A novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. accumulating poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon substrate

The objective of this paper was to report a bacterium designated as 88D, capable of producing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [P (3HB-co-3HV)] copolymer from a single carbon source, which was isolated from a municipal sewage treatment plant in Hyderabad, India. This microorganism, based on the phenotypical features and genotypic investigations, was identified as Bacillus sp. The optimal growth of Bacillus sp. 88D occurred between 28 and 30°C and at pH 7. The strain yielded a maximum of 64.62% dry cell weight (DCW) polymer in the medium containing glucose as carbon source, which was followed by 60.46% DCW polymer in glycerol containing medium. Bacillus sp. 88D produced P (3HB-co-3HV) from glucose or glycerol, when they were used as a single carbon substrate. This bacterium produced polyhydrxybutyrate (PHB) when sodium acetate was used as sole carbon substrate. The viscosity average molecular mass (Mv) of the copolymers ranged from 523 to 627 kDa. The physical, chemical and mechanical properties of the biopolymers were characterized.     

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).     

Production of copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate by fed-batch culture of Alcaligenes SP. SH-69

Summary Production of copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate [poly(3HB-co-3HV)] by fed-batch culture of Alcaligenes sp. SH-69 was investigated using glucose as a sole carbon source. Synthesis of poly(3HB-co-3HV) during the polymer accumulation stage was favored under dissolved oxygen tension at 20% and C/N ratio (mol glucose/mol ammonium) of 23.1. When conditions were optimal, 36 g liter^-1 of poly(3HB-co-3HV) containing 3.0 mol% of 3HV was produced. Decreasing C/N ratio resulted in an increase of 3HV fraction in the copolymer to a maximum level of 6.3 mol%.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> SP5212

Summary Production of poly(3-hydroxybutyric acid) [P(3HB)] by Rhodopseudomonas palustris SP5212 isolated in this laboratory has been optimized under phototrophic microaerophilic conditions. Cells grown in malate medium accumulated 7.7% (w/w) P(3HB) of cellular dry weight at the early stationary phase of growth. The accumulated P(3HB) however, attained 15% (w/w) of cellular dry weight when acetate (1.0%, w/v) was used as the sole carbon source under nitrogen-limiting conditions. Synthesis and accumulation of polymer was favoured by sulphate-free conditions and at a phosphate concentration sub-optimal for growth. The polymer content of cells was increased drastically (34% of cellular dry weight) when the acetate containing medium was supplemented with n-alkanoic acids. Compositional analysis by H¹ NMR revealed that these accumulated polymers were composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (3HV). The contents of 3HV in these copolymers ranged from 14 to 38 mol%.     

Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 μg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 μg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 μM Fe²⁺ to this medium increased the yield to 226 μg/g DCW.     

Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose,glucose and sugarcane bagasse hydrolysate

Fifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose. Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps. The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate. Burkholderia cepacia IPT 048 and B. sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources. Polymer contents and yields, respectively, reached 62% and 0.39 g g^–1 with strain IPT 101 and 53% and 0.29 g g^–1 with strain IPT 048. A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101. IPT 048 showed similar performances in the presence of either growth-limiting nutrient. In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l^–1 dry biomass, containing 60% P3HB. Polymer productivity and yield from this carbon source reached 0.47 g l^–1 h^–1 and 0.22 g g^–1, respectively.     

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C₆), 3-hydroxyoctanoate (HO or C₈), and/or 3-hydroxydecanoate (HD or C₁₀) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97–99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.     

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.     

