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.

Low-cost gel-filled microwell array device for screening marine microbial consortium.

In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 µm × 600 µm × 700 µm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples.

A Novel Small RNA on the Pseudomonas putida KT2440 Chromosome Is Involved in the Fitness Cost Imposed by IncP-1 Plasmid RP4.

Plasmids can provide advantageous traits to host bacteria, although they may impose a fitness cost. Chromosome-encoded factors are important for regulating the expression of genes on plasmids, and host chromosomes may differ in terms of their interactions with a given plasmid. Accordingly, differences in fitness cost loading and compensatory co-evolution may occur for various host chromosome/plasmid combinations. However, the mechanisms of compensatory evolution are highly divergent and require further insights. Here, we reveal novel evolutionally mechanisms of Pseudomonas putida KT2440 to improve the fitness cost imposed by the incompatibility P-1 (IncP-1) multidrug resistance plasmid RP4. A mixed culture of RP4-harboring and -free KT2440 cells was serially transferred every 24 h under non-selective conditions. Initially, the proportion of RP4-harboring cells decreased rapidly, but it immediately recovered, suggesting that the fitness of RP4-harboring strains improved during cultivation. Larger-sized colonies appeared during 144-h mixed culture, and evolved strains isolated from larger-sized colonies showed higher growth rates and fitness than those of the ancestral strain. Whole-genome sequencing revealed that evolved strains had one of two mutations in the same intergenic region of the chromosome. Based on the research of another group, this region is predicted to contain a stress-inducible small RNA (sRNA). Identification of the transcriptional start site in this sRNA indicated that one mutation occurred within the sRNA region, whereas the other was in its promoter region. Quantitative reverse-transcription PCR showed that the expression of this sRNA was strongly induced by RP4 carriage in the ancestral strain but repressed in the evolved strains. When the sRNA region was overexpressed in the RP4-free strain, the fitness decreased, and the colony size became smaller. Using transcriptome analysis, we also showed that the genes involved in amino acid metabolism and stress responses were differentially transcribed by overexpression of the sRNA region. These results indicate that the RP4-inducible chromosomal sRNA was responsible for the fitness cost of RP4 on KT2440 cells, where this sRNA is of key importance in host evolution toward rapid amelioration of the cost.

Ecological impact assessment of a bioaugmentation site on remediation of chlorinated ethylenes by multi-omics analysis.

Bioremediation may affect the ecological system around bioremediation sites. However, little is known about how microbial community structures change over time after the initial injection of degraders. In this study, we have assessed the ecological impact of bioaugmentation using metagenomic and metatranscriptomic approaches to remove trichlorinated ethylene/cis-dichloroethylene (TCE/cDCE) by Rhodococcus jostii strain RHA1 as an aerobic chemical compound degrader. Metagenomic analysis showed that the number of organisms belonging to the genus Rhodococcus, including strain RHA1, increased from 0.1% to 76.6% of the total microbial community on day 0 at the injection site. Subsequently, the populations of strain RHA1 and other TCE/cDCE-degrading bacteria gradually decreased over time, whereas the populations of the anaerobic dechlorinators Geobacter and Dehalococcoides increased at later stages. Metatranscriptomic analysis revealed a high expression of aromatic compound-degrading genes (bphA1-A4) in strain RHA1 after RHA1 injection. From these results, we concluded that the key dechlorinators of TCE/cDCE were mainly aerobic bacteria, such as RHA1, until day 1, after which the key dechlorinators changed to anaerobic bacteria, such as Geobacter and Dehalococcocides, after day 6 at the injection well. Based on the α-diversity, the richness levels of the microbial community were increased after injection of strain RHA1, and the microbial community composition had not been restored to that of the original composition during the 19 days after treatment. These results provide insights into the assessment of the ecological impact and bioaugmentation process of RHA1 at bioremediation sites.