One-stage Cultivation Research Articles

Using nitrogen starvation and excess phosphorus for two-stage algae cultivation to improve butanol production of lipid-extracted algae

Lipid-extracted algae (LEA) can be processed via acetone, butanol and ethanol fermentation to produce butanol as an additive to algal biofuel, but direct fermentation was ineffective. In this study, nitrogen and phosphorus regulation for algae cultivation was applied to modify the biochemical composition of algal biomass and hence improve butanol production from LEA. The results show that when nitrogen starvation and excess phosphorus were used during algae cultivation, a significant rise of starch productivity and decline of protein productivity for algal biomass were obtained, which resulted in the highest butanol productivity of 20.16 mg L−1 d−1, a 5.48-fold increase over traditional one-stage cultivation. Mechanism analysis suggested that nitrogen starvation enhanced lipid and starch contents and reduced protein content, which modified C/N ratio of LEA, prevented acid crash and improved butanol production; excess phosphorus modified amino acid contents of phenylalanine and arginine, which strengthened fluxes directing to butanol generation. Through this investigation, the approach of using nitrogen starvation and excess phosphorus to modify biochemical composition of algal biomass and subsequently improve butanol production of LEA was demonstrated to be applicable.

Enhanced production of astaxanthin and co-bioproducts from microalga Haematococcus sp. integrated with valorization of industrial wastewater under two-stage LED light illumination strategy

Microalgal-based biochemicals and biofuels integrated with phycoremediation of industrial wastewater can potentially increase the profitability and sustainability of the food–water–energy–environment nexus. A key challenge in the high-density cultivation of microalgae is to provide light of efficient wavelengths in photobioreactors. In this study, specific narrow bands of light-emitting diodes (LEDs) and their intensities were manipulated to enhance astaxanthin and co-bioproduct production by the microalga Haematococcus sp. Among the strategies tested, the two-stage LED light illumination involving red LED for 5 days and then blue LED for the next 5 days, with a continuous light intensity of 40 μ mol m −2 s −1 , was shown to be the most efficient in improving the performance of Haematococcus sp. These conditions led to the highest astaxanthin production, of 2.55 mg/L, which was 1.22- to 2.07-fold higher than that by one-stage cultivation, with no significant effect on pigments (chlorophylls and carotenoids) and lipid production. Using a two-stage strategy integrated with the valorization of seafood processing effluent, Haematococcus sp. grew well in the secondary effluent from a seafood processing plant, giving 1.33 g/L of biomass, with 3.39 mg/L of astaxanthin, 14.3 mg/L of chlorophylls, 6.22 mg/L of carotenoids, and 0.41 g/L of lipids. The produced astaxanthin can be used as a sustainable source of bioactive ingredients with desirable antioxidative properties, giving ABTS activity of 5.24 mg of TE/g of astaxanthin, DPPH activity of 2.04 mg of GAE/g of astaxanthin, and PFRAP activity of 0.87 mg of GAE/g of astaxanthin. Meanwhile, lipids contain long-chain fatty acids (C16–C18> 96%) and their biodiesel characteristics were also satisfactory when compared to those required by international biodiesel standards, indicating the potential for use as biodiesel feedstock. Our results suggested that the integration process was cost effective and environmentally friendly for microalgal-based wastewater treatment and a microalgal-based multiproduct biorefinery. • Two-stage cultivation using red–blue LEDs was effective for astaxanthin production. • Industrial effluent was valorized into valuable products and effectively treated. • Microalgae also produce lipids with high potential as biodiesel feedstocks. • This integrated process would contribute to the sustainability of biofuel production.

Chemical Triggering Cyanobacterial Glycogen Accumulation: Methyl Viologen Treatment Increases Synechocystis sp. PCC 6803 Glycogen Storage by Enhancing Levels of Gene Transcript and Substrates in Glycogen Synthesis.

