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Fulltext Editorial: Recent Advances in Bioremediation/Biodegradation by Extreme Microorganisms

Donati ER, Sani RK, Goh KM, Chan KG

Front Microbiol, 2019;10:1851.
PMID: 31474953 DOI: 10.3389/fmicb.2019.01851
Fulltext Genome analysis of a thermophilic exopolysaccharide-producing bacterium - Geobacillus sp. WSUCF1

Wang J, Goh KM, Salem DR, Sani RK

Sci Rep, 2019 02 07;9(1):1608.
PMID: 30733471 DOI: 10.1038/s41598-018-36983-z

Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.
Fulltext Genomics of extreme environments: unveiling the secrets of survival

Goh KM, González-Siso MI, Sani RK

Sci Rep, 2023 Dec 05;13(1):21441.
PMID: 38052842 DOI: 10.1038/s41598-023-48470-1

Life on Earth has displayed remarkable adaptability to the harshest environments, spanning polar regions, scorching deserts, abyssal oceans, lightless caves, noxious lakes, boiling hot springs, and nuclear waste sites. These resilient organisms, known as extremophiles or polyextremophiles, owe their survival due to their unique genetic adaptations. This collection, titled ‘Genomics of Extreme Environments’, comprises several articles published in the esteemed journal Scientific Reports. Each article within this collection investigated genetic signature and adaptation in different extreme environments, including the cold polar region, arid desert, oxygen-deprived Tibetan mountains and others. These studies provide invaluable understanding of how life thrives and evolves under extreme conditions, shedding light on genetic mechanisms and adaptation strategies.
Fulltext Editorial: Genetics, Genomics and -Omics of Thermophiles

Goh KM, Chan KG, Sani RK, Donati ER, Reysenbach AL

Front Microbiol, 2017;8:560.
PMID: 28421055 DOI: 10.3389/fmicb.2017.00560
Fulltext Complete genome sequence of Rhodothermaceae bacterium RA with cellulolytic and xylanolytic activities

Liew KJ, Teo SC, Shamsir MS, Sani RK, Chong CS, Chan KG, et al.

3 Biotech, 2018 Aug;8(8):376.
PMID: 30105201 DOI: 10.1007/s13205-018-1391-z

Rhodothermaceae bacterium RA is a halo-thermophile isolated from a saline hot spring. Previously, the genome of this bacterium was sequenced using a HiSeq 2500 platform culminating in 91 contigs. In this report, we report on the resequencing of its complete genome using a PacBio RSII platform. The genome has a GC content of 68.3%, is 4,653,222 bp in size, and encodes 3711 genes. We are interested in understanding the carbohydrate metabolic pathway, in particular the lignocellulosic biomass degradation pathway. Strain RA harbors 57 glycosyl hydrolase (GH) genes that are affiliated with 30 families. The bacterium consists of cellulose-acting (GH 3, 5, 9, and 44) and hemicellulose-acting enzymes (GH 3, 10, and 43). A crude cell-free extract of the bacterium exhibited endoglucanase, xylanase, β-glucosidase, and β-xylosidase activities. The complete genome information coupled with biochemical assays confirms that strain RA is able to degrade cellulose and xylan. Therefore, strain RA is another excellent member of family Rhodothermaceae as a repository of novel and thermostable cellulolytic and hemicellulolytic enzymes.
Heterologous expression, purification and biochemical characterization of a new endo-1,4-β-xylanase from Rhodothermaceae bacterium RA

Liew KJ, Ngooi CY, Shamsir MS, Sani RK, Chong CS, Goh KM

Protein Expr Purif, 2019 12;164:105464.
PMID: 31376486 DOI: 10.1016/j.pep.2019.105464

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.
Fulltext Global Transcriptomic Responses of Roseithermus sacchariphilus Strain RA in Media Supplemented with Beechwood Xylan

Liew KJ, Bruce NC, Sani RK, Chong CS, Yaakop AS, Shamsir MS, et al.

