Light-mediated control of gene expression in the anoxygenic phototrophic bacterium Rhodobacter capsulatus using photocaged inducers.

Photocaged inducer molecules, especially photocaged isopropyl-ß-d-1-thiogalactopyranoside (cIPTG), are well-established optochemical tools for light-regulated gene expression and have been intensively applied in Escherichia coli and other bacteria including Corynebacterium glutamicum, Pseudomonas putida or Bacillus subtilis. In this study, we aimed to implement a light-mediated on-switch for target gene expression in the facultative anoxygenic phototroph Rhodobacter capsulatus by using different cIPTG variants under both phototrophic and non-phototrophic cultivation conditions. We could demonstrate that especially 6-nitropiperonyl-(NP)-cIPTG can be applied for light-mediated induction of target gene expression in this facultative phototrophic bacterium. Furthermore, we successfully applied the optochemical approach to induce the intrinsic carotenoid biosynthesis to showcase engineering of a cellular function. Photocaged IPTG thus represents a light-responsive tool, which offers various promising properties suitable for future applications in biology and biotechnology including automated multi-factorial control of cellular functions as well as optimization of production processes.

Plant-Growth-Promoting Effect by Cell Components of Purple Non-Sulfur Photosynthetic Bacteria.

Rhodobacter sphaeroides, a purple non-sulfur photosynthetic bacterium (PNSB), was disrupted by sonication and fractionated by centrifugation into the supernatant and pellet. The effects of the supernatant and pellet on plant growth were examined using Brassica rapa var. perviridis (komatsuna) in the pot experiments. Both fractions showed growth-promoting effects: the supernatant at high concentrations (1 × 107 to 4 × 107 cfu-equivalent mL-1) and the pellet at a low concentration of 2 × 103 cfu-equivalent mL-1). We expected lipopolysaccharide (LPS) to be the active principle of the pellet fraction and examined the effects of LPS on the growth of B. rapa var. perviridis. The growth of the plants was significantly enhanced by the foliar feeding of R. sphaeroides LPS at concentrations ranging from 10 to 100 pg mL-1. The present study is the first report indicating that LPS acts as one of the active principles of the plant-growth-promoting effect of PNSB.

Extraction of Polyhydroxyalkanoates from Purple Non-Sulfur Bacteria by Non-Chlorinated Solvents.

In this study, non-chlorinated solvents such as cyclohexanone (CYC) and three ionic liquids, (ILs) (1-ethyl-3-methylimidazolium dimethylphosphate, [EMIM][DMP], 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][DEP] and 1-ethyl-3-methylimidazolium methylphosphite, [EMIM][MP]) were tested to extract polyhydroxyalkanoates (PHAs) from the purple non-sulfur photosynthetic bacterium (PNSB) Rhodovulumsulfidophilum DSM-1374. The photosynthetic bacterium was cultured in a new generation photobioreactor with 4 L of working volume using a lactate-rich medium. The extracted PHAs were characterized using a thermogravimetric analysis, differential scanning calorimetry, infrared spectroscopy, proton nuclear magnetic resonance and gel permeation chromatography. The most promising results were obtained with CYC at 125 °C with an extraction time of above 10 min, obtaining extraction yields higher than 95% and a highly pure poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) with around 2.7 mol% of hydroxylvalerate (HV). A similar yield and purity were obtained with chloroform (CHL) at 10 °C for 24 h, which was used as the referent solvent Although the three investigated ILs at 60 °C for 4 and 24 h with biomass/IL up to 1/30 (w/w) obtained PHAs strongly contaminated by cellular membrane residues, they were not completely solubilized by the investigated ILs.

Biosynthesis of pinene in purple non-sulfur photosynthetic bacteria.

BACKGROUND: Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. RESULTS: Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 µg/L of pinene was yielded. Then, genes of 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 µg/L. CONCLUSIONS: In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

Use of Purple Non-Sulfur Photosynthetic Bacteria (Rhodobacter sphaeroides) in Promoting Ciliated Protozoa Growth.

