Production of PHB and P (3HB-co-3HV) biopolymers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain OU303A isolated from municipal sewage sludge

Bacillus megaterium strain OU303A isolated from municipal sewage sludge was selected for the study of biosynthesis of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate P (HB-co-HV) copolymer. The strain yielded a maximum of 62.43% DCW polymer in the medium containing glycerol as carbon source, which was followed by 58.63% DCW polymer in glucose containing medium. We found that this strain was capable of producing 2.5% hydroxyvalerate copolymer from a single carbon substrate, glucose. The strain showed an increase in the amount of HV monomer content, when the precursor for the copolymer was included in the fermentation medium. The characterization of the biopolymers was carried out using FTIR, GC-MS, H¹ NMR and DSC. This is the first report of B. megaterium strain producing HV copolymer, without the addition of any precursor in the fermentation medium.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from structurally unrelated single carbon sources by newly isolated Pseudomonas sp. EL-2

Summary A Pseudomonas sp. EL-2 strain capable of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was isolated from activated sludge. For simulation of P(3HB-co-3HV) production in the cells, deficiency of nutrients such as NH₄ ⁺, SO₄ ^2- and Mg²⁺ was crucial and the maximum content of P(3HB-co-3HV) could reach 46% on NH₄ ⁺-deficient medium. This organism synthesized P(3HB-co-3HV) with 3HV monomer in the range from 1.9 to 49.3 mol% from unrelated single carbon sources such as glucose, fructose, propionate, or sorbitol. P(3HB-co-3HV)s containing a higher fraction of 3HV were produced by adding propionic acid to glucose medium.     

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch cultures of Pseudomonas sp EL-2

Pseudomonas sp EL-2 was cultivated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from a structurally unrelated carbon source, glucose, by a fed-batch culture technique. Variation of the carbon to nitrogen (C/N) ratio of the medium produced optimal P(3HB-co-3HV) production at a C/N ratio of 95. Production of P(3HB-co-3HV) was favored by a dissolved oxygen tension of 40%. A maximum biomass concentration of 38 g L⁻¹ containing 53% P(3HB-co-3HV) was achieved after 45 h of cultivation. This corresponds to a volumetric productivity of 0.84 g L⁻¹ h⁻¹. The copolymer contained 7.5 mol% 3-hydroxyvalerate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 36–40. Received 28 January 1999/ Accepted in revised form 11 September 1999     

Production and characterization of PHB from two novel strains of Bacillus spp. isolated from soil and activated sludge

The present study reports two bacteria, designated 87I and 112A, which were isolated from soil and activated sludge samples from Hyderabad, India, and that are capable of producing poly-3-hydroxybutyrate (PHB). Based on phenotypical features and genotypic investigations, these microorganisms were identified as Bacillus spp. Their optimal growth occurred between 28°C and 30°C and pH 7. Bacillus sp. 87I yielded a maximum of 70.04% dry cell weight (DCW) PHB in medium containing glucose as carbon source, followed by 55.5% DCW PHB in lactose-containing medium, whereas Bacillus sp. 112A produced a maximum of 67.73% PHB from glucose, 58.5% PHB from sucrose, followed by 50.5% PHB from starch as carbon substrates. The viscosity average molecular mass (M _v) of the polymers from Bacillus sp. 87I was 513 kDa and from Bacillus sp. 112A was 521 kDa. All the properties of the biopolymers produced by the two strains 87I and 112A were characterized.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer by <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> strain 7B

The ability of Azotobacter chroococcum strain 7B, producer of poly(3-hydroxybutyrate) (PHB), to synthesize its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was studied. It was demonstrated, for the first time, that A. chroococcum strain 7B was able to synthesize P(3HB-co-3HV) with various molar rates of HV in the polymer chain when cultivated on medium with sucrose and carboxylic acids as precursors of HV elements in the PHB chain, namely, valeric (13.1–21.6 mol %), propanoic (3.1 mol %), and hexanoic (2.1 mol %) acids. Qualitative and functional differences between PHB and P(3HB-co-3HV) were demonstrated by example of the release kinetic of methyl red from films made of synthesized polymers. Maximal HV incorporation into the polymer chain (28.8mol %) was recorded when the nutrient medium was supplemented with 0.1% peptone on the background of 20 mM valerate. These results suggest that that the studied strain can be regarded as a potential producer of not only PHB but also P(3HB-co-3HV).     