Microorganisms, 2020 Jun 29;8(7).
PMID: 32610703 DOI: 10.3390/microorganisms8070976

The majority of the members in order Rhodothermales are underexplored prokaryotic extremophiles. Roseithermus, a new genus within Rhodothermales, was first described in 2019. Roseithermus sacchariphilus is the only species in this genus. The current report aims to evaluate the transcriptomic responses of R. sacchariphilus strain RA when cultivated on beechwood xylan. Strain RA doubled its growth in Marine Broth (MB) containing xylan compared to Marine Broth (MB) alone. Strain RA harbors 54 potential glycosyl hydrolases (GHs) that are affiliated with 30 families, including cellulases (families GH 3, 5, 9, and 44) and hemicellulases (GH 2, 10, 16, 29, 31,43, 51, 53, 67, 78, 92, 106, 113, 130, and 154). The majority of these GHs were upregulated when the cells were grown in MB containing xylan medium and enzymatic activities for xylanase, endoglucanase, β-xylosidase, and β-glucosidase were elevated. Interestingly, with the introduction of xylan, five out of six cellulolytic genes were upregulated. Furthermore, approximately 1122 genes equivalent to one-third of the total genes for strain RA were upregulated. These upregulated genes were mostly involved in transportation, chemotaxis, and membrane components synthesis.
Fulltext Characterization of a glucose-tolerant β-glucosidase from Anoxybacillus sp. DT3-1

Chan CS, Sin LL, Chan KG, Shamsir MS, Manan FA, Sani RK, et al.

Biotechnol Biofuels, 2016;9(1):174.
PMID: 27555880 DOI: 10.1186/s13068-016-0587-x

In general, biofuel production involves biomass pretreatment and enzymatic saccharification, followed by the subsequent sugar conversion to biofuel via fermentation. The crucial step in the production of biofuel from biomass is the enzymatic saccharification. Many of the commercial cellulase enzyme cocktails, such as Spezyme(®) CP (Genencor), Acellerase™ 1000 (Genencor), and Celluclast(®) 1.5L (Novozymes), are ineffectively to release free glucose from the pretreated biomass without additional β-glucosidase.
Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1

Rai R, Samanta D, Goh KM, Chadha BS, Sani RK

Int J Biol Macromol, 2024 Feb;257(Pt 2):128679.
PMID: 38072346 DOI: 10.1016/j.ijbiomac.2023.128679

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable β-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.
pH-dependent genotypic and phenotypic variability in Oleidesulfovibrio alaskensis G20

Saxena P, Samanta D, Thakur P, Goh KM, Subramaniam M, Peyton BM, et al.

Appl Environ Microbiol, 2025 Mar 26.
PMID: 40135858 DOI: 10.1128/aem.02565-24

Sulfate-reducing bacteria (SRB) exhibit versatile metabolic adaptability with significant flexibility influenced by pH fluctuations, which play a critical role in biogeochemical cycles. In this study, we used a model SRB, Oleidesulfovibrio alaskensis G20, to determine the temporal effects of pH variations (pH 6, 7, and 8) on both growth dynamics and metabolic gene expressions. The specific growth rate at pH 6 (0.014 h-1) closely matched that at pH 7 (0.016 h-1), while pH 8 exhibited a lower growth rate (0.010 h-1). Lactate consumption peaked at pH 7 (0.35 mM lactate.h-1) and declined at pH 8 (0.09 mM lactate.h-1). Significant hydrogen production was evident under both acidic and alkaline conditions. Gene expression studies revealed that ATPases function as proton pumps, while hydrogenases mediate reversible proton-to-hydrogen conversion. Sulfate and energy metabolism act as electron acceptors and donors, while amino acid synthesis regulates basic and acidic amino acids to mitigate pH stress. Downregulation of FtsZ at pH 6 suggests impaired division, correlating with slightly longer lengths (~2 µm), while upregulation of divisome proteins at pH 8 suggests efficient division processes, aligning with shorter lengths (~1.8 µm). This study will facilitate the employment of O. alaskensis G20 in extreme pH environments, enhancing its effectiveness in optimizing bioremediation and anaerobic digestion processes.
IMPORTANCE: Sulfate-reducing bacteria (SRB) play essential roles in global sulfur and carbon cycling and are critical for bioremediation and anaerobic digestion processes. However, detailed studies on the genotypic and phenotypic responses of SRB under varying pH conditions are limited. This study addresses this gap by examining the pH-dependent genetic and metabolic adaptations of Oleidesulfovibrio alaskensis G20, revealing key mechanisms regulating hydrogenase and ATPase activities, cell division, and extracellular polymeric substance formation. These findings provide new insights into how SRB maintains pH homeostasis, showcasing their ability to survive and function in both acidic and alkaline environments. Furthermore, this study reveals critical genetic and phenotypic characteristics that will directly aid to engineer industrial effluent management systems, bioremediation, and dissolved heavy metal recovery. By elucidating the dynamic response of O. alaskensis G20 to varied pH environments, the research provides a foundation for enhancing the resilience and performance of SRB-based systems, paving the way for improved environmental and industrial applications.
Integrating multi-platform assembly to recover MAGs from hot spring biofilms: insights into microbial diversity, biofilm formation, and carbohydrate degradation

