Pseudomonas pharmafabricae sp. nov., Isolated From Pharmaceutical Wastewater.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated ZYSR67-ZT, was isolated from a pharmaceutical wastewater sample collected from a chemical factory in Zhejiang, China. The strain was motile by a single polar flagellum and grew at 4-42 °C (optimum, 35 °C), pH 5.0-9.0 (optimum, 6.0) and 0-5.0% (w/v) NaCl (optimum, 2.0%). Based on multilocus sequence analysis using 16S rRNA, gyrB, rpoB and rpoD, the strain ZYSR67-ZT formed a distinct phylogenetic group in the genus Pseudomonas. The average nucleotide identity values between strain ZYSR67-ZT and the closely related 10 type strains of the Pseudomonas species were 75.8-78.6%. The in silico DNA-DNA hybridization values indicated that strain ZYSR67-ZT and the type strains of the Pseudomonas shared 21.4-23.1% DNA relatedness. The predominant isoprenoid quinone system was ubiquinone-9 while ubiquinone-8 was present in trace amounts. The major fatty acids (> 10%) identified were C12:0, C16:0, C18:1 ω7c and summed features 3 (C16:1 ω7c and/or iso-C15:0 2OH). The major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content was 62.6 mol%. On the basis of morphological, physiological and chemotaxonomic characteristics, together with the results of phylogenetic analysis, strain ZYSR67-ZT was proposed to represent a novel species of the genus Pseudomonas, named Pseudomonas pharmafabricae sp. nov.. The type strain is ZYSR67-ZT (= CGMCC 1.15498T = JCM 31306T).

Isolation of Viable but Non-culturable Bacteria from Printing and Dyeing Wastewater Bioreactor Based on Resuscitation Promoting Factor.

Printing and dyeing wastewater with high content of organic matters, high colority, and poor biochemical performance is hard to be degraded. In this study, we isolated viable but non-culturable (VBNC) bacteria from printing and dyeing wastewater with the culture media contained resuscitation promoting factor (Rpf) protein secreted by Micrococcus luteus, counted the culturable cells number with the most probable number, sequenced 16S rRNA genes, and performed polymerase chain reaction-denaturing gradient gel electrophoresis. It is obviously that the addition of Rpf in the enrichment culture could promote growth and resuscitation of bacteria in VBNC state to obtain more fastidious bacteria significantly. The identified bacteria were assigned to nine genera in the treatment group, while the two strains of Ochrobactrum anthropi and Microbacterium sp. could not be isolated from the control group. The function of isolated strains was explored and these strains could degrade the dye of Congo red. This study provides a new sight into the further study including the present state, composition, formation mechanism, and recovery mechanism about VBNC bacteria in printing and dyeing wastewater, which would promote to understand bacterial community in printing and dyeing wastewater, and to obtain VBNC bacteria from ecological environment.

Arthrobacter liuii sp. nov., resuscitated from Xinjiang desert soil.

A Gram-stain positive, aerobic, non-motile actinobacterium, designated DSXY973(T), was isolated from soil samples collected from Xinjiang desert using medium supplemented with resuscitation-promoting factor, and subjected to a polyphasic taxonomic investigation. Phylogenetic analysis based on 16S rRNA gene sequences revealed that DSXY973(T) belonged to the genus Arthrobacter and was most closely related to Arthrobacter oryzae JCM 15922(T) with 97.1 % similarity. The DNA G+C content was 67.6 %. Cells of strain DSXY973(T) mainly contained MK-9(H2), and the cell wall contained l-lysine as the primary diamino acid. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C15 : 0. Strain DSXY973(T) was positive for catalase and negative for oxidase activity. On the basis of its phylogenetic position and phenotypic properties, strain DSXY973(T) represents a novel species of the genus Arthrobacter, for which the name Arthrobacter liuii sp. nov. is proposed. The type strain is DSXY973(T) ( = CGMCC1.12778(T) = JCM 19864(T)).

Enhanced degradation of biphenyl from PCB-contaminated sediments: the impact of extracellular organic matter from Micrococcus luteus.