Two-stage cultivation is effective for glycogen production by cyanobacteria. Cells were first grown under adequate nitrate supply (BG11) to increase biomass and subsequently transferred to nitrogen deprivation (-N) to stimulate glycogen accumulation. However, the two-stage method is time-consuming and requires extensive energy. Thus, one-stage cultivation that enables both cell growth and glycogen accumulation is advantageous. Such one-stage method could be achieved using a chemical triggering glycogen storage. However, there is a limited study on such chemicals. Here, nine compounds previously reported to affect cyanobacterial cellular functions were examined in Synechocystis sp. PCC 6803. 2-Phenylethanol, phenoxyethanol, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and methyl viologen can stimulate glycogen accumulation. The oxidative stress agent, methyl viologen significantly increased glycogen levels up to 57% and 69% [w/w dry weight (DW)] under BG11 and -N cultivation, respectively. One-stage cultivation where methyl viologen was directly added to the pre-grown culture enhanced glycogen storage to 53% (w/w DW), compared to the 10% (w/w DW) glycogen level of the control cells without methyl viologen. Methyl viologen treatment reduced the contents of total proteins (including phycobiliproteins) but caused increased transcript levels of glycogen synthetic genes and elevated levels of metabolite substrates for glycogen synthesis. Metabolomic results suggested that upon methyl viologen treatment, proteins degraded to amino acids, some of which could be used as a carbon source for glycogen synthesis. Results of oxygen evolution and metabolomic analysis suggested that photosynthesis and carbon fixation were not completely inhibited upon methyl viologen treatment, and these two processes may partially generate upstream metabolites required for glycogen synthesis.

Engineering type I methanotrophic bacteria as novel platform for sustainable production of 3-hydroxybutyrate and biodegradable polyhydroxybutyrate from methane and xylose

Methylotuvimicrobium alcaliphilum20Z recombinant strain co-utilizing methane and xylose from anthropogenic activities and lignocellulose biomassis a promising cell factory platform. In this study, the production of (R)-3-hydroxybutyrate and poly (3-hydroxybutyrate) inM. alcaliphilum20Z was demonstrated. The production of (R)-3-hydroxybutyrate was optimized by introducing additional thioesterase, and a tunable genetic module. The final recombinant strain produced the highest titer of 334.52 ± 2 mg/L (R)-3-hydroxybutyrate (yield of 1,853 ± 429 mg/g dry cell weight). The poly (3-hydroxybutyrate) yielded 1.29 ± 0.08% (w/w) from methane and xylose in one-stage cultivation. Moreover, the study demonstrated the importance of pathway reversibility as an effective design strategy for balancing the driving force and intermediate accumulation. This is the first demonstration of the production ofbiodegradablepoly (3-hydroxybutyrate) from methane in type I methanotrophs, which is a key step toward sustainable biomanufacturing and carbon–neutral society.

High-efficiency production of biomass and biofuel under two-stage cultivation of a stable microalga Botryococcus braunii mutant generated by ethyl methanesulfonate-induced mutation

Green microalga Botryococcus braunii is a eukaryotic photosynthetic microorganism considered as a rich source of biofuel compounds, such as hydrocarbons and lipids, despite its rather slow growth. To improve the biomass, hydrocarbon, and lipid productivity, an ethyl methanesulfonate-induced mutagenesis was carried out on the indigenous microalga, Botryococcus braunii AARL G036. Among those mutants, EMS-mutant E1.0H15 gave the highest biomass and hydrocarbon productivity, which was higher than 1.17, 1.45, and 1.16-fold, respectively, when compared with the wild type (WT). Subsequently, two-stage cultivation with crucial stress factors was successfully exploited to boost the biofuel productivity of EMS-mutant E1.0H15 by increasing the lipid content by 1.22-fold (up to 48.6%) and by 1.20-fold for the hydrocarbon content (up to 71.6%) compared to one-stage cultivation. More importantly, after 24-month maintenance, the high biofuel productivity in EMS-mutant E1.0H15 remains unchanged. In addition, the fatty acid composition (>96% C16–C18) and fuel properties of the mutated microalgal lipid-derived biodiesel indicated good oxidation stability (17.5 h) and high heating values (38.6 MJ kg−1), which were in accordance with those of the international standards, i.e., EN-14214 and ASTM D675140. This strategy could contribute greatly to the further development of the microalgal strain, allowing improvement and cultivation for the desired biofuel properties.

Selenite as a Lipid Inductor in Marine Microalga Dunaliella tertiolecta: Comparison of One-Stage and Two-Stage Cultivation Strategies.

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00-40.00mg L-1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00mg L-1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00mg L-1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta.

