Dataset Information

Isolation and characterisation of Methylocystis spp. for poly-3-hydroxybutyrate production using waste methane feedstocks.

ABSTRACT: Waste plastic and methane emissions are two anthropogenic by-products exacerbating environmental pollution. Methane-oxidizing bacteria (methanotrophs) hold the key to solving these problems simultaneously by utilising otherwise wasted methane gas as carbon source and accumulating the carbon as poly-3-hydroxybutyrate, a biodegradable plastic polymer. Here we present the isolation and characterisation of two novel Methylocystis strains with the ability to produce up to 55.7?±?1.9% poly-3-hydroxybutyrate of cell dry weight when grown on methane from different waste sources such as landfill and anaerobic digester gas. Methylocystis rosea BRCS1 isolated from a recreational lake and Methylocystis parvus BRCS2 isolated from a bog were whole genome sequenced using PacBio and Illumina genome sequencing technologies. In addition to potassium nitrate, these strains were also shown to grow on ammonium chloride, glutamine and ornithine as nitrogen source. Growth of Methylocystis parvus BRCS2 on Nitrate Mineral Salt (NMS) media with 0.1% methanol vapor as carbon source was demonstrated. The genetic tractability by conjugation was also determined with conjugation efficiencies up to 2.8?×?10^-2 and 1.8?×?10^-2 for Methylocystis rosea BRCS1 and Methylocystis parvus BRCS2 respectively using a plasmid with ColE1 origin of replication. Finally, we show that Methylocystis species can produce considerable amounts of poly-3-hydroxybutyrate on waste methane sources without impaired growth, a proof of concept which opens doors to their use in integrated bio-facilities like landfills and anaerobic digesters.

SUBMITTER: Rumah BL

PROVIDER: S-EPMC7788130 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Isolation and characterisation of Methylocystis spp. for poly-3-hydroxybutyrate production using waste methane feedstocks.

Rumah Bashir L BL Stead Christopher E CE Claxton Stevens Benedict H BH Minton Nigel P NP Grosse-Honebrink Alexander A Zhang Ying Y

AMB Express 20210106 1

Waste plastic and methane emissions are two anthropogenic by-products exacerbating environmental pollution. Methane-oxidizing bacteria (methanotrophs) hold the key to solving these problems simultaneously by utilising otherwise wasted methane gas as carbon source and accumulating the carbon as poly-3-hydroxybutyrate, a biodegradable plastic polymer. Here we present the isolation and characterisation of two novel Methylocystis strains with the ability to produce up to 55.7 ± 1.9% poly-3-hydroxybu ...[more]

PMID: 33409745

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Project description:BackgroundDue to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production.ResultsWe constructed a poly (3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJ?phaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5 days of culture. The HJ?phaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJ?phaZ (phaA3), HJ?phaZ (phaB2), and HJ?phaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJ?phaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJ?phaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88?±?0.08 g l-1 and with 100 mM ammonium sulfate; 0.99?±?0.05 g l-1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively.ConclusionIn this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly (3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria.

Project description:BackgroundMicrobial polyesters, also known as polyhydroxyalkanoates (PHAs), closely resemble physical and mechanical features of petroleum derived plastics. Recombinant Escherichia coli strains are being used in industrial production of PHAs in Stirred Tank Bioreactors (STRs). However, use of Air-Lift Reactors (ALRs) has been known to offer numerous technical operating options over STRs, and as such has been successfully implemented in many bioprocesses. Halomonas boliviensis is a halophilic bacterium that is known to assimilate various carbohydrates and convert them into a particular type of PHA known as poly(3-hydroxybutyrate) (PHB). Owing to this capability, it has been used to synthesize the polyester using hydrolysates of starch or wheat bran in stirred tank bioreactors.ResultsThis research article firstly describes the production of PHB in shake flasks by H. boliviensis using different combinations of carbohydrates and partially hydrolyzed starch as carbon sources. The highest PHB yields, between 56 and 61 % (wt.), were achieved when either starch hydrolysate or a mixture of glucose and xylose were used as carbon sources. The starch hydrolysate obtained in this study was then used as carbon source in an ALR. The largest amount of PHB, 41 % (wt.), was attained after 24 hrs of cultivation during which maltose in the hydrolysate was assimilated more rapidly than glucose during active cell growth; however, the rate of assimilation of both the carbohydrates was found to be similar during synthesis of PHB. An incomplete pentose phosphate pathway, which lacks 6-phosphogluconate dehydrogenase, was deduced from the genome sequence of this bacterium and may result in the characteristic assimilation of glucose and maltose by the cells.ConclusionsThis study showed that the production of PHB by H. boliviensis using cheap substrates such as starch hydrolysate in a simple production system involving an ALR is feasible. Both maltose and glucose in the hydrolysate induce cell growth and PHB synthesis; most likely the cells balance adequately CoA and NAD(P)H during the assimilation of these carbohydrates. The combination of cheap substrates, simple production systems and the use of non-strict sterile conditions by the halophile H. boliviensis are desirable traits for large scale production of PHB, and should lead to a competitive bioprocess.

Project description:BACKGROUND:Recently, it has been indicated that anaerobic co-digestion of waste activated sludge with other waste streams at wastewater treatment plants is a promising strategy for enhancing methane production and materials recovery. The enhanced methane production can be used as a renewable source of energy in wastewater treatment plants. It can also reduce the amount of greenhouse gas emission in landfilling of the waste streams. RESULTS:According to the results obtained in this study, anaerobic co-digestion of waste activated sludge with mixed fruit waste and cheese whey improves methane production and the quality of digested sludge in comparison to the anaerobic digestion of waste activated sludge individually. It was indicated that carbon/nitrogen ratio (C/N) in the mixture of waste activated sludge, fruit waste and cheese whey improved considerably, leading to better anaerobic organisms' activity during digestion. With assessing the activity of protease and cellulase, as the main enzymes hydrolyzing organic matter in anaerobic digestion, it was indicated that co-digestion of waste activated sludge with mixed fruit waste and cheese whey enhances the activity of these enzymes by 22 and 9% respectively. At the end of digestion, the amount of cumulative methane production significantly increased by 31% in the reactor with 85% waste activated sludge and 15% mixed fruit waste and cheese whey, compared to the reactor with 100% waste activated sludge. In addition, chemical oxygen demand (COD) and volatile solid (VS) in digested sludge was improved respectively by 9 and 7% when mixed fruit waste and cheese whey was used. CONCLUSIONS:This study revealed that mixed fruit waste and cheese whey is potentially applicable to anaerobic digestion of waste activated sludge, as fruit waste and cheese whey have high C/N ratio that enhance low C/N in waste activated sludge and provide a better diet for anaerobic organisms. This is of significant importance because not only could higher amount of renewable energy be generated from the enhanced methane production in wastewater treatment plants, but also capital costs of the companies whose waste streams are being transported to wastewater treatments plants could be reduced considerably.

Dataset Information

Isolation and characterisation of Methylocystis spp. for poly-3-hydroxybutyrate production using waste methane feedstocks.

Publications

Isolation and characterisation of Methylocystis spp. for poly-3-hydroxybutyrate production using waste methane feedstocks.

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