Recent advances in the bioremediation of polychlorinated biphenyl (PCB)-contaminated environments have focused on the development of approaches to stimulate the activities of indigenous bacterial communities. In this study, extracellular organic matter (EOM) from Micrococcus luteus was used to enhance the biphenyl-degrading capability of potentially functional microorganisms. The obtained results suggest that EOM significantly enhanced the biphenyl (BP)-degradation capability. Under a concentration of 3,500 mg/L BP, BP-degradation efficiency reached 60.8 % at a dosage of 10 % EOM (v/v), whereas the degradation efficiencies of control group (with inactivated EOM addition) and blank group (with lactate minimal medium) were only 21.5 and 6.2 %, respectively. Denaturing gradient gel electrophoresis (DGGE) profiles demonstrated that EOM played a key role in shifts in the composition and diversity of bacterial community. The Illumina high-throughput sequencing analysis indicated that the genera of Rhodococcus and Pseudomonas closely related to BP/PCB-degradation were greatly abundant after EOM addition. Together with polymerase chain reaction (PCR)-DGGE analysis, the link between the enhanced BP-degrading capability and the stimulation and resuscitation function of EOM in uncultured bacteria belonging to phylum Actinobacteria was tentatively established. These results suggest that EOM from M. luteus as an additive holds great potential for the efficient and cost-effective bioremediation of PCB-contaminated environment.

Rhodococcus biphenylivorans sp. nov., a polychlorinated biphenyl-degrading bacterium.

A Gram-positive, aerobic, non-motile and rod-coccus shaped novel actinobacterial strain, designated as TG9(T), was isolated from a polychlorinated biphenyl (PCB)-contaminated sediment in Taizhou city, Zhejiang province, eastern China. The isolate was observed to grow at 10-45 °C (optimum 28-32 °C), pH 5.0-11.0 (optimum pH 7.0-8.0) and with 0-9.0 % (w/v) NaCl (optimum 0-3.0 %). Comparison of the 16S rRNA gene sequences of strain TG9(T) and other members of the genus Rhodococcus showed that strain TG9(T) shared highest similarities with Rhodococcus pyridinivorans DSM 44555(T) (99.4 %), R. rhodochrous DSM 43241(T) (99.2 %), R. gordoniae DSM 44689(T) (99.2 %) and R. artemisiae DSM 45380(T) (98.2 %). However, low levels of DNA-DNA relatedness (15-48 %), which are below the 70 % limit for prokaryotic species identification, were obtained by DNA-DNA hybridization. Strain TG9(T) was found to contain meso-diaminopimelic acid as the diagnostic diamino acid and arabinose and galactose in the whole-cell hydrolysate. Mycolic acids were found to be present. The major fatty acids were identified as C16:0, C16:1 ω7c and/or iso-C15:0 2-OH, 10-methyl C18:0 and C18:1 ω9c. The only menaquinone detected was MK-8 (H2). The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, glycolipid and traces of some unknown lipids. The genomic DNA G+C content of strain TG9(T) was determined to be 62.8 %. The combined phenotypic and genotypic data show that the strain represents a novel species of the genus Rhodococcus for which the name Rhodococcus biphenylivorans sp. nov. is proposed, with the type strain TG9(T) (=CGMCC 1.12975(T) = KCTC 29673(T) = MCCC 1K00286(T)).

Rhodococcus soli sp. nov., an actinobacterium isolated from soil using a resuscitative technique.

A Gram-positive, aerobic, non-motile, non-spore forming strain, designated DSD51W(T), was isolated using a resuscitative technique from a soil sample collected from Kyoto park, Japan, and characterized by using a polyphasic approach. The morphological and chemotaxonomic properties of the isolate were typical of those of members of the genus Rhodococcus. Strain DSD51W(T) was found to form a coherent cluster with Rhodococcus hoagii ATCC 7005(T), Rhodococcus equi NBRC 101255(T), Rhodococcus defluvii Call(T) and Rhodococcus kunmingensis YIM 45607(T) as its closest phylogenetic neighbours in 16S rRNA gene sequence analysis. However, the DNA-DNA hybridization values with the above strains were 58.2 ± 2.2, 58.4 ± 1.9, 45.1 ± 1.4 and 40.3 ± 4.7 %, respectively. In combination with differences in physiological and biochemical properties, strain DSD51W(T) can be concluded to represent a novel species of the genus Rhodococcus, for which the name Rhodococcus soli sp. nov. is proposed, with the type strain DSD51W(T) (=KCTC 29259(T) = JCM 19627(T) = DSM 46662(T) = KACC 17838(T)).

Prauserella shujinwangii sp. nov., from a desert environment.

A Gram-positive, spore-forming, rod-shaped actinomycete, designated XJ46(T), was isolated from Xinjiang Uyghur Autonomous Region, China and subjected to a polyphasic taxonomic analysis. Morphological and chemotaxonomic characteristics of XJ46(T) were identified as being similar to those of members of the genus Prauserella. The phylogenetic tree based on 16S rRNA gene sequences showed that XJ46(T) shared the highest similarity (95.9%) with Prauserella marina MS498(T). Based on its phenotypic characteristics, chemotaxonomic analysis and 16S rRNA gene sequence analysis, strain XJ46(T) is proposed to represent a novel species of the genus Prauserella, named Prauserella shujinwangii sp. nov. The type strain is XJ46(T) ( =CGMCC 4.7125(T) =JCM 19736(T)).