Improving culture conditions for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam

The production of polyhydroxyalkanoate (PHA) by Bacillus sp. ND153, a bacterium strain isolated from a mangrove forest in Vietnam, was studied. Bacillus sp. ND153 was grown on HM-1 medium with different carbon sources (e.g. glucose, sucrose, maltose, dextrin, and starch). Glucose was found to be the most suitable carbon source for PHA accumulation, whereas starch and dextrin favored cell growth over PHA accumulation. Optimization of the culture medium for PHA production was investigated by applying factorial design, and a maximum PHA content of 79 % (w/w) was obtained with low concentrations of NH₄Cl and MgSO₄ and a high concentration of KH₂PO₄ in the medium. Propionate was used as the precursor for the production of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and the amount of 3-hydroxyvalerate (3HV) in the polymer showed an increasing linear trend with the increase in propionate concentration from 0.2 g l^?1 to 1.0 g l^?1. Thus, the production of PHBV by Bacillus sp. ND153, with 3HV fraction ranging from 1 mol% to 30 mol%, was noted to be high, and the characteristics of fast cell growth and accumulation of PHA exhibited by Bacillus sp. ND153 make it a promising choice for biopolyester production.     

n-Amyl alcohol as a substrate for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacteria

Abstract n -Amyl alcohol was examined as a source for the synthesis of the 3-hydroxyvalerate (3HV) unit of the biopolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), by Alcaligenes sp., Pseudomonas sp. and several methylotrophic bacteria. A. eutrophus and Ps. lemoignei synthesized P(3HB-co-3HV) from glucose and n -amyl alcohol under nitrogen-deficient conditions. Many of methylotrophic bacteria grown on methanol synthesized the copolyester from methanol and n -amyl alcohol under nitrogen-deficient conditions. The content and composition of the polyester varied from strain to strain. Paracoccus denitrificans differed from all others in having a higher content of 3-hydroxyvalerate units in the copolyester synthesized.     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) P(3HB-<Emphasis Type="Italic">co</Emphasis>-3HV) with a broad range of 3HV content at high yields by <Emphasis Type="Italic">Burkholderia sacchari</Emphasis> IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y _3HV/prop) increasing from 1.10 to 1.34 g g⁻¹. Copolymer productivity of 1 g l⁻¹ h⁻¹ was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y _3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).     

Accumulation of PHA and its copolyesters by Methylobacterium sp. KCTC 0048

Summary Methylobacterium sp. KCTC 0048 isolated from soil, could synthesize a variety of copolyesters when secondary carbon substrates were added to nitrogen-limited cultures containing methanol as a major carbon and energy source. The copolyester of 3-hydroxy-butyrate and 3-hydroxyvalerate, P(3HB-co-3HV) accumulated when valeric acid, pentanol or heptanoic acid was added to the nitrogen-limited medium containing methanol. The copolyester of 3-hydroxybutyrate and 4-hydroxybutyrate, P(3HB-co-4HB) was synthesized from 4-hydroxybutyrate, 1,4-butanediol, or -butyrolactone, and the copolyester of 3-hydroxybutyrate and 3-hydroxypropionate (P(3HB-co-3HP)), from 3-hydroxypropionate as the secondary carbon substrates, respectively.     

Degradation of microbial polyester poly(3-hydroxybutyrate) in environmental samples and in culture

Poly(3-hydroxybutyrate) [P(3HB)] test-pieces prepared from the polymer produced by Azotobacter chroococcum were degraded in natural environments like soil, water, compost and sewage sludge incubated under laboratory conditions. Degradation in terms of % weight loss of the polymer was maximum (45%) in sewage sludge after 200 days of incubation at 30°C. The P(3HB)-degrading bacterial cultures (36) isolated from degraded test-pieces showed different degrees of degradation in polymer overlayer method. The extent of P(3HB) degradation increases up to 12 days of incubation and was maximum at 30°C for majority of the cultures. For most efficient cultures the optimum concentration of P(3HB) for degradation was 0.3% (w/v). Supplementation of soluble carbon sources like glucose, fructose and arabinose reduced the degradation while it was almost unaffected with lactose. Though the cultures degraded P(3HB) significantly, they were comparatively less efficient in utilizing copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB-co-3HV)].     

Isolation and characterization of a new <Emphasis Type="Italic">Bacillus</Emphasis> sp. 50-3 with highly alkaline keratinase activity from <Emphasis Type="Italic">Calotes versicolor</Emphasis> faeces

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkali-stable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.     

补料培养提高真养产碱杆菌产3-羟基丁酸和3-羟基戊酸共聚物生产强度的研究

对Alcaligenes eutrophus进行高密度培养,研究表明在发酵过程中进行有效控制,可以较大幅度地提高3－羟基丁酸和3－羟基戊酸共聚物[P(3HB-co-3HV)]的生产强度。实验中选择使用限氮的方法积累P(3HB-co-3HV),分别采用丙酸和戊酸为3HV前体,对摇瓶种子生长状态,停氮时机对菌体生产P(3HB-co-3HV)的影响以及补酸（3HV前体）策略进行了研究,在6．6L罐中,以葡萄糖为碳源,以丙酸为3HV前体培养50h,细胞干重,PHA产量,PHA含量分别达到149．9g/L,149.9g/L,83.3%(其中3HV组分占PHA的12．4mol%),生产强度达到2．50（g.h^-1.L^-1);以戊酸为3HV前体培养45h,细胞干重,PHA产量,PHA含量分别达到160．2g/L,119.0g/L,74.2%（其中3HV组分占PHA的17．7mol%)生产强度达到2．64（g.h^-1.L^-1)。     

