Marine Photosynthetic Bacterium Rhodovulum Research Articles

Engineered Nanogel Particles Enhance the Photoautotrophic Biosynthesis of Polyhydroxyalkanoate in Marine Photosynthetic Bacteria

Improving polyhydroxyalkanoate (PHA, a biodegradable plastic) production under photoautotrophic cultivation is challenging for sustainable bioproduction. In this study, we demonstrated the use of engineered nanogel particles to enhance PHA accumulation in the marine photosynthetic bacterium Rhodovulum sulfidophilum under photoautotrophic culture. We screened the effect of 13 engineered nanogel particles on the cell growth and PHA accumulation of R. sulfidophilum. The addition of anionic nanogel particles significantly enhanced PHA accumulation in R. sulfidophilum up to 157-fold compared to that without nanogel particles. By performing 13C tracer experiments and gas chromatography–mass spectrometry analysis, we confirmed that HCO3– was assimilated throughout the central carbon metabolism and that the accumulated PHA was indeed incorporated from HCO3–. Our results indicate successful PHA production with the supplementation of engineered nanogel particles under photoautotrophic cultivation in R. sulfidophilum. Furthermore, the strategy of using engineered nanoparticles demonstrated in this study may be applicable to other microbial cell factories to produce other commodity metabolites.

PufC Gene Targeted PCR Primers for Identification and Classification of Marine Photosynthetic Bacterium Rhodovulum sulfidophilum

pufC Gene Targeted PCR Primers for Identification and Classification of Marine Photosynthetic Bacterium Rhodovulum sulfidophilum

Optimal iron concentrations for growth-associated polyhydroxyalkanoate biosynthesis in the marine photosynthetic purple bacterium Rhodovulum sulfidophilum under photoheterotrophic condition.

Polyhydroxyalkanoates (PHAs) are a group of natural biopolyesters that resemble petroleum-derived plastics in terms of physical properties but are less harmful biologically to the environment and humans. Most of the current PHA producers are heterotrophs, which require expensive feeding materials and thus contribute to the high price of PHAs. Marine photosynthetic bacteria are promising alternative microbial cell factories for cost-effective, carbon neutral and sustainable production of PHAs. In this study, Rhodovulum sulfidophilum, a marine photosynthetic purple nonsulfur bacterium with a high metabolic versatility, was evaluated for cell growth and PHA production under the influence of various media components found in previous studies. We evaluated iron, using ferric citrate, as another essential factor for cell growth and efficient PHA production and confirmed that PHA production in R. sulfidophilum was growth-associated under microaerobic and photoheterotrophic conditions. In fact, a subtle amount of iron (1 to 2 μM) was sufficient to promote rapid cell growth and biomass accumulation, as well as a high PHA volumetric productivity during the logarithmic phase. However, an excess amount of iron did not enhance the growth rate or PHA productivity. Thus, we successfully confirmed that an optimum concentration of iron, an essential nutrient, promotes cell growth in R. sulfidophilum and also enhances PHA utilization.

Identification of novel long chain N-acylhomoserine lactones of chain length C20 from the marine phototrophic bacterium Rhodovulum sulfidophilum.

AGC: automatic gain control; AHL: N-acylhomoserine lactone; CD: cyclodextrin; CID: collision induced dissociation; DDA: data dependent acquisition; EPI: enhanced product ion; FISh: fragment ion search; HCD: high energy collisional dissociation; HSL: homoserine lactone; IT: injection time; LC: liquid chromatography; MS: mass spectrometry; PRM: parallel reaction monitoring; RP: reverse phase; SRM: selected reaction monitoring; TLC: thin layer chromatography; UHPLC: ultra high performance liquid chromatography.

Hydrogen production from glucose by a mutant strain of Rhodovulum sulfidophilum P5 in single-stage photofermentation

Single-stage hydrogen production from glucose was investigated using the marine photosynthetic bacterium Rhodovulum sulfidophilum TH-79, a Tn7 transposon mutant of strain P5. The mutation in strain TH-79 did not affect its cell growth in glucose medium compared with the parent strain. TH-79 displayed improved photoheterotrophic hydrogen production performance when the medium contained glucose or galactose as the sole carbon source. The mutant produced about 7.07 mol H2/mol glucose, which is similar to the yields of more complicated integration systems. A one-stage photofermentation system using a seawater culture medium appears to be a promising alternative to the integration of dark- and photofermentation systems.