Gordonia jinhuaensis sp. nov., a novel actinobacterium, isolated from a VBNC (viable but non-culturable) state in pharmaceutical wastewater.

A Gram-stain positive, aerobic, non-motile and rod-shaped actinobacterial strain, designated as ZYR 51(T), was isolated from pharmaceutical wastewater in Jinhua city, Zhejiang province, Eastern China. Isolation was aided by using a resuscitation-promoting factor, suggesting the strain was recovered from a viable but non-culturable state. Strain ZYR 51(T) was characterized by a polyphasic taxonomic approach. Growth was found to occur at 10-45 °C, pH 6.0-10.0 and 0-9 % NaCl (w/v). Based on the 16S rRNA gene sequence, phylogenetic analysis clearly demonstrated that strain ZYR 51(T) belongs to the genus Gordonia and showed low level similarities (below 97 %) with all other members of this genus. The strain was found to possess meso-diaminopimelic acid (meso-DAP), along with MK-9(H2) as the predominant menaquonine. Mycolic acids were found to be present. C16:0 (34.9 %), 10-methyl C18:0 (30.3 %), iso-C18:0(8.2 %), and summed feature 3 (C16:1 ω6c and/or C16:1ω7c as define by MIDI; 18.8 %) were identified as the major cellular fatty acids. The polar lipid profile of strain ZYR 51(T) was found to consist of diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and some unknown lipids. The genomic DNA G+C content of strain ZYR 51(T) was determined to be 67.7 mol%. The combined genotypic and phenotypic data showed that the strain represents a novel species of the genus Gordonia, for which the name Gordonia jinhuaensis sp. nov. is proposed, with the type strain is ZYR 51(T) (=CGMCC 1.12827(T) = NBRL B-59111(T) = NBRC 110001(T)).

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.

[Potential of viable but non-culturable bacteria in polychlorinated biphenyls degradation--a review].

The state of "viable but non-culturable" (VBNC) is a survival strategy adopted by microorganisms when exposed to environmental stress. With the increasingly serious problem of xenobiotics pollution, enhanced microbial processes that exploit the potential of microbes to remediate polychlorinated biphenyl-contaminated environments have been developed. Microorganisms represent a significant advance with respect to the transformation and degradation of polychlorinated biphenyls in the environment. It is of great importance to study the potential function of VBNC bacteria in polluted environment. In this paper, current research status of VBNC bacteria is summarized, and resuscitation of VBNC bacteria to potentially stimulate microbial degradation of pollutants is discussed in detail. Furthermore, we put forward a novel approach to explore the potential of VBNC bacteria for polychlorinated biphenyls degradation using resuscitation promoting factor (Rpf) of Micrococcus luteus. The novel efficient method is helpful for excavating and obtaining highly desirable polychlorinated biphenyls degrading microorganisms. Moreover, the prospect of VBNC bacteria to other environmental remediation fields, such as flocculation and nitrification deodorant, is addressed.

Study on the flocculability of the Arthrobacter sp., an actinomycete resuscitated from the VBNC state.

A bioflocculant with high flocculating activity, LC13-SF, produced by strain LC13(T) which was in a viable but nonculturable (VBNC) state, and which was woken up by Rpf (resuscitation promoting factor), was systematically investigated with regard to its fermentation conditions and flocculating activity. The key parameters influencing the bioflocculant LC13-SF were investigated through measuring the optical density at 660 (OD(660)) of the fermentation liquid and the optical density at 550 (OD(550)) of the centrifugal supernatant. The flocculating efficiency and the Zeta potentials were chosen as the response variables for the study of the flocculating activity. The results showed that the optimal conditions for bioflocculant LC13-SF production were a fermentation time of 72 h, an initial pH of 7.0, a fermentation temperature of 30°C and a shaking speed of 150 r/min. The optimized flocculating process was as follows: a final volume percentage of bioflocculant LC13-SF and 0.5% (w/w) CaCl(2) were 1.5 and 5%, respectively in a 4 g/L Kaolin suspension, and the system pH was adjusted to 8.0. Under these conditions, the flocculating efficiency and the absolute value of the Zeta potential reached 94.83% and 4.37, respectively.

[Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp. DS471].

OBJECTIVE: The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene, secreted by Micrococcus luteus IAM 14879, in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria. METHODS: Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified, cloned into a pET15b expression vector, and transformed into Escherichia coli BL21 (DE3). Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria, Rhodococcus sp. DS471, were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879. RESULTS: The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli, was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully, and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control. CONCLUSION: Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.

[Research progress of VBNC bacteria--a review].