Simultaneous enhancement on renewable bioactive compounds from Porphyridium cruentum via a novel two-stage cultivation

Porphyridium cruentum is a versatile cell factory to produce various bioactive and vital substances for food, feed, cosmetics and pharmaceuticals. In this study, a novel two-stage cultivation was the first time to establish for microalgal growth and bioactive compounds production by regulating light-emitting diodes wavelengths using marine microalga Porphyridium cruentum. Firstly, the best light light-emitting diodes wavelength(s) was/were optimized for microalgal growth, and B-phycoerythrin, polyunsaturated fatty acids and exopolysaccharides production by microalga. Results showed that green wavelength significantly enhanced microalgal cell growth (2.93 g/L), and B-phycoerythrin (51.65 mg/g biomass) and exopolysaccharides (255.62 mg/L) synthesis, while white wavelength promoted total fatty acids (7.51%) and polyunsaturated fatty acids (48.21 mg/g biomass) accumulation. Then, a two-stage cultivation was developed and re-evaluated. It was found that microalga grown in the two-stage cultivation (12 days for green wavelength and then 6 days for white wavelength) had similar biomass and achieved higher bioactive substances contents compared with the one-stage cultivation with green wavelength. The developed two-stage cultivation could address the issue about the decrease in B-phycoerythrin content by the one-stage cultivation. Overall, this two-stage cultivation was an efficient strategy for microalgal growth and renewable bioactive substances production by P. cruentum.

Revalorization of adsorbed residual oil in spent bleaching clay as a sole carbon source for polyhydroxyalkanoate (PHA) accumulation in Cupriavidus necator Re2058/pCB113

Spent bleaching clay (SBC) containing ~33 wt% adsorbed residual oil from the refining of palm oil in the milling industry was evaluated for use as a sole carbon source for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] production by the recombinant strain Cupriavidus necator Re2058/pCB113. The biosynthesis of polyhydroxyalkanoate (PHA) was conducted in a one-stage cultivation process using shaken flasks. Palm olein and the extracted oil from SBC were used for comparison with the adsorbed residual oil (ARO) in SBC to evaluate the efficiency of the latter as a carbon source. The ARO in SBC can be utilized by the bacterium for the growth and accumulation of PHA. Cell growth and PHA accumulation were observed to be the best when 20 g/L ARO was supplied as a carbon source, resulting in the production of 11.8 g/L dry cell weight containing 39.8 wt% P(3HB-co-15 mol% 3HHx). The biomass yield showed that ARO in SBC is a suitable carbon feedstock for this strain. Thus, these results suggest that ARO in SBC, an inexpensive carbon source, can be used to produce low-cost commercialized PHA in bulk. Polyhydroxyalkanoate (PHA) is a biopolyester similar to some commodity plastics. The production cost of PHA is high partly due to the cost of feedstock. Spent bleaching clay (SBC) is a byproduct of the palm oil refining process. Adsorbed residual oil in SBC was successfully utilized by Cupriavidus necator Re2058/pCB113 in shaken flask culture for PHA production. Both the SBC and the resulting PHA were characterized by SEM, rheometer, TGA, DSC, GC and 1H-NMR to demonstrate the production of a PHA copolymer containing 3-hydroxyhexanoate.

Nitrate concentration-shift cultivation to enhance protein content of heterotrophic microalga Chlorella vulgaris: Over-compensation strategy

Protein production from microalgae requires both high cell density during cultivation and high protein content in cells. Heterotrophic microalgae can achieve high cell density, and yet are confronted with the problem of low protein content. Based on over-compensation strategy, a new concentration-shift method was proposed to cultivate heterotrophic Chlorella vulgaris, aiming to increase protein content. With a prior starvation period, microalgae utilized more nitrate and accumulated more proteins compared to one-stage cultivation. Considering the convenience of operation, nitrate-added culture was adopted for producing heterotrophic microalgae, rather than sterile centrifugal culture. Operating parameters including nitrate concentration in N-deficient medium, N-starved time and nitrate concentration in N-rich medium were optimized, which were 0.18gl−1, 38h and 2.45gl−1, respectively. Under the optimized conditions, protein content in heterotrophic Chlorella reached 44.3%. Furthermore, the heterotrophic microalga was suggested to be a potential single-cell protein source according to the amino acid composition.

