Microbial community dynamics and functional potentials in the conversion of oil palm wastes into biomethane.

Oil palm processing generates substantial waste materials rich in organic content, posing various environmental challenges. Anaerobic digestion (AD), particularly for palm oil mill effluent (POME), offers a sustainable solution, by converting waste into valuable biomethane for thermal energy or electricity generation. The synergistic activities of the AD microbiota directly affect the biomethane production, and the microbial community involved in biomethane production in POME anaerobic digestion has been reported. The composition of bacterial and archaeal communities varies under different substrate and physicochemical conditions. This review discusses the characteristics of POME, explores the microbial members engaged in each stage of AD, and elucidates the impacts of substrate and physicochemical conditions on the microbial community dynamics, with a specific focus on POME. Finally, the review outlines current research needs and provides future perspectives on optimizing the microbial communities for enhanced biomethane production from oil palm wastes.

Genomic Analysis to Elucidate the Lignocellulose Degrading Capability of a New Halophile Robertkochia solimangrovi.

Robertkochia solimangrovi is a proposed marine bacterium isolated from mangrove soil. So far, the study of this bacterium is limited to taxonomy only. In this report, we performed a genomic analysis of R. solimangrovi that revealed its lignocellulose degrading ability. Genome mining of R. solimangrovi revealed a total of 87 lignocellulose degrading enzymes. These enzymes include cellulases (GH3, GH5, GH9 and GH30), xylanases (GH5, GH10, GH43, GH51, GH67, and GH115), mannanases (GH2, GH26, GH27 and GH113) and xyloglucanases (GH2, GH5, GH16, GH29, GH31 and GH95). Most of the lignocellulolytic enzymes encoded in R. solimangrovi were absent in the genome of Robertkochia marina, the closest member from the same genus. Furthermore, current work also demonstrated the ability of R. solimangrovi to produce lignocellulolytic enzymes to deconstruct oil palm empty fruit bunch (EFB), a lignocellulosic waste found abundantly in palm oil industry. The metabolic pathway taken by R. solimangrovi to transport and process the reducing sugars after the action of lignocellulolytic enzymes on EFB was also inferred based on genomic data. Collectively, genomic analysis coupled with experimental studies elucidated R. solimangrovi to serve as a promising candidate in seawater based-biorefinery industry.

Improvement and screening of astaxanthin producing mutants of newly isolated Coelastrum sp. using ethyl methane sulfonate induced mutagenesis technique.

Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

Genome sequence of an uncharted halophilic bacterium Robertkochia marina with deciphering its phosphate-solubilizing ability.

The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30DT is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57 Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land.

Draft genome sequence of Parvularcula flava strain NH6-79 T, revealing its role as a cellulolytic enzymes producer.

To date, the genus Parvularcula consists of 6 species and no potential application of this genus was reported. Current study presents the genome sequence of Parvularcula flava strain NH6-79 T and its cellulolytic enzyme analysis. The assembled draft genome of strain NH6-79 T consists of 9 contigs and 7 scaffolds with 3.68 Mbp in size and GC content of 59.87%. From a total of 3,465 genes predicted, 96 of them are annotated as glycoside hydrolases (GHs). Within these GHs, 20 encoded genes are related to cellulosic biomass degradation, including 12 endoglucanases (5 GH10, 4 GH5, and 3 GH51), 2 exoglucanases (GH9) and 6 ß-glucosidases (GH3). In addition, highest relative enzyme activities (endoglucanase, exoglucanase, and ß-glucosidase) were observed at 27th hour when the strain was cultured in the carboxymethyl cellulose/Avicel®-containing medium for 45 h. The combination of genome analysis with experimental studies indicated the ability of strain NH6-79 T to produce extracellular endoglucanase, exoglucanase, and ß-glucosidase. These findings suggest the potential of Parvularcula flava strain NH6-79 T in cellulose-containing biomass degradation and that the strain could be used in cellulosic biorefining process.

Genome sequence data of Mangrovimonas sp. strain CR14 isolated from mangrove forest at Tanjung Piai National Park, Malaysia.