Biodegradation of kraft-lignin by <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from sludge of pulp and paper mill

Eight bacterial strains were isolated on kraft lignin (KL) containing mineral salt medium (L-MSM) agar with glucose and peptone from the sludge of pulp and paper mill. Out of these, ITRC-S8 was selected for KL degradation, because of its fast growth at highest tested KL concentration and use of various lignin-related low molecular weight aromatic compounds (LMWACs) as sole source of carbon and energy. The bacterium was identified by biochemical tests as Gram-positive, rod-shaped and non-motile. Subsequent 16S rRNA gene sequencing showed 95% base sequence homology and it was identified as Bacillus sp. In batch experiments, a decrease in pH was observed initially followed by an increase till it reached an alkaline pH, which did not alter the culture growth significantly. The bacterium reduced the colour and KL content of 500 mg l⁻¹ KL in MSM, in the presence of glucose and peptone, at pH 7.6, temperature 30°C, agitation of 120 rpm and 6 days of incubation by 65 and 37% respectively. Significant reduction in colour and KL content in subsequent incubations is indicative of a co-metabolism mechanism, possibly due to initial utilization of added co-substrates for energy followed by utilization of KL as a co-metabolic. The degradation of KL by bacterium was confirmed by GC-MS analysis indicating formation of several LMWACs such as t-cinnamic acid, 3, 4, 5-trimethoxy benzaldehyde and ferulic acid as degradation products, which were not present in the control (uninoculated) sample. This favours the idea of biochemical modification of the KL polymer to a single monomer unit.     

Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1

Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and by-products of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GC-MS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH₄)₂SO₄ fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDS-PAGE), but was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N³⁻). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca²⁺, Mg²⁺, Na⁺, and K⁺. The kinetic parameters, V_max and K_m, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 × 10⁻¹ ??mol min⁻¹ and 6.73 × 10⁻¹ mM, respectively.     

High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli.

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Incorporation of polyethylene glycol in polyhydroxyalkanoic acids accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 (MTCC 3853), a free-living nitrogen-fixing bacterium accumulates poly(3-hydroxybutyric acid) [PHB, 69% of cell dry weight (CDW)] when grown on glucose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV with 19.2 mol% 3HV] when grown on glucose and valerate. Use of ethylene glycol (EG) and/or polyethylene glycols (PEGs) of low molecular weight as sole carbon source were detrimental to A. chroococcum growth and polymer yields. PEG-200, however, in the presence of glucose was incorporated into the polyhydroxyalkanoate (PHA) polymer. Addition of PEG-200 (150 mM) to culture medium during mid-log phase growth favored increased incorporation of EG units (12.48 mol%) into the PHB polymer. In two-step culture experiments, where valerate and PEG simultaneously were used in fresh medium, EG was incorporated most effectively in the absence of glucose, leading to the formation of a copolymer containing 18.05 mol% 3HV and 14.78 mol% EG. The physico-mechanical properties of PEG-containing copolymer (PHBV–PEG) were compared with those of the PHB homopolymer and the PHBV copolymer. The PHBV–PEG copolymer appeared to have less crystallinity and greater flexibility than the short-chain-length (SCL) PHA polymers.     

High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Improved production of rifamycin SV by <Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis> MM2 by the feeding method of carbon source

The purpose of this research was to study the enhancement of rifamycin SV (RSV) yield according to the feeding method of a carbon source for Amycolatopsis mediterranei MM2. RSV produced during fermentation dropped sharply after reaching a maximum, and it was found that this trend of RSV production resulted from simultaneous production and degradation of RSV. To reduce RSV degradation during incubation, the effect of carbon source on cell growth was investigated. Based on the results, glucose was a better carbon source than glycerol for cell growth, although glycerol was better than glucose for RSV production, as reported in our previous study. To confirm this, RSV yield and dry cell weight (DCW) were measured according to the feeding method of carbon source using a 5-L size bioreactor. Results showed that initial cell growth rate and RSV yield were significantly increased regardless of the addition method of glucose into glycerol, provided glucose was present in the initial stage of cell growth.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high molar fractions of 3-hydroxyvalerate by a threonine-overproducing mutant of Alcaligenes sp. SH-69

A threonine overproducing mutant of Alcaligenes sp. SH-69 was isolated and its ability to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), was investigated. The 3HV fraction in poly(3HB-co-3HV) produced from glucose as the sole carbon source exceeded 22 mol%, which is approximately six times higher than that achieved by the wild type under the same culture conditions. Furthermore, the addition of a relatively low concentration (10 mM) of propionic acid, valeric acid or levulinic acid to the glucose medium greatly increased the molar fraction of 3HV in the copolyester, to 38–77 mol%. The results suggest that metabolic engineering of the biosynthetic pathways supplying polyhydroxyalkanoate monomers, such as the threonine biosynthetic pathway, can lead to new poly(3HB-co-3HV)-producing strains.     

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.