Synthesis of high 4-hydroxybutyrate copolymer by Cupriavidus sp. transformants using one-stage cultivation and mixed precursor substrates strategy.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is noted for its high biocompatibility, which makes it an excellent candidate for biopharmaceutical applications. The wild-type Cupriavidus sp. USMAA1020 strain is able to synthesize P(3HB-co-4HB) copolymers with different 4HB monomer compositions (up to 70mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and γ-butyrolactone were applied for the production of P(3HB-co-4HB) with different 4HB molar fraction. A sharp increase in 4HB monomer composition was attained by introducing additional copies of PHA synthase gene (phaC), responsible for P(3HB-co-4HB) polymerization. The phaC of Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 were cloned and heterologously introduced into host, wild-type Cupriavidus sp. USMAA1020. The gene dosage treatment resulted in the accumulation of 93mol% 4HB by the transformant strains when grown in similar conditions as the wild-type USMAA1020. The PHA synthase activities for both transformants were almost two-fold higher than the wild-type. The ability of the transformants to produce copolymers with high 4HB monomer composition was also tested in large scale production system using 5L and 30L bioreactors with a constant oxygen mass transfer rate. The 4HB monomer composition could be maintained at a range of 83-89mol%. The mechanical and thermal properties of copolymers improved with increasing 4HB monomer composition. The copolymers produced could be tailored for specific biopharmaceutical applications based on their properties.

Enhancing biomass and oil accumulation of Monoraphidium sp. FXY-10 by combined fulvic acid and two-step cultivation

The combination of combined fulvic acid (FA) and two-step cultivation was proposed as a novel strategy to enhance biomass and oil accumulation of Monoraphidium sp. FXY-10 compared with the traditional monoculture of microalgae. In this study, the effect of FA on the growth and lipid accumulation of Monoraphidium sp. FXY-10 was investigated using one- and two-stage cultivation. In the heterotrophic one-stage cultivation, 1.5-fold biomass was enhanced with 80mg L−1 FA (6.41g L−1) compared with the control biomass (4.21g L−1). In the heterotrophic–photoautotrophic two-stage cultivation, different FA concentrations were used to stimulate lipid accumulation. The lipid content increased from 30.78% to 54.65% when 25mg L−1 FA was added to the culture. The main fatty acids in the FA-triggered Monoraphidium sp. FXY-10 were C16/C18 fatty acids (97%), which are appropriate for biodiesel production. Results showed that the proposed strategy is useful for biomass and oil production of Monoraphidium sp. FXY-10.

A one-stage cultivation process for lipid- and carbohydrate-rich biomass of Scenedesmus obtusiusculus based on artificial and natural water sources

A one-stage cultivation process of the microalgae Scenedesmus obtusiusculus with medium based on natural water sources was developed to enhance lipids and carbohydrates. A medium based on artificial sea water, Baltic Sea water and river water with optimized nutrient concentrations compared to the standard BG-11 for nitrate (−75%), phosphate and iron (−90%) was used for cultivation. Although nitrate exhaustion over cultivation resulted in nitrate limitation, growth of the microalgae was not reduced. The lipid content increased from 6.0% to 19.9%, an increase in oleic and stearic acid was observed. The unsaponifiable matter of the lipid fraction was reduced from 19.5% to 11.4%. The carbohydrate yield rose from 45% to 50% and the protein content decreased from 32.4% to 15.9%. Using natural water sources with optimized nutrient concentrations could open the opportunity to modulate biomass composition and to reduce the cultivation costs.

A one-stage cultivation process for the production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) from olive mill wastewater by Haloferax mediterranei

Olive mill wastewater (OMW), a highly polluting waste from the olive oil industry, was utilized as sole carbon source for the production of polyhydroxyalkanoate (PHA) by extremely halophilic Haloferax Mediterranei (H. mediterranei) in a one stage cultivation step. H. mediterranei showed remarkable cell growth and tolerated the inhibitory effect of polyphenols present in medium containing 25% of OMW. H. mediterranei cultivation conditions were optimized in medium containing 15% OMW by investigating several parameters that affect the production of PHA. The highest polymer yield (0.2g/L) and PHA content (43% PHA/cell dry mass) were achieved at 37°C, 170rpm and 22% salt concentration. Analysis of the produced PHA revealed the production of copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) containing 6.5mol% 3-hydroxyvalerate (3HV). The production of PHBHV was observed without the need for fermentation step or adding external carbon source. The PHBHV displayed reduced melting points at 140.1°C and 154.4°C when compared to homopolymer polyhydroxybutyrate.