Screening and hydrogen-producing characters of a highly efficient H2-producing mutant of Rhodovulum sulfidophilum P5

In this study, transposon mutagenesis technology was utilized to enhance the hydrogen production capability of a wild marine photosynthetic bacterium Rhodovulum sulfidophilum P5. A mutant strain TH-253 that exhibited high hydrogen yield and weaker light absorption ability was screened. Under strong light conditions, the mutant produced more hydrogen than that of the WT. Under optimum light intensity (120 μmol photons/m(2)s), the mutant achieved its highest hydrogen yield (1,436 ± 44 mL H2/L, about 3.21 ± 0.10 mol H2/mol acetate), which was 40.37% higher that of the WT. In continuous operation mode, the hydrogen yield (3.59 ± 0.11 mol H2/mol acetate) and average hydrogen production rate (16.91 ± 0.46 mL H2/Lh) of the mutant were 43.40% and 45.07% higher than those of the WT, respectively. The mutant strain TH-253 may be used as an appropriate starting strain for future photosynthesis-based large scale hydrogen production.

Production of polyhydroxybutyrate by the marine photosynthetic bacterium Rhodovulum sulfidophilum P5

The effects of different NaCl concentrations, nitrogen sources, carbon sources, and carbon to nitrogen molar ratios on biomass accumulation and polyhydroxybutyrate (PHB) production were studied in batch cultures of the marine photosynthetic bacterium Rhodovulum sulfidophilum P5 under aerobic-dark conditions. The results show that the accumulation of PHB in strain P5 is a growth-associated process. Strain P5 had maximum biomass and PHB accumulation at 2%–3% NaCl, suggesting that the bacterium can maintain growth and potentially produce PHB at natural seawater salinity. In the nitrogen source test, the maximum biomass accumulation (8.10±0.09 g/L) and PHB production (1.11±0.13 g/L and 14.62%±2.2 of the cell dry weight) were observed when peptone and ammonium chloride were used as the sole nitrogen source. NH4+-N was better for PHB production than other nitrogen sources. In the carbon source test, the maximum biomass concentration (7.65±0.05 g/L) was obtained with malic acid as the sole carbon source, whereas the maximum yield of PHB (5.03±0.18 g/L and 66.93%±1.69% of the cell dry weight) was obtained with sodium pyruvate as the sole carbon source. In the carbon to nitrogen ratios test, sodium pyruvate and ammonium chloride were selected as the carbon and nitrogen sources, respectively. The best carbon to nitrogen molar ratio for biomass accumulation (8.77±0.58 g/L) and PHB production (6.07±0.25 g/L and 69.25%±2.05% of the cell dry weight) was 25. The results provide valuable data on the production of PHB by R. sulfidophilum P5 and further studies are on-going for best cell growth and PHB yield.

Characterization of extracellular DNA production and flocculation of the marine photosynthetic bacterium Rhodovulum sulfidophilum

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids involved in its flocculation. Previously, we showed that the RNA fraction of these extracellular nucleic acids released into the culture medium contains mainly non-aminoacylated fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Here, we report the characterization of extracellular DNA itself and its production during cultivation. No differences were detected in nucleotide sequence between the intracellular DNA and extracellular soluble DNA on Southern blotting. Whole intracellular DNA seemed to be released from the cell. The bacterial floc was degraded by deoxyribonuclease or ribonuclease treatment, indicating that at least the extracellular DNA and RNAs in the floc are involved in the maintenance of the floc. When cultivated in nutritionally rich medium, the bacteria formed small flocs and produced large amounts of extracellular DNA, which were solubilized in the medium. In nutritionally poor medium, however, huge flocs of cells appeared and almost no extracellular soluble DNA was observed in the medium. As the floc was degraded by deoxyribonuclease treatment, it seems likely that the extracellular soluble DNA observed in the rich medium may be incorporated into the large floc and play a role in floc maintenance in poor medium. Addition of an inhibitor of quorum sensing, alpha-cyclodextrin, inhibited huge floc maintenance in the nutritionally poor medium. In the presence of alpha-cyclodextrin, the floc was rapidly degraded and extracellular soluble DNA production increased.