Production of polyhydroxyalkanoate from starch by the native isolate <Emphasis Type="Italic">Bacillus cereus</Emphasis> CFR06

A bacterial strain that produces amylase and polyhydroxyalkanoate (PHA) was isolated, identified, and classified under the Bacillus cereus group based on 16S rRNA gene sequences and specific reaction in poly-myxin egg yolk Mannitol bromothymol blue agar (PEMBA) medium and in combination with microbiological and biochemical tests. The complete ORF of phaC gene was cloned by PCR technique and nucleotide sequences were determined. Results indicated that the phaC gene had 99% homology with phaC of B. cereus (AE016877.1), 98% with B. thuringiensis (AY331151.1), and 94% with several strains of B. anthracis and B. cereus group including Bacillus sp. INT005. However, only 90% sequence homology with phaC of B. megaterium (AF109909.2) was observed. The PHA production using different fermentable sugars was tested and it was found that the CFR06 was able to accumulate 36–60% of PHA in cell dry weight (CDW). Zymogram of amylase indicated that native strain produces an extracellular enzyme of ∼80 kDa. The potency of the organism to hydrolyze starch due to the intrinsic amylase activity was considered, and starch was used as the sole carbon source for growth and PHA production. GC, FTIR, and ¹H NMR analysis of the polymer indicated that the strain was a potent polyhydroxybutyrate (PHB) producer. The bacterium accumulated about 48% PHA in CDW in a starch containing medium.     

Production of red pigments byMonascus purpureus in submerged culture

For the purpose of mass producingMonascus red pigments optimum medium composition and environmental conditions were investigated in submerged flask cultures. The optimum carbon and nitrogen sources were determined to be 30 g/L of glucose and 1.5 g/L of monosodium glutamate (MSG). Of the three metals examined, Fe²⁺ showed the stronges stimulatory effect on pigment production and some stimulatory effect was also found in Mn²⁺. Optimum pH and agitation speed were determined to be 6.5 and 700 rpm, respectively. Under the optimum culture conditions batch fermentation showed that the maximum biomass yield and specific productivity of red pigments were 0.20 g DCW/g glucose and, 32.5 OD₅₀₀ g DCW⁻¹ h⁻¹, respectively.     

Production of polyhydroxyalkanoates by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> UWD using swine wastewater: Effect of supplementing glucose,yeast extract,and inorganic salts

This study examined the effect of adding glucose, yeast extract, and inorganic salts to swine wastewater (SWW) in a batch culture on the production of a biodegradable plastic, polyhydroxyalkanoate (PHA). A bacterial strain, Azotobacter vinelandii UWD, was used to produce PHA without limiting the non-carbon nutrients. The addition of glucose (30 g/L) to the SWW medium increased the level of cell growth (4.4∼7.0 times) and PHA production (3.8∼8.5 times) depending upon the dilution of SWW. A 50% dilution of SWW was found to be optimal considering the dry cell weight (9.40 g/L), PHA content (58 wt%), and hydroxyvalerate (HV) mol fraction in the PHA (4.3 mol%). A 75% SWW medium was more advantageous for producing PHA with a higher HV fraction (7.1 mol%) at the expense of losing 22% of PHA production. The undiluted SWW medium produced less than one third of the PHA compared with the 50% SWW medium, but the HV fraction was the highest (10.8 mol%). Regarding the effect of the glucose concentration, at 20 g/L glucose, the dry cell weight and level of PHA production increased to 9.34 g/L (0.63 g PHA/g dry cell weight) and 5.90 g/L, respectively. At 50 g/L glucose, there was no significant increase in PHA production. For the glucose-supplemented (30 g/L) 50% SWW medium, the addition of a nitrogen source (1 g/L of yeast extract) did not increase the level of cell growth or PHA production because the C:N ratio (23:1) was already close to the optimal value (22:1). Better aeration increased the productivity of PHA. External nitrogen supplements (1 g/L of yeast extract) and other essential mineral salts was not necessary for bacterial growth because they were contained in the SWW. These results suggest that SWW is an excellent feedstock for producing larger amounts of the value-added material, PHA, if it is combined with carbohydrate-rich organic waste.     