The viable but non-culturable (VBNC) is a microbial state, in which microbial cells are metabolically active but cannot be cultivated by routine methods. In this article, we address the formation mechanism, change and variety, resuscitation, research significance and application prospects of VBNC state in bacteria. Furthermore, we report our research findings on VBNC state of bacteria in the past 10 years, including resuscitation, culturable, phylogenetic relationship and potential functions.

Purification effect evaluation of the designed new volcanic soil adsorption material containing bioreactor for eutrophic water treatment.

The purpose of this study was to investigate the purification effect of a new adsorption material containing bioreactor and the critical role of viable but non-culturable (VBNC) bacteria in a eutrophication ecosystem. Major water quality parameters of the prepared eutrophic water were determined, and the microbial community was analyzed during 2 years. The results showed that removal rates of total phosphorus (TP), total nitrogen (TN), chlorophyll-a (Chl-a), and chemical oxygen demand (COD) were 90.7-95.9%, 84.5-92.4%, 87.9-95.8%, and 68.3-82.7%, respectively, indicating the high efficiency of the bioreactor in the eutrophic water treatment. Although the bioreactor had been operated for 2 years, water from the treatment group was much clearer and odorless than from the control group, exhibiting the long service life of the bioreactor. Stopping operation in August caused significant decrease of the removal rates of major water quality parameters (p < 0.05). This operational stop event and high temperature in summer exerted a dual effect on the bioreactor, whereas the impact could be minimized when the bioreactor was running. Moreover, the total bacteria under +Rpf (active resuscitation-promoting factor) treatment were higher than under -Rpf (inactive resuscitation-promoting factor) treatment, implying that Rpf could resuscitate VBNC bacteria in the eutrophication ecosystem. Nine strains of VBNC bacteria were isolated based on the BLAST results of the 16S rRNA gene. Also, these bacteria might contribute to the eutrophic water treatment based on their functions of phosphorus collecting and denitrification. These results provided new insights for engineering technology innovations, and consequently these findings had benefits in eutrophic water treatment.

Extracellular organic matter from Micrococcus luteus containing resuscitation-promoting factor in sequencing batch reactor for effective nutrient and phenol removal.

Culture supernatant containing resuscitation-promoting factor (SRpf) from Micrococcus luteus was added to the sequencing batch reactor (SBR) for effective treatment of phenol-containing wastewater. SRpf acclimation significantly improved combined removal of phenol and nutrients. Moreover, the Illumina high-throughput sequencing analysis revealed that the SRpf boosted bacteria diversity, which enhanced the stability of the system under phenol stress. Addition of SRpf increased the abundances of Actinobacteria and Proteobacteria phyla which are involved in nutrient and phenol removal. Specifically, SRpf promoted Nitrosomonas and Nitrospira which participate in nitrification, family Comamonadaceae, genera Dechloromonas and Pseudomonas involved in denitrification, and Acinetobacter, Pseudomonas and Rhodocyclus which remove phosphorus elements. Moreover, the abundances of Bacillus and Klebsiella responsible for phenol removal as well as Pseudomonas and Acinetobacter were significantly increased after SRpf acclimation. These results show that SBR combined with SRpf acclimation provide optimal nutrient and phenol removal.

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.

Draft Genome Sequence of Lactobacillus brevis Strain 3M004, a Probiotic with Potential Quorum-Sensing Regulation.

We present here the draft genome sequence of Lactobacillus brevis strain 3M004, a probiotic that has potential for regulating quorum sensing. The strain was obtained from a type of aquafeed. The assembly consists of 2,459,326 bp and contains 33 contigs, with a G+C content of 45.10%.

A novel approach to enhance biological nutrient removal using a culture supernatant from Micrococcus luteus containing resuscitation-promoting factor (Rpf) in SBR process.

A culture supernatant from Micrococcus luteus containing resuscitation-promoting factor (SRpf) was used to enhance the biological nutrient removal of potentially functional bacteria. The obtained results suggest that SRpf accelerated the start-up process and significantly enhanced the biological nutrient removal in sequencing batch reactor (SBR). PO4 (3-)-P removal efficiency increased by over 12 % and total nitrogen removal efficiency increased by over 8 % in treatment reactor acclimated by SRpf compared with those without SRpf addition. The Illumina high-throughput sequencing analysis showed that SRpf played an essential role in shifts in the composition and diversity of bacterial community. The phyla of Proteobacteria and Actinobacteria, which were closely related to biological nutrient removal, were greatly abundant after SRpf addition. This study demonstrates that SRpf acclimation or addition might hold great potential as an efficient and cost-effective alternative for wastewater treatment plants (WWTPs) to meet more stringent operation conditions and legislations.

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.