Photosynthetic bacterium (PSB) was isolated from sediment samples of Yamagawa Bay, Kagoshima, Japan. Phylogenetic analysis results of PSB isolate were closely related to Rhodobacter sphaeroides, purple non-sulfur photosynthetic bacteria (PNSB). Pink-colored smooth edges of single bacterial colonies were observed after 3-5 days of incubation period on Basic I medium agar plates. Rhodobacter sphaeroides microscopic examination showed a short rod cell (1-2 µm length) with round ends. Sediment and water samples used for ciliates cultivation were collected from Kuwano-ura Bay, Koshiki Island, Japan. Ciliates were cultivated using fish meal with radish leaves medium (MI), with sediment into MI (MII) and algae media (MIII). The use of the algae media (MIII) in cultivation mixture produced the highest total number of ciliates. Big size ciliates were identified as Euplotes minuta and Cyclidium varibonneti, while small size was identified as Micrometopion nutans, based on PCR-DGGE. When ciliates were cultured with the PSB isolate, Rhodobacter sphaeroides as a feed, ciliates grow to 2,081 individual ml-1 72 hrs later. These findings indicate that PNSB can be used to promote ciliates growth.

Photo-hydrogen and lipid production from lactate, acetate, butyrate, and sugar manufacturing wastewater with an alternative nitrogen source by Rhodobacter sp. KKU-PS1.

Photo-hydrogen and lipid production from individual synthetic volatile fatty acids (VFAs) and sugar manufacturing wastewater (SMW) by Rhodobacter sp. KKU-PS1 with sodium glutamate or Aji-L (i.e., waste from the process of crystallizing monosodium glutamate) as a nitrogen source was investigated. Using individual synthetic VFAs, the maximum hydrogen production was achieved with Aji-L as a nitrogen source rather than sodium glutamate. The maximum hydrogen production was 1,727, 754 and 1,353 mL H2/L, respectively, using 25 mM of lactate, 40 mM of acetate and 15mM of butyrate as substrates. Under these conditions, lipid was produced in the range of 10.6-16.9% (w/w). Subsequently, photo-hydrogen and lipid production from SMW using Aji-L as nitrogen source was conducted. Maximal hydrogen production and hydrogen yields of 1,672 mL H2/L and 1.92 mol H2/mol substrate, respectively, were obtained. Additionally, lipid content and lipid production of 21.3% (w/w) and 475 mg lipid/L were achieved. The analysis of the lipid and fatty acid components revealed that triacyglycerol (TAG) and C18:1 methyl ester were the main lipid and fatty acid components, respectively, found in Rhodobacter sp. KKU-PS1 cells.

A Screening Method for the Isolation of Polyhydroxyalkanoate-Producing Purple Non-sulfur Photosynthetic Bacteria from Natural Seawater.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2) showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions.

Bioaugmentation of Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 to enhance bio-hydrogen production of Rhodobacter sphaeroides KKU-PS5.

BACKGROUND: Bioaugmentation or an addition of the desired microorganisms or specialized microbial strains into the anaerobic digesters can enhance the performance of microbial community in the hydrogen production process. Most of the studies focused on a bioaugmentation of native microorganisms capable of producing hydrogen with the dark-fermentative hydrogen producers while information on bioaugmentation of purple non-sulfur photosynthetic bacteria (PNSB) with lactic acid-producing bacteria (LAB) is still limited. In our study, bioaugmentation of Rhodobacter sphaeroides KKU-PS5 with Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 was conducted as a method to produce hydrogen. Unfortunately, even though well-characterized microorganisms were used in the fermentation system, a cultivation of two different organisms in the same bioreactor was still difficult because of the differences in their metabolic types, optimal conditions, and nutritional requirements. Therefore, evaluation of the physical and chemical factors affecting hydrogen production of PNSB augmented with LAB was conducted using a full factorial design followed by response surface methodology (RSM) with central composite design (CCD). RESULTS: A suitable LAB/PNSB ratio and initial cell concentration were found to be 1/12 (w/w) and 0.15 g/L, respectively. The optimal initial pH, light intensity, and Mo concentration obtained from RSM with CCD were 7.92, 8.37 klux and 0.44 mg/L, respectively. Under these optimal conditions, a cumulative hydrogen production of 3396 ± 66 mL H2/L, a hydrogen production rate (HPR) of 9.1 ± 0.2 mL H2/L h, and a hydrogen yield (HY) of 9.65 ± 0.23 mol H2/mol glucose were obtained. KKU-PS5 augmented with TISTR 895 produced hydrogen from glucose at a relatively high HY, 9.65 ± 0.23 mol H2/mol glucose, i.e., 80 % of the theoretical yield. CONCLUSIONS: The ratio of the strains TISTR 895/KKU-PS5 and their initial cell concentrations affected the rate of lactic acid production and its consumption. A suitable LAB/PNSB ratio and initial cell concentration could balance the lactic acid production rate and its consumption in order to avoid lactic acid accumulation in the fermentation system. Through use of appropriate environmental conditions for bioaugmentation of PNSB with LAB, a hydrogen production could be enhanced.