Mangrovimonas sp. strain CR14 is a halophilic bacterium affiliated with family Flavobacteriaceae which was successfully isolated from mangrove soil samples obtained from Tanjung Piai National Park, Johor. The whole genome of strain CR14 was sequenced on an Illumina HiSeq 2500 platform (2 × 150 bp paired end). Herein, we report the genome sequence of Mangrovimonas sp. strain CR14 in which its assembled genome consisted 20 contigs with a total size of 3,590,195 bp, 3209 coding sequences, and an average 36.08% G + C content. Genome annotation and gene mining revealed that this bacterium demonstrated proteolytic activity which could be potentially applied in detergent industry. This whole-genome shotgun data of Mangrovimonas sp. strain CR14 has been deposited at DDBJ/ENA/GenBank under the accession JAAFZY000000000. The version described in this paper is version JAAFZY010000000.

Genome analysis of cellulose and hemicellulose degrading Micromonospora sp. CP22.

In this study, a bacterial strain CP22 with ability to produce cellulase, xylanase and mannanase was isolated from the oil palm compost. Based on the 16S rRNA gene analysis, the strain was affiliated to genus Micromonospora. To further investigate genes that are related to cellulose and hemicellulose degradation, the genome of strain CP22 was sequenced, annotated and analyzed. The de novo assembled genome of strain CP22 featured a size of 5,856,203 bp with G + C content of 70.84%. Detailed genome analysis on lignocellulose degradation revealed a total of 60 genes consisting of 47 glycoside hydrolase domains and 16 carbohydrate esterase domains predicted to be involved in cellulolytic and hemicellulolytic deconstruction. Particularly, 20 genes encode for cellulases (8 endoglucanases, 3 exoglucanases and 9 ß-glucosidases) and 40 genes encode for hemicellulases (15 endo-1,4-ß-xylanase, 3 ß-xylosidase, 3 α-arabinofuranosidase, 10 acetyl xylan esterase, 6 polysaccharide deacetylase, 1 ß-mannanase, 1 ß-mannosidase and 1 α-galactosidase). Thirty-two genes encoding carbohydrate-binding modules (CBM) from six different families (CBM2, CBM4, CBM6, CBM9, CBM13 and CBM22) were present in the genome of strain CP22. These CBMs were found in 27 cellulolytic and hemicellulolytic genes, indicating their potential role in enhancing the substrate-binding capability of the enzymes. CBM2 and CBM13 are the major CBMs present in cellulases and hemicellulases (xylanases and mannanases), respectively. Moreover, a GH10 xylanase was found to contain 3 CBMs (1 CBM9 and 2 CBM22) and these CBMs were reported to bind specifically to xylan. This genome-based analysis could facilitate the exploration of this strain for lignocellulosic biomass degradation.

Robertkochia solimangrovi sp. nov., isolated from mangrove soil, and emended description of the genus Robertkochia.

To date, there is sparse information for the genus Robertkochia with Robertkochia marina CC-AMO-30DT as the only described member. We report here a new species isolated from mangrove soil collected at Malaysia Tanjung Piai National Park and perform polyphasic characterization to determine its taxonomic position. Strain CL23T is a Gram-negative, yellow-pigmented, strictly aerobic, catalase-positive and oxidase-positive bacterium. The optimal growth conditions were determined to be at pH 7.0, 30-37 °C and in 1-2 % (w/v) NaCl. The major respiratory quinone was menaquinone-6 (MK-6) and the highly abundant polar lipids were four unidentified lipids, a phosphatidylethanolamine and two unidentified aminolipids. The 16S rRNA gene similarity between strain CL23T and R. marina CC-AMO-30DT is 96.67 %. Strain CL23T and R. marina CC-AMO-30DT clustered together and were distinguished from taxa of closely related genera in 16S rRNA gene phylogenetic analysis. Genome sequencing revealed that strain CL23T has a genome size of 4.4 Mbp and a G+C content of 40.72 mol%. Overall genome related indexes including digital DNA-DNA hybridization value and average nucleotide identity are 17.70 % and approximately 70%, below the cutoffs of 70 and 95%, respectively, indicated that strain CL23T is a distinct species from R. marina CC-AMO-30DT. Collectively, based on the phenotypic, chemotaxonomic, phylogenetic and genomic evidences presented here, strain CL23T is proposed to represent a new species with the name Robertkochia solimangrovi sp. nov. (KCTC 72252T=LMG 31418T). An emended description of the genus Robertkochia is also proposed.

Complete genome sequence of Sphingomonas paucimobilis AIMST S2, a xenobiotic-degrading bacterium.

