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Growth Optimisation and Kinetic Profiling of Diesel Biodegradation by a Cold-Adapted Microbial Consortium Isolated from Trinity Peninsula, Antarctica.

ABSTRACT: Pollution associated with petrogenic hydrocarbons is increasing in Antarctica due to a combination of increasing human activity and the continent's unforgiving environmental conditions. The current study focuses on the ability of a cold-adapted crude microbial consortium (BS24), isolated from soil on the north-west Antarctic Peninsula, to metabolise diesel fuel as the sole carbon source in a shake-flask setting. Factors expected to influence the efficiency of diesel biodegradation, namely temperature, initial diesel concentration, nitrogen source type and concentration, salinity and pH were studied. Consortium BS24 displayed optimal cell growth and diesel degradation activity at 1.0% NaCl, pH 7.5, 0.5 g/L NH₄Cl and 2.0% v/v initial diesel concentration during one-factor-at-a-time (OFAT) analyses. The consortium was psychrotolerant based on the optimum growth temperature of 10‒15 °C. In conventionally optimised media, the highest total petroleum hydrocarbons (TPH) mineralisation was 85% over a 7-day incubation. Further optimisation of conditions predicted through statistical response-surface methodology (RSM) (1.0% NaCl, pH 7.25, 0.75 g/L NH₄Cl, 12.5 °C and 1.75% v/v initial diesel concentration) boosted mineralisation to 95% over a 7-day incubation. A Tessier secondary model best described the growth pattern of BS24 in diesel-enriched medium, with maximum specific growth rate, μ_max, substrate inhibition constant, K_i and half saturation constant, K_s, being 0.9996 h^-1, 1.356% v/v and 1.238% v/v, respectively. The data obtained suggest the potential of microbial consortia such as BS24 in bioremediation applications in low-temperature diesel-polluted soils.

SUBMITTER: Roslee AFA

PROVIDER: S-EPMC8228002 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A soil bacterial consortium that was grown on diesel fuel and consisted of more than 10 members from different genera was maintained through repetitive subculturing and was utilized as a practical model to investigate a bacterial community that was continuously exposed to petroleum hydrocarbons. Through metagenomics analyses, consortium member isolation, growth assays, and metabolite identification which supported the linkage of genomic data and functionality, two pioneering genera, Sphingobium and Pseudomonas, whose catabolic capabilities were differentiated, were found to be responsible for the creation of specialized ecological niches that were apparently occupied by other bacterial members for survival within the consortium. Coexisting genera Achromobacter and Cupriavidus maintained their existence in the consortium through metabolic dependencies by utilizing hydrocarbon biotransformation products of pioneer metabolism, which was confirmed through growth tests and identification of biotransformation products of the isolated strains. Pioneering Sphingobium and Pseudomonas spp. utilized relatively water-insoluble hydrocarbon parent compounds and facilitated the development of a consortium community structure that resulted in the creation of niches in response to diesel fuel exposure which were created through the production of more-water-soluble biotransformation products available to cocolonizers. That these and other organisms were still present in the consortium after multiple transfers spanning 15 years provided evidence for these ecological niches. Member survival through occupation of these niches led to robustness of each group within the multispecies bacterial community. Overall, these results contribute to our understanding of the complex ecological relationships that may evolve during prokaryotic hydrocarbon pollutant biodegradation.IMPORTANCE There are few metagenome studies that have explored soil consortia maintained on a complex hydrocarbon substrate after the community interrelationships were formed. A soil bacterial consortium maintained on diesel fuel was utilized as a practical model to investigate bacterial community relationships through metagenomics analyses, consortium member isolation, growth assays, and metabolite identification, which supported the linkage of genomic data and functionality. Two pioneering genera were responsible for the biodegradation of aromatics and alkanes by initiating biotransformation and thereby created specialized niches that were populated by other members. A model that represents these relationships was constructed, which contributes to our understanding of the complex ecological relationships that evolve during prokaryotic hydrocarbon pollutant biodegradation.

Project description:BackgroundCold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.ResultsA gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures.ConclusionBy taking advantage of its high activity at low temperatures and its acidic pH optimum, this recombinant chitinase will be valuable in various biotechnological applications under low temperature and acidic pH conditions.

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Growth Optimisation and Kinetic Profiling of Diesel Biodegradation by a Cold-Adapted Microbial Consortium Isolated from Trinity Peninsula, Antarctica.

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