Extracellular tRNAs of the Marine Photosynthetic BacteriumRhodovulum sulfidophilumAre Not Aminoacylated

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces nucleic acids extracellularly. We have identified these extracellular RNAs as fully mature sized tRNAs and fragments of 16S and 23S rRNAs. Most of the tRNAs have mature 3'-terminal CCA sequences. In the present study we found that these extracellular tRNAs were not aminoacylated, although almost all intracellular tRNAs are aminoacylated.

Astaxanthin formation in the marine photosynthetic bacterium Rhodovulum sulfidophilum expressing crtI, crtY, crtW and crtZ

This study reports the production of astaxanthin in the photosynthetic bacterium Rhodovulum sulfidophilum, which has adequate precursor pools and storage capabilities for heterologous carotenoid production. Chemical mutagenesis was carried out using ethylmethane sulfonate to produce mutants with a modified carotenoid biosynthesis pathway downstream of phytoene. Stable green- and gray-colored mutants were selected. Green mutants contained neurosporene or chloroxanthin as their major carotenoid (>90%), while the gray mutants accumulated phytoene. We previously demonstrated the production of beta-carotene in Rhodovulum sulfidophilum by cloning the Erythrobacter longus crtI (phytoene dehydrogenase) and crtY (lycopene cyclase) genes. In the present study, an expression vector for astaxanthin production was constructed that contained the Paracoccus crtW (beta-carotene oxygenase) and crtZ (beta-carotene hydroxylase) genes in addition to the E. longus crtI and crtY genes. A transconjugant, which can synthesize astaxanthin, was successfully generated (2.0 microg g(-1) DCW).

Characterization of Extracellular RNAs Produced by the Marine Photosynthetic Bacterium Rhodovulum sulfidophilum

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids that are involved in its flocculation. These were found to be produced concomitantly with cell growth. The RNA fraction of these extracellular nucleic acids was subjected to cDNA analysis by applying a micro RNA cloning method and found to contain mainly fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Analyses of modified bases and genes of the RNAs revealed no structural difference between the intracellular and extracellular RNAs. This is the first report of structural analyses of bacterial extracellular RNAs.

Extracellular RNAs produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum

A marine photosynthetic bacterium Rhodovulum sulfidophilum secretes nucleic acids. Previously we have shown that these extracellular nucleic acids are a mixture of double-strand DNAs and single-strand RNAs. Here, we have more closely analyzed the RNA fraction of these extracellular nucleic acids. We determined the sequences of secreted RNAs. They have same sequences with parts of Rhodovulum sulfidophilum DSM 2351 16SrRNA or parts of ribosomal RNA operon of related species. Some of them were tRNAs. Interestingly, these tRNAs were mature tRNAs that had mature sizes, mature 5'-ends as well as 3'-CCA sequences. Additionally, we have found that these sequences were coded on genome, as expected. All of the secreted RNAs we detected may be parts of intracellular RNAs.

Extracellular DNA and RNA produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum.

A marine photosynthetic bacterium Rhodovulum sulfidophilum secretes nucleic acids that are involved in flocculating ability. These extracellular nucleic acids have not been well characterized. Here, we have analyzed these nucleic acids and revealed that the extracellular nucleic acids are a mixture of double-stranded DNAs and single-stranded RNAs. The DNAs have sizes of more than 30 kbp and at least a part of these DNAs is probably an amplified portion of genomic DNA. The RNAs seems to be tRNA like molecules from size estimation.

