Bacteria, where a clear intensification towards Clostridia class members was evident. Independent of temperature, some similarities were found in digestates when comparing with animal manure, including low volatile fatty acids concentrations and a high fraction of Euryarchaeota in the total microbial community, in which members of Methanosarcinales dominated for both temperature conditions; these indicators could be considered a sign of process stability.
Efficient approaches for the utilization of waste sewage sludge have been widely studied. One of them is to use it for the bioenergy production, specifically methane gas which is well-known to be driven by complex bacterial interactions during the anaerobic digestion process. Therefore, it is important to understand not only microorganisms for producing methane but also those for controlling or regulating the process. In this study, azithromycin analogs belonging to macrolide, ketolide, and lincosamide groups were applied to investigate the mechanisms and dynamics of bacterial community in waste sewage sludge for methane production. The stages of anaerobic digestion process were evaluated by measuring the production of intermediate substrates, such as protease activity, organic acids, the quantification of bacteria and archaea, and its community dynamics. All azithromycin analogs used in this study achieved a high methane production compared to the control sample without any antibiotic due to the efficient hydrolysis process and the presence of important fermentative bacteria and archaea responsible in the methanogenesis stage. The key microorganisms contributing to the methane production may be Clostridia, Cladilinea, Planctomycetes, and Alphaproteobacteria as an accelerator whereas Nitrosomonadaceae and Nitrospiraceae may be suppressors for methane production. In conclusion, the utilization of antibiotic analogs of macrolide, ketolide, and lincosamide groups has a promising ability in finding the essential microorganisms and improving the methane production using waste sewage sludge.