Complete genomes of xenobiotic-degrading microorganisms provide valuable resources for researchers to understand molecular mechanisms involved in bioremediation. Despite the well-known ability of Sphingomonas paucimobilis to degrade persistent xenobiotic compounds, a complete genome sequencing is lacking for this organism. In line with this, we report the first complete genome sequence of Sphingomonas paucimobilis (strain AIMST S2), an organophosphate and hydrocarbon-degrading bacterium isolated from oil-polluted soil at Kedah, Malaysia. The genome was derived from a hybrid assembly of short and long reads generated by Illumina HiSeq and MinION, respectively. The assembly resulted in a single contig of 4,005,505 bases which consisted of 3,612 CDS and 56 tRNAs. An array of genes involved in xenobiotic degradation and plant-growth promoters were identified, suggesting its' potential role as an effective microorganism in bioremediation and agriculture. Having reported the first complete genome of the species, this study will serve as a stepping stone for comparative genome analysis of Sphingomonas strains and other xenobiotic-degrading microorganisms as well as gene expression studies in organophosphate biodegradation.

Insights into microbial community structure and diversity in oil palm waste compost.

Empty fruit bunch (EFB) and palm oil mill effluent (POME) are the major wastes generated by the oil palm industry in Malaysia. The practice of EFB and POME digester sludge co-composting has shown positive results, both in mitigating otherwise environmentally damaging waste streams and producing a useful product (compost) from these streams. In this study, the bacterial ecosystems of 12-week-old EFB-POME co-compost and POME biogas sludge from Felda Maokil, Johor were analysed using 16S metagenome sequencing. Over ten phyla were detected, with Chloroflexi being the predominant phylum, representing approximately 53% of compost and 23% of the POME microbiome reads. The main bacterial lineage found in the compost and POME was Anaerolinaceae (Chloroflexi) with 30% and 18% of the total gene fragments, respectively. The significant differences between compost and POME communities were abundances of Syntrophobacter, Sulfuricurvum and Coprococcus. No methanogens were identified due to the bias in general 16S primers to eubacteria. The preponderance of anaerobic species in the compost and high abundance of secondary metabolite fermenting bacteria is due to an extended composting time, with anaerobic collapse of the pile due to the tropical heat. Predictive functional profiles of the metagenomes using 16S rRNA marker genes suggest that the presence of enzymes involved in degradation of polysaccharides such as glucoamylase, endoglucanase and arabinofuranosidase, all of which were strongly active in POME. Eubacterial species associated with cellulytic methanogenesis were present in both samples.

Bioremediation of palm oil mill effluent (POME) using indigenous Meyerozyma guilliermondii.

Despite being a key Malaysian economic contributor, the oil palm industry generates a large quantity of environmental pollutant known as palm oil mill effluent (POME). Therefore, the need to remediate POME has drawn a mounting interest among environmental scientists. This study has pioneered the application of Meyerozyma guilliermondii with accession number (MH 374161) that was isolated indigenously in accessing its potential to degrade POME. This strain was able to treat POME in shake flask experiments under aerobic condition by utilising POME as a sole source of carbon. However, it has also been shown that the addition of suitable carbon and nitrogen sources has significantly improved the degradation potential of M. guilliermondii. The remediation of POME using this strain resulted in a substantial reduction of chemical oxygen demand (COD) of 72%, total nitrogen of 49.2% removal, ammonical nitrogen of 45.1% removal, total organic carbon of 46.6% removal, phosphate of 60.6% removal, and 92.4% removal of oil and grease after 7 days of treatment period. The strain also exhibited an extracellular lipase activity which promotes better wastewater treatment. Additionally, Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS) analyses have specifically shown that M. guilliermondii strain can degrade hydrocarbons, fatty acids, and phenolic compounds present in the POME. Ultimately, this study has demonstrated that M. guilliermondii which was isolated indigenously exhibits an excellent degrading ability. Therefore, this strain is suitable to be employed in the remediation of POME, contributing to a safe discharge of the effluent into the environment.

Solid-state fermentation of oil palm frond petiole for lignin peroxidase and xylanase-rich cocktail production.

