Pseudomonas and Pseudarthrobacter are the key players in synergistic phenanthrene biodegradation at low temperatures.

Hydrocarbon contamination, including contamination with polycyclic aromatic hydrocarbons (PAHs), is a major concern in Antarctica due to the toxicity, recalcitrance and persistence of these compounds. Under the Antarctic Treaty, nonindigenous species are not permitted for use in bioremediation at polluted sites in the Antarctic region. In this study, three bacterial consortia (C13, C15, and C23) were isolated from Antarctic soils for phenanthrene degradation. All isolated bacterial consortia demonstrated phenanthrene degradation percentages ranging from 45 to 85% for 50 mg/L phenanthrene at 15 â„ƒ within 5 days. Furthermore, consortium C13 exhibited efficient phenanthrene degradation potential across a wide range of environmental conditions, including different temperature (4-30 â„ƒ) and water availability (without polyethylene glycol (PEG) 6000 or 30% PEG 6000 (w/v)) conditions. Sequencing analysis of 16S rRNA genes revealed that Pseudomonas and Pseudarthrobacter were the dominant genera in the phenanthrene-degrading consortia. Moreover, six cultivable strains were isolated from these consortia, comprising four strains of Pseudomonas, one strain of Pseudarthrobacter, and one strain of Paeniglutamicibacter. These isolated strains exhibited the ability to degrade 50 mg/L phenanthrene, with degradation percentages ranging from 4 to 22% at 15 â„ƒ within 15 days. Additionally, the constructed consortia containing Pseudomonas spp. and Pseudarthrobacter sp. exhibited more effective phenanthrene degradation (43-52%) than did the individual strains. These results provide evidence that Pseudomonas and Pseudarthrobacter can be potential candidates for synergistic phenanthrene degradation at low temperatures. Overall, our study offers valuable information for the bioremediation of PAH contamination in Antarctic environments.

Simultaneous immobilization enhances synergistic interactions and crude oil removal of bacterial consortium.

Oil spillage has serious adverse effects on marine environments. The degradation of crude oil by microorganisms may be an effective and sustainable approach. In this study, the removal of crude oil from seawater by immobilized bacterial consortium was performed and the enhancement of crude oil degradation efficiency by varying immobilization methods and inoculum volume ratio was examined. The nonpathogenic and heavy metal-tolerant bacterial consortium of Sphingobium naphthae MO2-4 and Priestia aryabhattai TL01-2 was immobilized by biofilm formation on aquaporousgels. The simultaneous immobilization of strains MO2-4 and TL01-2 showed better crude oil removal efficiency than independent immobilization, which indicated positive interactions among consortium members in the mixed-culture immobilized systems. Moreover, the immobilized consortium at a 2:1 (MO2-4:TL01-2) inoculum volume ratio showed the best crude oil removal capacity. The immobilized consortium removed 77% of 2000 mg L-1 crude oil in seawater over 7 days. The immobilized consortium maintained crude oil removal efficacy in semicontinuous experiments. In addition, the immobilized consortium was used to remediate seawater contaminated with 1000 mg L-1 crude oil in a 20 L wave tank. After 28 days, the crude oil degradation efficiency of immobilized consortium was approximately 70%, and crude oil degradation through natural attenuation was not observed. Moreover, the genomic features of strains MO2-4 and TL01-2 are reported. Genomic analyses of both strains confirmed the presence of many genes involved in hydrocarbon degradation, heavy metal resistance, biosurfactant synthesis, and biofilm formation, supporting the biodegradation results and characterizing strain properties. The results of this work introduce the potential benefit of simultaneous immobilization of bacterial consortia to improve efficiency of crude oil biodegradation and has motivated further investigations into large-scale remediation of crude oil-contaminated seawater.

Conversion of golden oyster mushroom, Pleurotus citrinopileatus to sugar syrup using enzymatic hydrolysis as a substrate for novel bacterial cellulose (Nata) fermentation.