Biosorption of cadmium ions using a photosynthetic bacterium, Rhodobacter sphaeroides S and a marine photosynthetic bacterium, Rhodovulum sp. and their biosorption kinetics

We examined the biosorption characteristics of cadmium ions onto a photosynthetic bacterium, Rhodobacter sphaeroides S and a marine photosynthetic bacterium Rhodovulum sp. PS88 in a batch culture system. Both photosynthetic bacteria are capable of cadmium removal with 30 g/ l sodium chloride and divalent cations (Mg 2+ and Ca 2+) in the culture medium. In particular, the strain PS88 hows a high removal ratio and high specific removal rate of cadmium ions from the culture medium under aerobic-dark (heterotrophic) and anaerobic-light (photoheterotrophic) conditions. The adsorption of cadmium onto strains PS88 and S is dependent on the cadmium concentration, and follows the Freundlich adsorption isotherm. In addition, biosorption isotherms for cadmium show that the strain PS88 exhibits higher values of the empirical constant for the cadmium adsorption capacity, Kr, than that of the strain S under both aerobic-dark ( K f=17.44) and anaerobic-light ( K f=1.270) conditions.

Biosorption of Cadmium Ions Using a Photosynthetic Bacterium, Rhodobacter sphaeroides S and a Marine Photosynthetic Bacterium, Rhodovulum sp. and Their Biosorption Kinetics

We examined the biosorption characteristics of cadmium ions onto a photosynthetic bacterium, Rhodobacter sphaeroides S and a marine photosynthetic bacterium Rhodovulum sp. PS88 in a batch culture system. Both photosynthetic bacteria are capable of cadmium removal with 30 g/ l sodium chloride and divalent cations (Mg 2+ and Ca 2+ ) in the culture medium. In particular, the strain PS88 hows a high removal ratio and high specific removal rate of cadmium ions from the culture medium under aerobic-dark (heterotrophic) and anaerobic-light (photoheterotrophic) conditions. The adsorption of cadmium onto strains PS88 and S is dependent on the cadmium concentration, and follows the Freundlich adsorption isotherm. In addition, biosorption isotherms for cadmium show that the strain PS88 exhibits higher values of the empirical constant for the cadmium adsorption capacity, Kr, than that of the strain S under both aerobic-dark ( K f =17.44) and anaerobic-light ( K f =1.270) conditions.

Extracellular formation of 5-aminolevulinic acid by intact cells of the marine photosynthetic bacterium Rhodovulum sp. under various pH conditions

Extracellular formation of 5-aminolevulinic acid (ALA) by Rhodovulum sp. PS88 correlated with the consumption of the undissociated form of levulinic acid (LA) in an intact cell system. The concentration of the undissociated form of LA governed the extracellular formation of ALA at various culture pH values. This phenomenon might be caused by inhibition of ALA dehydratase by the undissociated form of LA after uptake into the cells as observed in Rhodobacter sphaeroides.

Growth and flocculation of a marine photosynthetic bacterium Rhodovulum sp.

A marine photosynthetic bacterium (PS88), identified as Rhodovulum sp., with flocculating ability was isolated from the sea sediment mud of a shrimp cultivation farm in Thailand. This bacterium flocculated in glutamate/malate medium during aerobic dark or anaerobic light cultivation. The flocculating ability was enhanced with the increase of NaCl concentration to 6% (w/v). When PS88 was grown in glutamate/malate medium containing 3.5% NaCl, protein, RNA and DNA were produced exocellularly and there was flocculation. The yields of DNA, RNA and protein were 8.3, 62.5 and 48.5 mg/g dry cell, respectively. The flocculated cells were deflocculated by treatment with a nucleolytic enzyme such as RNase or DNase, while amylase, protease, trypsin, cellulase and pectinase had no deflocculating effect. These results suggest that the exocellular nucleic acids are active in flocculation.

High density cell culture of a marine photosynthetic bacterium Rhodovulum sp. with self-flocculated cells

High density cell cultivation of a marine photosynthetic bacterium, Rhodovulum sp. PS88 with self-flocculated cells was established by using a single-tower fermenter. High density cell culture with continuous cultivation was yielded 43 g dry matter l−1 with acetate as a substrate consumed at 22.5 g/l day. © Rapid Science Ltd. 1998