In current practice, oil palm frond leaflets and stems are re-used for soil nutrient recycling, while the petioles are typically burned. Frond petioles have high commercialization value, attributed to high lignocellulose fiber content and abundant of juice containing free reducing sugars. Pressed petiole fiber is the subject of interest in this study for the production of lignocellulolytic enzyme. The initial characterization showed the combination of 0.125 mm frond particle size and 60% moisture content provided a surface area of 42.3 m2/g, porosity of 12.8%, and density of 1.2 g/cm3, which facilitated fungal solid-state fermentation. Among the several species of Aspergillus and Trichoderma tested, Aspergillus awamori MMS4 yielded the highest xylanase (109 IU/g) and cellulase (12 IU/g), while Trichoderma virens UKM1 yielded the highest lignin peroxidase (222 IU/g). Crude enzyme cocktail also contained various sugar residues, mainly glucose and xylose (0.1-0.4 g/L), from the hydrolysis of cellulose and hemicellulose. FT-IR analysis of the fermented petioles observed reduction in cellulose crystallinity (I900/1098), cellulose-lignin (I900/1511), and lignin-hemicellulose (I1511/1738) linkages. The study demonstrated successful bioconversion of chemically untreated frond petioles into lignin peroxidase and xylanase-rich enzyme cocktail under SSF condition.

Biodecolorization of recalcitrant dye as the sole sourceof nutrition using Curvularia clavata NZ2 and decolorization ability of its crude enzymes.

Extensive use of recalcitrant azo dyes in textile and paper industries poses a direct threat to the environment due to the carcinogenicity of their degradation products. The aim of this study was to investigate the efficiency of Curvularia clavata NZ2 in decolorization of azo dyes. The ability of the fungus to decolorize azo dyes can be evaluated as an important outcome as existing effluent treatment is unable to remove the dyes effectively. C. clavata has the ability to decolorize Reactive Black 5 (RB5), Acid Orange 7 (AO7), and Congo Red azo dyes, utilizing these as sole sources of carbon and nitrogen. Ultraviolet-visible (UV-vis) spectroscopy and Fourier infrared spectroscopy (FTIR) analysis of the extracted RB5's metabolites along with desorption tests confirmed that the decolorization process occurred due to degradation and not merely by adsorption. Enzyme activities of extracellular enzymes such as carboxymethylcellulase (CMCase), xylanase, laccase, and manganese peroxidase (MnP) were also detected during the decolorization process. Toxicity expressed as inhibition of germination was reduced significantly in fungal-treated azo dye solution when compared with the control. The cultivation of C. clavata under sequential batch system also recorded a decolorization efficiency of above 90%. The crude enzyme secreted by C. clavata also showed excellent ability to decolorize RB5 solutions with concentrations of 100 ppm (88-92%) and 1000 ppm (70-77%) without redox mediator. This proved that extracellular enzymes produced by C. clavata played a major role in decolorization of RB5.

The impact of biochars on sorption and biodegradation of polycyclic aromatic hydrocarbons in soils--a review.

Amending polycyclic aromatic hydrocarbon (PAH)-contaminated soils with biochar may be cheaper and environmentally friendly than other forms of organic materials. This has led to numerous studies on the use of biochar to either bind or stimulate the microbial degradation of organic compounds in soils. However, very little or no attention have been paid to the fact that biochars can give simultaneous impact on PAH fate processes, such as volatilization, sorption and biodegradation. In this review, we raised and considered the following questions: How does biochar affect microbes and microbial activities in the soil? What are the effects of adding biochar on sorption of PAHs? What are the effects of adding biochar on degradation of PAHs? What are the factors that we can manipulate in the laboratory to enhance the capability of biochars to degrade PAHs? A triphasic concept of how biochar can give simultaneous impact on PAH fate processes in soils was proposed, which involves rapid PAH sorption into biochar, subsequent desorption and modification of soil physicochemical properties by biochar, which in turn stimulates microbial degradation of the desorbed PAHs. It is anticipated that biochar can give simultaneous impact on PAH fate processes in soils.

Isolation, screening, and identification of potential cellulolytic and xylanolytic producers for biodegradation of untreated oil palm trunk and its application in saccharification of lemongrass leaves.

This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g(-1)), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g(-1)) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strain's xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to the xylanase glycoside hydrolase family 11 (GH11) with a protein size of 24.49 kD. Saccharification of lemongrass leaves using crude cellulases and xylanase gives the maximum reducing sugars production of 6.84 g/L with glucose as the major end product and traces of phenylpropanic compounds (vanillic acid, p-coumaric acid, and ferulic acid).

Decolorization of palm oil mill effluent using growing cultures of Curvularia clavata.