Liew KJ, Shahar S, Shamsir MS, Shaharuddin NB, Liang CH, Chan KG, et al.

Environ Microbiome, 2024 May 06;19(1):29.
PMID: 38706006 DOI: 10.1186/s40793-024-00572-7

BACKGROUND: Hot spring biofilms provide a window into the survival strategies of microbial communities in extreme environments and offer potential for biotechnological applications. This study focused on green and brown biofilms thriving on submerged plant litter within the Sungai Klah hot spring in Malaysia, characterised by temperatures of 58-74 °C. Using Illumina shotgun metagenomics and Nanopore ligation sequencing, we investigated the microbial diversity and functional potential of metagenome-assembled genomes (MAGs) with specific focus on biofilm formation, heat stress response, and carbohydrate catabolism.
RESULTS: Leveraging the power of both Illumina short-reads and Nanopore long-reads, we employed an Illumina-Nanopore hybrid assembly approach to construct MAGs with enhanced quality. The dereplication process, facilitated by the dRep tool, validated the efficiency of the hybrid assembly, yielding MAGs that reflected the intricate microbial diversity of these extreme ecosystems. The comprehensive analysis of these MAGs uncovered intriguing insights into the survival strategies of thermophilic taxa in the hot spring biofilms. Moreover, we examined the plant litter degradation potential within the biofilms, shedding light on the participation of diverse microbial taxa in the breakdown of starch, cellulose, and hemicellulose. We highlight that Chloroflexota and Armatimonadota MAGs exhibited a wide array of glycosyl hydrolases targeting various carbohydrate substrates, underscoring their metabolic versatility in utilisation of carbohydrates at elevated temperatures.
CONCLUSIONS: This study advances understanding of microbial ecology on plant litter under elevated temperature by revealing the functional adaptation of MAGs from hot spring biofilms. In addition, our findings highlight potential for biotechnology application through identification of thermophilic lignocellulose-degrading enzymes. By demonstrating the efficiency of hybrid assembly utilising Illumina-Nanopore reads, we highlight the value of combining multiple sequencing methods for a more thorough exploration of complex microbial communities.
Biochar from pyrolyzed Tibetan Yak dung as a novel additive in ensiling sweet sorghum: An alternate to the hazardous use of Yak dung as a fuel in the home

Bai Y, Rafiq MK, Li S, Degen AA, Mašek O, Sun H, et al.

J Hazard Mater, 2021 02 05;403:123647.
PMID: 33264862 DOI: 10.1016/j.jhazmat.2020.123647

Yak dung is used as fuel in Tibetan homes; however, this use is hazardous to health. An alternative use of the dung that would be profitable and offset the loss as a fuel would be very beneficial. Sweet sorghum silage with yak dung biochar as an additive was compared with a control silage with no additives and three silages with different commercial additives, namely Lactobacillus buchneri, Lactobacillus plantarum and Acremonium cellulase. Biochar-treated silage had a significantly greater concentration of water-soluble carbohydrates than the other silages (76 vs 12.4-45.8 g/kg DM) and a greater crude protein content (75.5 vs 61.4 g/kg DM), lactic acid concentration (40.7 vs 27.7 g/kg DM) and gross energy yield (17.8 vs 17.4 MJ/kg) than the control silage. Biochar-treated and control silages did not differ in in vitro digestibility and in total gas (507 vs 511 L/kg DM) and methane production (57.9 vs 57.1 L/kg DM). Biochar inhibited degradation of protein and water-soluble carbohydrates and enhanced lactic acid production, which improved storability of feed. It was concluded that yak dung biochar is an efficient, cost-effective ensiling additive. The profit could offset the loss of dung as fuel and improve the health of Tibetan people.