Linxian Ding

Exploring the potential environmental functions of viable but non-culturable bacteria

A conventional plate count is the most commonly employed method to estimate the number of living bacteria in environmental samples. In fact, judging the level of viable culture by plate count is limited, because it is often several orders of magnitude less than the number of living bacteria actually present. Most of the bacteria are in “viable but non-culturable” (VBNC) state, whose cells are intact and alive and can resuscitate when surrounding conditions are more favorable. The most exciting recent development in resuscitating VBNC bacteria is a bacterial cytokine, namely, the resuscitation-promoting factor (Rpf), secreted by Micrococcus luteus, which promotes the resuscitation and growth of high G+C Gram-positive organisms, including some species of the genus Mycobacterium. However, most of studies deal with VBNC bacteria only from the point of view of medicine and epidemiology. It is therefore of great significance to research whether these VBNC state bacteria also possess some useful environmental capabilities, such as degradation, flocculation, etc. Further studies are needed to elucidate the possible environmental role of the VBNC bacteria, rather than only considering their role as potential pathogens from the point view of epidemiology and public health. We have studied the resuscitation of these VBNC bacteria in polluted environments by adding culture supernatant containing Rpf from M. luteus, and it was found that, as a huge microbial resource, VBNC bacteria could provide important answers to dealing with existing problems of environmental pollution. This mini-review will provide new insight for considering the potentially environmental functions of VBNC bacteria.

A novel approach to stimulate the biphenyl-degrading potential of bacterial community from PCBs-contaminated soil of e-waste recycling sites

SRpf, culture supernatants from Micrococcus luteus containing the resuscitation-promoting factor (Rpf), was used to enhance the biphenyl-degrading capability of potential microorganisms. The obtained results suggest that the enrichment culture produced by the addition of SRpf (enrichment culture treatment group, ECT) enhanced the biphenyl degradation efficiency, cell growth and bacterial diversity significantly. Biphenyl at concentration of 1500 mg/L was almost completely degraded in 24 h using SRpf at a dosage of 15% (v/v). Six strains unique to the ECT were isolated in pure cultures. This study provides a new insight into bacterial degradation of biphenyl for PCBs-bioremediation, and could be developed as a novel efficient method for obtaining highly desirable pollutant-degrading microorganisms.

Identification, characterization and molecular analysis of the viable but nonculturable Rhodococcus biphenylivorans

Numerous bacteria, including pollutant-degrading bacteria can enter the viable but nonculturable state (VBNC) when they encounter harsh environmental conditions. VBNC bacteria, as a vast majority of potent microbial resource can be of great significance in environmental rehabilitation. It is necessary to study the VBNC state of pollutant-degrading bacteria under various stress conditions. The aim of this study was to determine whether Rhodococcus biphenylivorans could enter the VBNC state under oligotrophic and low temperature conditions, and to examine the changes of morphology, enzymatic activity and gene expressions that might underline such state. The obtained results indicated that R. biphenylivorans TG9T could enter into the VBNC state and recover culturability under favorable environmental conditions. Results from Illumina high throughput RNA-sequencing revealed that the up-regulated genes related to ATP accumulation, protein modification, peptidoglycan biosynthesis and RNA polymerase were found in the VBNC cells, and the down-regulated genes mainly encoded hypothetical protein, membrane protein and NADH dehydrogenase subunit, which render VBNC cells more tolerant to survive under inhospitable conditions. This study provides new insights into prevention and control of the VBNC state of pollutant-degrading bacteria for their better capabilities in environmental rehabilitation.

Culture-dependent and culture-independent characterization of potentially functional biphenyl-degrading bacterial community in response to extracellular organic matter from Micrococcus luteus

Biphenyl (BP)‐degrading bacteria were identified to degrade various polychlorinated BP (PCB) congers in long‐term PCB‐contaminated sites. Exploring BP‐degrading capability of potentially useful bacteria was performed for enhancing PCB bioremediation. In the present study, the bacterial composition of the PCB‐contaminated sediment sample was first investigated. Then extracellular organic matter (EOM) from Micrococcus luteus was used to enhance BP biodegradation. The effect of the EOM on the composition of bacterial community was investigated by combining with culture‐dependent and culture‐independent methods. The obtained results indicate that Proteobacteria and Actinobacteria were predominant community in the PCB‐contaminated sediment. EOM from M. luteus could stimulate the activity of some potentially difficult‐to‐culture BP degraders, which contribute to significant enhancement of BP biodegradation. The potentially difficult‐to‐culture bacteria in response to EOM addition were mainly Rhodococcus and Pseudomonas belonging to Gammaproteobacteria and Actinobacteria respectively. This study provides new insights into exploration of functional difficult‐to‐culture bacteria with EOM addition and points out broader BP/PCB degrading, which could be employed for enhancing PCB‐bioremediation processes.