A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass

The use of alga biomass for biogas generation has been studied for over fifty years but until today, several distinct features, like inefficient degradation and low C/N ratios, limit the applicability of algal biomass for biogas production in larger scale. In this work we investigated a novel, one-stage combined cultivation/fermentation strategy including inherently progressing nitrogen starvation conditions to generate improved microalgal biomass substrates. For this strategy, comparable low amounts of nitrogen fertilizers were applied during cultivation and no additional enzymatic, chemical or physical pretreatments had to be performed. The results of this study demonstrate that progressing nitrogen limitation leads to continuously increasing C/N ratios of the biomass up to levels of 24–26 for all three tested alga strains (Chlamydomonas reinhardtii, Parachlorella kessleri and Scenedesmus obliquus). Importantly, the degradation efficiency of the algal cells increased with progressing starvation, leading to strain-specific cell disintegration efficiencies of 35%–100% during the fermentation process. Nitrogen limitation treatment resulted in a 65% increase of biogas yields for C. reinhardtii biomass (max. 698±23mL biogas g−1 VS) when compared to replete conditions. For P. kessleri and S. obliquus, yields increased by 94% and 106% (max. 706±39mL and 586±36mL biogas g−1 VS, respectively).From these results we conclude that this novel one-stage cultivation strategy with inherent nitrogen limitation can be used as a pretreatment for microalgal biomass generation, in order to produce accessible substrates with optimized C/N ratios for the subsequent anaerobic fermentation process, thus increasing methane production and avoiding the risk of ammonia inhibition effects within the fermenter.

Effect of oxygen supply on Monascus pigments and citrinin production in submerged fermentation

The influence of oxygen supply on Monascus pigments and citrinin production by Monascus ruber HS.4000 in submerged fermentation was studied. For Monascus cultivation with high pigments and low citrinin production, the initial growth phase, mid-stage phase, and later-stage production phase were separated by shifting oxygen supply. The optimal condition for the fermentation process in shake-flask fermentation was a three-stage rotating rate controlled strategy (0-48 h at 150 rpm, 48-108 h at 250 rpm, 108-120 h at 200 rpm) with medium volume of 100 mL added to 250 mL Erlenmeyer flasks at 30°C for 120 h cultivation. Compared to constant one-stage cultivation (medium volume of 100 mL, rotating rate of 250 rpm), the pigments were reduced by 40.4%, but citrinin was reduced by 64.2%. The most appropriate condition for the fermentation process in a 10 L fermentor is also a three-stage aeration process (0-48 h at 300 L/h, 48-96 h at 500 L/h, 96-120 h at 200 L/h) with agitation of 300 rpm at 30°C for 120 h cultivation, and 237.3 ± 5.7 U/mL pigments were produced in 120 h with 6.05 ± 0.19 mg/L citrinin in a 10 L fermentor. Compared to aeration-constant (500 L/h) cultivation, pigment production was increased by 29.6% and citrinin concentration was reduced by 79.5%.

Evaluation of unrefined glycerine pitch as an efficient renewable carbon resource for the biosynthesis of novel yellow-pigmented P(3HB-co-4HB) copolymer towards green technology

Discharging the unrefined glycerine, a by-product from biodiesel production is the simplistic solution adopted for its management which has led to its price reduction in the market worldwide and created serious environmental impact. Therefore, we have explored the application of unrefined glycerine pitch as direct fermentative substrate in the biosynthesis of novel yellow-pigmented poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer by Cupriavidus sp. USMAHM13 through onestage cultivation. Utilization of glycerine pitch (10 g/L) together with 1,4-butanediol (5 g/L) had resulted in the highest achievement of 2.91 g/L of P(3HB-co-40%4HB) copolymer which was naturally dyed with the yellow pigment through the co-extraction process. Enhancement of 4HB monomer accumulation was also attained through the addition of ammonium acetate as nitrogen source. It was revealed that utilization of recovered crude glycerine from glycerine pitch was more preferred compared to the other recovered components. Utilization of glycerine pitch in the biosynthesis of P(3HB-co-4HB) copolymer would not only contribute to the efficient waste management but also would promote the development of cost-efficiency microbial fermentation.