Enzymatic hydrolysis of the golden oyster mushroom (Pleurotus citrinopileatus) generated a new bacterial cellulose (BC). The sugar syrup obtained from the hydrolysis of mushroom powder by commercial enzymes gave maximum total soluble solids (TSS) content at 8.83 ± 0.29°Brix, while 8.82 ± 0.06 mg GAE/g substrate of total phenolic content (TPC) was obtained when using initial substrate and enzyme concentrations at 125 g/L and 5.0%, respectively. Glutamic acid, aspartic acid, alanine and valine were determined as the main amino acids found in P. citrinopileatus hydrolysis at 524.74 ± 0.03, 247.09 ± 0.04, 176.82 ± 0.07 and 174.57 ± 0.01 mg/100 g sample, respectively. Thin-layer chromatography revealed that the obtained sugar syrup was glucose. The hydrolyzed mushroom fermented with Komagataeibacter xylinus AGR 60 at 30 ± 2 °C for 9 days produced optimal conditions at 4.0°Brix of the initial mushroom syrup and 12.0% (v/v) of the starter culture. Maximum BC thickness was 0.88 ± 0.03 cm with 7.90 ± 0.07 g dry weight, equivalent to 39.50 ± 0.35 g/L and 4.39 ± 0.04 g/L/day for BC production (P) and BC production rate (R p), respectively. The obtained BC was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, small-angle X-ray scattering and wide-angle X-ray diffraction. These showed the structure and functional properties as a natural source of fiber from the fermentation of a novel substrate.

Physiological changes in Rhodococcus ruber S103 immobilized on biobooms using low-cost media enhance stress tolerance and crude oil-degrading activity.

For economic feasibility, sugarcane molasses (0.5%, w/v) containing K2HPO4 (0.26%, w/v) and mature coconut water, low value byproducts, were used in cultivation of Rhodococcus ruber S103 for inoculum production and immobilization, respectively. Physiological changes of S103 grown in low-cost media, including cell hydrophobicity, saturated/unsaturated ratio of cellular fatty acids and biofilm formation activity, enhanced stress tolerance and crude oil biodegradation in freshwater and even under high salinity (5%, w/v). Biobooms comprised of S103 immobilized on polyurethane foam (PUF) was achieved with high biomass content (1010 colony-forming units g-1 PUF) via a scale-up process in a 5-L modified fluidized-bed bioreactor within 3 days. In a 500-L mesocosm, natural freshwater was spiked with crude oil (72 g or 667 mg g-1 dry biobooms), and a simulated wave was applied. Biobooms could remove 100% of crude oil within only 3 days and simultaneously biodegraded 60% of the adsorbed oil after 7 days when compared to boom control with indigenous bacteria. In addition, biobooms had a long shelf-life (at least 100 days) with high biodegradation activity (85.2 ± 2.3%) after storage in 10% (w/v) skimmed milk at room temperature. This study demonstrates that the low-cost production of biobooms has potential for future commercial bioremediation.

Bioaugmentation with zeolite-immobilized bacterial consortium OPK results in a bacterial community shift and enhances the bioremediation of crude oil-polluted marine sandy soil microcosms.

A pyrene-degrading consortium OPK containing Mycolicibacterium strains PO1 and PO2, Novosphingobium pentaromativorans PY1 and Bacillus subtilis FW1 effectively biodegraded medium- and long-chain alkanes as well as mixed hydrocarbons in crude oil. The detection of alkB and CYP153 genes in the genome of OPK members supports its phenotypic ability to effectively degrade a broad range of saturated hydrocarbons in crude oil. Zeolite-immobilized OPK was developed as a ready-to-use bioproduct and it exhibited 74% removal of 1000 mg L-1 crude oil within 96 h in sterilized seawater without nutrient supplementation and maintained high crude oil-removal activity under a broad range of pH values (5.0-9.0), temperatures (30-40 °C) and salinities (20-60‰). In addition, the immobilized OPK retained a high crude oil removal efficacy in semicontinuous experiments and showed reusability for at least 5 cycles. Remarkably, bioaugmentation with zeolite-immobilized OPK in sandy soil microcosms significantly increased crude oil (10,000 mg kg-1 soil) removal from 45% to 80.67% within 21 days compared to biostimulation and natural attenuation. Moreover, bioaugmentation with exogenous immobilized OPK stimulated an increase in the relative abundances of Alcanivorax genus, indigenous hydrocarbon-degrading bacteria, which in turn enhanced removal efficiency of crude oil contamination from sandy soil microcosms. The results indicate positive interactions between the bioaugmented immobilized consortium, harboring Mycolicibacterium as a key player, and indigenous Alcanivorax, which exhibited crucial functions for improving crude oil removal efficacy. The knowledge obtained forms an important basis for further synthesis and handling of a promising bio-based product for enhancing the in situ bioremediation of crude oil-polluted marine environments.