Agricultural wastewater that produces color are of environmental and health concern as colored effluent can produce toxic and carcinogenic by-products. From this study, batch culture optimization using response surface methods indicated that the fungus isolated from the pineapple solid waste, Curvularia clavata was able to decolorize sterile palm oil mill effluent (POME) which is mainly associated with polyphenol and lignin. Results showed successful decolorization of POME up to 80 % (initial ADMI [American Dye Manufacturing Index] of 3,793) with 54 % contributed by biosorption and 46 % by biodegradation after 5 days of treatment. Analysis using HPLC and GC-MS showed the degradation of color causing compound such as 3-methoxyphenyl isothiocynate and the production of new metabolites. Ecotoxicity test indicated that the decolorized effluent is safe for discharge. To determine the longevity of the fungus for a prolonged decolorization period, sequential batch decolorization studies were carried out. The results showed that lignin peroxidase and laccase were the main ligninolytic enzymes involved in the degradation of color. Carboxymethyl cellulase (CMCase) and xylanase activities were also detected suggesting possible roles of the enzymes in promoting growth of the fungus which consequently contributed to improved decolorization of POME. In conclusion, the ability of C. clavata in treating color of POME indicated that C. clavata is of potential use for decolorization and degradation of agricultural wastewater containing polyphenolic compounds.

Optimization of decolorization of palm oil mill effluent (POME) by growing cultures of Aspergillus fumigatus using response surface methodology.

The conventional treatment process of palm oil mill effluent (POME) produces a highly colored effluent. Colored compounds in POME cause reduction in photosynthetic activities, produce carcinogenic by-products in drinking water, chelate with metal ions, and are toxic to aquatic biota. Thus, failure of conventional treatment methods to decolorize POME has become an important problem to be addressed as color has emerged as a critical water quality parameter for many countries such as Malaysia. Aspergillus fumigatus isolated from POME sludge was successfully grown in POME supplemented with glucose. Statistical optimization studies were conducted to evaluate the effects of the types and concentrations of carbon and nitrogen sources, pH, temperature, and size of the inoculum. Characterization of the fungus was performed using scanning electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and Brunauer, Emmet, and Teller surface area analysis. Optimum conditions using response surface methods at pH 5.7, 35 °C, and 0.57 % w/v glucose with 2.5 % v/v inoculum size resulted in a successful removal of 71 % of the color (initial ADMI of 3,260); chemical oxygen demand, 71 %; ammoniacal nitrogen, 35 %; total polyphenolic compounds, 50 %; and lignin, 54 % after 5 days of treatment. The decolorization process was contributed mainly by biosorption involving pseudo-first-order kinetics. FTIR analysis revealed that the presence of hydroxyl, C-H alkane, amide carbonyl, nitro, and amine groups could combine intensively with the colored compounds in POME. This is the first reported work on the application of A. fumigatus for the decolorization of POME. The present investigation suggested that growing cultures of A. fumigatus has potential applications for the decolorization of POME through the biosorption and biodegradation processes.

Characterization of aerobic granular sludge treating high strength agro-based wastewater at different volumetric loadings.

Understanding the relationship between microbial community and mechanism of aerobic granulation could enable wider applications of granules for high-strength wastewater treatment. The majority of granulation studies principally determine the engineering aspects of granules formation with little emphasis on the microbial diversity. In this study, three identical reactors namely R1, R2 and R3 were operated using POME at volumetric loadings of 1.5, 2.5 and 3.5 kg COD m(-3) d(-1), respectively. Aeration was provided at a volumetric flow rate of 2.5 cms(-1). Aerobic granules were successfully developed in R2 and R3 while bioflocs dominated R1 until the end of experiments. Fractal dimension (D(f)) averaged at 1.90 suggesting good compactness of granules. The PCR-DGGE results indicated microbial evolutionary shift throughout granulation despite different operating OLRs based on decreased Raup and Crick similarity indices upon mature granule formation. The characteristics of aerobic granules treating high strength agro-based wastewater are determined at different volumetric loadings.

Genome sequence of Hydrogenophaga sp. strain PBC, a 4-aminobenzenesulfonate-degrading bacterium.

Hydrogenophaga sp. strain PBC is an effective degrader of 4-aminobenzenesulfonate isolated from textile wastewater. Here we present the assembly and annotation of its genome, which may provide further insights into its metabolic potential. This is the first announcement of the draft genome sequence of a strain from the genus Hydrogenophaga.

Genome sequence of Ralstonia sp. strain PBA, a bacterium involved in the biodegradation of 4-aminobenzenesulfonate.

Ralstonia sp. strain PBA was isolated from textile wastewater in a coculture with Hydrogenophaga sp. strain PBC. Here we present the assembly and annotation of its genome, which may provide further insights into the mechanism of its interaction with strain PBC during 4-aminobenzenesulfonate degradation.