Studies on the Microbial Synthesis and Characterization of Polyhydroxyalkanoates Containing 4-Hydroxyvalerate Using γ-Valerolactone

In this study, the ability of Cupriavidus sp. USMAA2-4 to synthesize polyhydroxyalkanoates (PHA) containing 4-hydroxyvalerate monomer (4HV) was studied through one-stage cultivation using γ-valerolactone as the carbon precursor. The presence of 4HV monomer unit in the polymer was detected through gas chromatography analysis, proving the capability of this wild strain bacterium to produce poly(3-hydrxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [P(3HB-co-3HV-co-4HV)] terpolymer. Existence of a 4HV monomer unit in the PHA produced was further confirmed through (13)C and (1)H NMR analysis. P(3HB-co-88 % 3HV-co-1 % 4HV) terpolymer with the highest PHA content of 63 wt% was obtained through combination of 0.14 wt% C of γ-valerolactone with 0.42 wt% C of oleic acid. Various compositions of P(3HB-co-3HV-co-4HV) terpolymer with 3HV and 4HV compositions ranging from 11 to 94 mol% and from 1 to 4 mol%, respectively, were acquired by manipulating γ-valerolactone and oleic acid concentrations. The molecular weight and the thermal and mechanical properties of four different compositions of terpolymers-P(3HB-co-91 % 3HV-co-1 % 4HV), P(3HB-co-55 % 3HV-co-2 % 4HV), P(3HB-co-27 % 3HV-co-2 % 4HV), and P(3HB-co-9 % 3HV-co-1 % 4HV)-were characterized. Among these terpolymers, P(3HB-co-27 % 3HV-co-2 % 4HV) terpolymer with a molecular weight of 5.7 (10(5) Da) exhibited the highest elongation to break (264 %). The monomer unit compositional distributions of these terpolymers were investigated through acetone-water fractionation analysis. The results suggested that these produced terpolymers had broad 3HV compositional distribution and narrow 4HV compositional distribution.

Yellow‐pigmented Cupriavidus sp., a novel bacterium capable of utilizing glycerine pitch for the sustainable production of P(3HB‐co‐4HB)

AbstractBackgroundDevelopment of bacterial strains that can utilize cheap and renewable carbon substrate will make the prices of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) [P(3HB‐co‐4HB)] competitive with synthetic plastics. Disposal of glycerine pitch, which is a scheduled waste from the oleochemicals industry, has raised awareness among communities as burning the waste literally means producing acrolein which is well known for its toxicity and adverse effects. Therefore, discovering potential application of glycerine pitch as carbon substrate is believed to overcome the waste management problem and reduce the P(3HB‐co‐4HB) production cost.ResultsCupriavidus sp. USMAHM13, a novel yellow‐pigmented bacterium produced the highest 4HB composition (43 mol%) with cell dry weight and PHA content of 6.0 g L‐1 and 49 wt%, respectively, through combination of glycerine pitch (5 g L‐1) and 1,4‐butanediol (5 g L‐1) via one‐stage cultivation. The yellow pigment extracts were analyzed using UV–VIS spectrophotometry and high performance liquid chromatography – mass spectrometry (HPLC‐MS), which revealed that the yellow pigment might represent a new compound in the carotenoid group.ConclusionBiosynthesis of P(3HB‐co‐4HB) by a novel yellow‐pigmented Cupriavidus sp. using glycerine pitch together with 1,4‐butanediol enhanced cost efficiency and sustainability in the production of P(3HB‐co‐4HB).

Over-expression of 3-ketoacyl-ACP synthase III or malonyl-CoA-ACP transacylase gene induces monomer supply for polyhydroxybutyrate production in Escherichia coli HB101

A Pseudomonas sp. 61-3 malonyl-CoA-ACP transacylase gene (fabDPs) was cloned by Southern analysis using an equivalent gene of Escherichia coli (fabDEc) as a probe. Some recombinant E. coli HB101 strains harboring fabDPs, fabDEc, or E. coli 3-ketoacyl-ACP synthase III gene (fabHEc) with Aeromonas caviae polyhydroxyalkanoate synthase gene (phaCAc) were constructed and grown on one-stage cultivation in Luria-Bertani broth containing glucose as carbon source. These strains accumulated 5 to 11 wt% of poly (3-hydroxybutyrate) (PHB) within cells. Over-expression of fabHEc, fabDEc, or fabDPs has been suggested to lead the monomer-supply of (R)-3-hydroxybutyryl-CoA for PHB synthesis in E. coli cells.