Chromate reduction by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> TKW3 isolated from marine sediments

Summary Bacillus megaterium strain TKW3 was isolated from multiple-metal-contaminated marine sediments of Tokwawan, Hong Kong SAR. This facultative aerobe utilized arabinose, mannitol, N-acetylglucosamine, maltose, caprate, citrate, butyrate or lactate as the sole source of carbon and energy for growth.B. megaterium TKW3 reduced highly toxic and soluble Cr⁶⁺ (as CrO₄²⁻) into almost non-toxic and insoluble Cr³⁺ under aerobic conditions. Complete reduction of 0.20 mM Cr⁶⁺ by B. megaterium TKW3 was achieved within 360 h. Initial Cr⁶⁺ concentration below 0.90 mM or inoculum less than 10⁷ cells ml⁻¹ did not have significant effect on ⁶⁺ reduction, while the residue Cr⁶⁺ concentration was the lowest at 10⁷ cells ml⁻¹. Cr⁶⁺ reduction by this strain was inhibited by high levels of NaCl (55%). B. megaterium TKW3 was also resistant to other oxyanions including 0.34 mM Cr₂O₇²⁻ 0.32 mM AsO₄³⁻, 0.58 mM SeO₃²⁻ and 0.53 mM SeO₄²⁻, and reduced soluble Se⁴⁺ (as SeO₃²⁻) to insoluble red amorphous Se⁰. B. megaterium TKW3 might have potential application in bioremediation of Cr-laden sediments associated with other oxyanions.     

Eubacterium aggreganssp. nov., a New Homoacetogenic Bacterium from Olive Mill Wastewater Treatment Digestor

A strictly anaerobic, homoacetogenic, Gram-positive, non spore-forming bacterium, designated strain SR12^T(T=type strain), was isolated from an anaerobic methanogenic digestor fed with olive mill wastewater. Yeast extract was required for growth but could also be used as sole carbon and energy source. Strain SR12^Tutilized a few carbohydrates (glucose, fructose and sucrose), organic compounds (lactate, crotonate, formate and betaine), alcohols (methanol), the methoxyl group of some methoxylated aromatic compounds, and H₂+CO₂. The end-products of carbohydrate fermentation were acetate, formate, butyrate, H₂and CO₂. End-products from lactate and methoxylated aromatic compounds were acetate and butyrate. Strain SR12^Twas non-motile, formed aggregates, had a G+C content of 55 mol % and grew optimally at 35°C and pH 7.2 on a medium containing glucose. Phylogenetically, strain SR12^Twas related toEubacterium barkeri, E. callanderi, andE. limosumwithE. barkerias the closest relative (similarity of 98%) with which it bears little phenotypic similarity or DNA homology (60%). On the basis of its phenotypic, genotypic, and phylogenetic characteristics, we propose to designate strain SR12^TasEubacterium aggreganssp. nov. The type strain is SR12^T(=DSM 12183).     

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).     

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.     

Production and Characterization of an Exopolysaccharide by Yeast

Antarctic yeast strains were investigated for exopolysaccharide biosynthesis and the Sporobolomyces salmonicolor AL₁ strain was selected. It was studied for exopolysaccharide biosynthesis on different carbon and nitrogen sources. The investigations showed that sucrose and ammonium sulphate were suitable culture medium components for polymer biosynthesis. Exopolysaccharide formation by the yeast strain was accompanied by a decrease in the culture medium pH value from the initial pH 5.3 to pH 1.7–2.0. During the biosynthetic process, the dynamic viscosity of the culture broth increased to the maximum value of 15.37 mPas and the polysaccharide yield reached 5.63 g/l on a culture medium containing 5.00% sucrose and 0.25% ammonium sulphate at a temperature of 22 °C for 120 h. The crude polysaccharide obtained from Sp. salmonicolor AL₁ featured high purity (90.16% of carbon content) and consisted of glucose (54.1%), mannose (42.6%) and fucose (3.3%). Pure mannan containing 98.6% of mannose was isolated from it.     

Production of Fusarium solani f. sp. pisi cutinase in Fusarium venenatum A3/5

Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l⁻¹ in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l⁻¹ or 8 mg l⁻¹) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

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     

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.