Bioballs carrying a syntrophic Rhodococcus and Mycolicibacterium consortium for simultaneous sorption and biodegradation of fuel oil in contaminated freshwater.

Nonpathogenic effective bacterial hydrocarbon degraders, Rhodococcus ruber S103, Mycolicibacterium parafortuitum J101 and Mycolicibacterium austroafricanum Y502, were isolated from mixed polycyclic aromatic hydrocarbon (PAH)-enriched river sediments. They possessed broad substrate specificities toward various PAHs and aliphatic compounds as sole carbon sources. These strains exhibited promising characteristics, including biosurfactant production, high cell hydrophobicity, biofilm formation and no antagonistic interactions, and contained genes encoding hydrocarbon-degrading enzymes. The mixed bacterial consortium combining S103, J101 and Y502, showed more effective syntrophic degradation of two types of refined petroleum products, diesel and fuel oils, than monocultures. The defined consortium immobilized on plastic balls achieved over 50% removal efficiency of high fuel oil concentration (3000 mg L-1) in a synthetic medium and contaminated freshwater. Furthermore, the immobilized cells simultaneously degraded more than 46% of total fuel oil adsorbed on plastic balls in both culture systems. SEM imaging confirmed that the immobilized consortium exhibited biofilm formation with the bacterial community covering most of the bioball surface, resulting in high bacterial survival against toxic contaminants. The results of this study showed the potential use of the cooperative interaction between Rhodococcus and Mycolicibacterium as immobilized bioballs for the bioremediation of fuel oil-contaminated environments. Additionally, this research has motivated further investigations into the development of bioremediation products for fuel oil degradation.

Enhanced ultrafine nanofibril biosynthesis of bacterial nanocellulose using a low-cost material by the adapted strain of Komagataeibacter xylinus MSKU 12.

Bacterial nanocellulose (BNC) is a renewable and biodegradable biopolymer which has currently received considerable attention due to the rapid increase in environmental issues. In this study, a cost-effective strategy for BNC production was successfully improved in the adapted strain, C30, which was obtained from Komagataeibacter xylinus MSKU 12 by a repetitive cultivation in a low-cost coconut water containing acetic acid and ethanol (CW-AE medium) at 37 °C. The adaptive procedure allowed the strain C30 to be adapted to grow and produce BNC with a higher yield in a limiting nutrient CW-AE medium, than that in a standard HS-AE medium. This strain could produce a high yield of BNC (9.69 g/L dry weight) in a low-cost medium, a modified CW-AE medium supplemented with sucrose and ammonium sulfate. Moreover, SEM images showed that BNC pellicle produced by the strain C30 in the modified CW-AE medium exhibited finer nanofibrils with a narrower range of width compared with those of MSKU 12 while no significant differences in their physicochemical characteristics were detected among these BNCs produced. Therefore, this finding demonstrates, not only the potential strain for the cost-effective BNC production at high temperature, but also the superior ultrafine nanofibrils production useful for further applications.

Komagataeibacter diospyri sp. nov., a novel species of thermotolerant bacterial nanocellulose-producing bacterium.

Thermotolerant bacterial nanocellulose-producing strains, designated MSKU 9T and MSKU 15, were isolated from persimmon and sapodilla fruits, respectively. These strains were aerobic, Gram-stain-negative, had rod-shaped cells, were non-motile and formed white-cream colonies. Phylogeny based on the 16S rRNA gene sequences revealed that MSKU 9T and MSKU 15 represented members of the genus Komagataeibacter and formed a monophyletic branch with K. swingsii JCM 17123T and K. europaeus DSM 6160T. The genomic analysis revealed that overall genomic relatedness index values of MSKU 9T with K. swingsii JCM 17123T and K. europaeus DSM 6160T were ~90 % average nucleotide identity (ANI) and ≤58.2 % digital DNA-DNA hybridization (dDDH), respectively. MSKU 9T and MSKU 15 can be differentiated from the closely related K. swingsii JCM 17123T by their growth on 30 % d-glucose and ability to utilize and to form acid from raffinose and sucrose as carbon sources, and from K. europaeus DSM 6160T by their ability to grow without acetic acid. The genomic DNA G+C contents of MSKU 9T and MSKU 15 were 60.4 and 60.2 mol%, respectively. The major fatty acids of MSKU 9T and MSKU 15 were summed feature 8 (C18 : 1 ω7c and/or C18  : 1ω6c). The respiratory quinone was determined to be Q10. On the basis of the results of the polyphasic taxonomic analysis, MSKU 9T (=TBRC 9844T=NBRC 113802T) represents a novel species of the genus Komagataeibacter, for which the name Komagataeibacter diospyri sp. nov. is proposed.