Induction of Viable but Nonculturable State in Rhodococcus and Transcriptome Analysis Using RNA-seq.

Viable but nonculturable (VBNC) bacteria, which maintain the viability with loss of culturability, universally exist in contaminated and non-contaminated environments. In this study, two strains, Rhodococcus sp. TG13 and TN3, which were isolated from PCB-contaminated sediment and non-contaminated sediment respectively, were investigated under low temperature and oligotrophic conditions. The results indicated that the two strains TG13 and TN3 could enter into the VBNC state with different incubation times, and could recover culturability by reversal of unfavourable factors and addition of resuscitation-promoting factor (Rpf), respectively. Furthermore, the gene expression variations in the VBNC response were clarified by Illumina high throughput RNA-sequencing. Genome-wide transcriptional analysis demonstrated that up-regulated genes in the VBNC cells of the strain TG13 related to protein modification, ATP accumulation and RNA polymerase, while all differentially expressed genes (DEGs) in the VBNC cells of the strain TN3 were down-regulated. However, the down-regulated genes in both the two strains mainly encoded NADH dehydrogenase subunit, catalase, oxidoreductase, which further verified that cold-induced loss of ability to defend oxidative stress may play an important role in induction of the VBNC state. This study further verified that the molecular mechanisms underlying the VBNC state varied with various bacterial species. Study on the VBNC state of non-pathogenic bacteria will provide new insights into the limitation of environmental micro-bioremediation and the cultivation of unculturable species.

Optimization of protein production by Micrococcus luteus for exploring pollutant-degrading uncultured bacteria

The screening of pollutant-degrading bacteria are limited due to most of bacteria in the natural environment cannot be cultivated. For the purpose of resuscitating and stimulating “viable but non-culturable” (VBNC) or uncultured bacteria, Micrococcus luteus proteins are more convenient and cost-effective than purified resuscitation-promoting factor (Rpf) protein. In this study, medium composition and culture conditions were optimized by using statistical experimental design and analysis to enhance protein production by M. luteus. The most important variables influencing protein production were determined using the Plackett-Burman design (PBD) and then central composite design (CCD) was adopted to optimize medium composition and culture conditions to achieve maximum protein yield. Results showed that the maximum protein yield of 25.13 mg/L (vs. 25.66 mg/L predicted) was obtained when the mineral solution, Lithium L-lactate, initial pH and incubation time were set at 1.5 ml/L, 8.75 g/L, 7.5 and 48 h, respectively. The predicated values calculated with the model were very close to the experimental values. Protein production was obviously increased with optimization fitting well with the observed fluorescence intensity. These results verified the feasibility and accuracy of this optimization strategy. This study provides promising information for exploring highly desirable pollutant-degrading microorganisms.

Resuscitation of viable but non-culturable bacteria to enhance the cellulose-degrading capability of bacterial community in composting

Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation‐promoting factors (Rpfs) due to entering a viable but non‐culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose‐degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl‐cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.

Revealing potential functions of VBNC bacteria in polycyclic aromatic hydrocarbons biodegradation

The bioremediation of polycyclic aromatic hydrocarbon (PAH)‐contaminated sites is not running smoothly, because of the lower activity of PAH‐degrading bacteria in actual bioremediation applications. The phenomenon of “viable but nonculturable” (VBNC) state may be a main limiting factor for their poor biodegradation capabilities of PAHs. Due to their abilities of entering into the VBNC state, most of bacterial populations with PAH‐degradation potential remain unculturable. Resuscitation of VBNC bacteria will enhance the degradation capability of indigenous bacteria which will eventually obtain their better capabilities in environmental bioremediation. Although evidences have been presented indicating that resuscitation of VBNC bacteria in polychlorinated biphenyl (PCB)‐contaminated environments not only significantly enhanced PCB degradation, but also obtained novel highly efficient PCB‐degrading bacteria, scanty information is available on the VBNC bacteria in PAH‐contaminated sites. VBNC bacteria, as a vast majority of potential microbial resource could be the repository of novel highly efficient PAH‐biodegraders. Therefore, studies need to be done on resuscitation of VBNC bacteria to overcome key bottlenecks in bioremediation of PAH‐contaminated sites. This mini‐review provides a new insight into the potential functions of VBNC bacteria in PAHs biodegradation.