Superfine bacterial nanocellulose produced by reverse mutations in the bcsC gene during adaptive breeding of Komagataeibacter oboediens.

Nonsense mutation in the bcsC gene occurred in the ethanol-adapted strain of Komagataeibacter oboediens MSKU 3, E3 strain, resulting in the loss of the function to produce BNC. In this study, we tried to restore the BNC-producing ability of E3 strain by the following adaptive mutation through repetitive static culture, and obtained four BNC-producing revertant strains, of which the bcsC gene had InDel mutations near the frameshift mutation region in E3 strain, resulting in several amino acid alterations compared with the BcsC of MSKU 3. Each revertant produced BNCs with different productivity on the static culture. Interestingly, one of the revertants, R37-9, produced BNC with a finer structure and narrower range of fibrils width, compared to others. The genome of R37-9 strain revealed only one amino acid substitution in the bcsC gene. Thus, we concluded that N713D mutation occurred in the bcsC gene is responsible for the finer fibrils structure.

In Vitro Thermal and Ethanol Adaptations to Improve Vinegar Fermentation at High Temperature of Komagataeibacter oboediens MSKU 3.

High temperature and high ethanol concentrations obviously affect vinegar fermentation. The thermotolerant and ethanol-resistant strains are expected to become one of the technologies for effective vinegar fermentation. This study aimed to further improve thermotolerant Komagataeibacter oboediens MSKU 3 through thermal and ethanol adaptations for acetic acid fermentation. The MSKU 3 strain was independently cultured by a repetitive cultivation in gradually increasing temperature from 37 to 39 °C for thermal adaptation, while adaptation to ethanol was carried out from concentrations of 3 to 5.5% (v/v) at 37 °C. Acetic acid fermentation revealed that the thermo-adapted T4 strain could produce 2.82% acidity with 3% ethanol at 39 °C, whereas the ethanol-adapted E3 strain could produce 3.54% acidity with 5.5% ethanol at 37 °C, in contrast to the parental strain, MSKU 3, in which no fermentation occurs at either 39 °C or 5.5% ethanol. Furthermore, genome mapping analysis of T4 and E3 strains against the genome of parental strain MSKU 3 revealed several mutated genes that are associated with thermotolerance or ethanol adaptation. The occurrence of these adaptation-associated mutations during adaptive evolution was also analyzed. Therefore, adapted strains T4 and E3 revealed the potential of Komagataeibacter oboediens strain improvement to further enhance vinegar fermentation with high ethanol concentration at high temperature.

Mutated fabG gene encoding oxidoreductase enhances the cost-effective fermentation of jasmine rice vinegar in the adapted strain of Acetobacter pasteurianus SKU1108.

A low-nutrient adapted strain, Acetobacter pasteurianus G-40, was successfully obtained by repetitive cultivation of A. pasteurianus 7E-13 under selective pressure. The adapted strain could grow well and produce 3.45-fold higher amounts of acetic acid than 7E-13 in jasmine rice wine containing 6% ethanol at 37 °C in a shaking flask. The G-40 strain also exhibited higher amounts of acetic acid (5.16%) in 2-L jar fermentor compared with 7E-13, where the bio-conversion yield to acetic acid from ethanol was 71% and 55.5% in the adapted strain and parental strain, respectively. In addition, genome sequence analysis of G-40 revealed that the strain has mutations in the 6 genes, of which the fabG gene encoding oxidoreductase is largely mutated by the partial recombination with a highly homologous fabG homolog present in the large plasmid of the strain. Over-expression of the mutated fabG gene and also the replacement of the original fabG gene in the chromosome with the mutated one obviously enhanced growth and acetic acid production of 7E-13 in the rice wine without any nutrient supplementation, indicating that the mutation in the fabG gene is mainly involved in higher fermentation ability under low-nutrient conditions. Thus, the results suggest that the adapted G-40 strain has proven useful for the cost-effective fermentation of rice vinegar.