Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Production of poly-β-hydroxybutyrate on molasses by recombinant Escherichia coli

Beet molasses successfully replaced glucose as sole carbon source to produce poly--hydroxybutyrate by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The fermentation with molasses was cheaper than with glucose. The final dry cell weight, PHB content and PHB productivity were 39.5 g/L, 80% (w/w) and 1 g/Lh, respectively, in a 5 L stirred tank fermenter after 31.5 h fed-batch fermentation with constant pH and dissolved O2 content. © Rapid Science Ltd. 1998     

Biosynthesis of poly-3-hydroxybutyrate by Sphaerotilus natans

A sheathed bacterium Sphaerotilus natans could not survive at 4°C for 2 months, and mutants that exhibited different colony phenotypes were obtained only by repeating the short period of storage at 4 °C. The ability of these mutants and the parent strain to produce poly-3-hydroxybutyrate (PHB) was compared in batch cultures. The parent strain accumulated 30% (w/w) PHB, while one of the mutants defective in sheath formation, designated as T2, accumulated over 50% PHB. Because T2 did not require strict air or nitrogen limitation for polymer accumulation, its production was growth-associated, allowing one-stage fermentation. In a pH-controlled fermentation using a jar fermentor, 10 g/l glucose was converted into 2.0 g/l PHB in 24 h.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

Production of poly-β-hydroxybutyrate by thermophilic cyanobacterium, Synechococcus sp. MA19, under phosphate-limited conditions

Synechococcus sp. MA19, grown autotrophically under phosphate-limited conditions at 50 °C, produced poly--hydroxybutyrate (PHB) when intracellular phosphate content was 0.043–0.076mmol per g of cellular components. In the culture for 260h using Ca₃(PO₄)₂ as a phosphate source, strain MA19 accumulated PHB at 55% (w/w) of the dry cells and the amount of PHB produced was 2.4gl^–1 which was almost twice that without Ca₃(PO₄)₂ addition.     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.     

Production of poly(3-hydroxybutyrate) from whey by fed-batch culture of recombinant Escherichia coli in a pilot-scale fermenter

Production of poly(3-hydroxybutyrate) [P(3HB)] from wheyby fed-batch culture of recombinant Escherichia coli CGSC 4401 harboring a plasmid containing the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes was examined in a 30 l fermenter supplying air only. With lactose below 2 g l^–1, cells grew to 12 g dry cell l^–1 with 9% (w/w) P(3HB) content. Accumulation of P(3HB) could be triggered by increasing lactose to 20 g l^–1. By employing this strategy, 51 g dry cell l^–1 was obtained with a 70% (w/w) P(3HB) content after 26 h. The productivity was 1.35 g P(3HB) l^–1 h^–1. The same fermentation strategy was used in a 300 l fermenter, and 30 g dry cell l^–1 with 67% (w/w) P(3HB) content was obtained in 20 h.     

Suitability of commercial beet molasses fractions as substrates fro polyhydroxyalkanoate production byAzotobacter vinelandii UWD

Summary Beet molasses that had been fractionated commercially by ion exclusion resulted in two waste-streams: extract molasses (EM) and concentrated separator by-product (CSB). Only EM at 4–5% w/v contained sufficient sugar to promote polyhydroxybutyrate (PHB) formation byAzotobacter vinelandii UWD, but the yield of PHB/protein was less than that obtained in unfractionated beet molasses. EM and especially CSB added at 0.5–2.0% w/v to media containing a variety of sugar sources promoted an increased yield of PHB/protein. The best use of these beet molasses fractions was, therefore, as a minor addition to media containing sugars to increase PHB yield, but not as a primary substrate for PHB production.     

Biosynthesis of PHB from a new isolated <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain: Outlook on future developments with endospore forming bacteria

Diverse Bacillus strains are known as producers of polyhydroxyalkanoates (PHAs) under nutrient-limiting conditions. However, these limiting conditions have the same nutritional characteristics that stimulate spore generation in Gram-positive microorganisms. In the present work, a new isolated Bacillus megaterium strain was characterized based on 16S rRNA gene sequences (1,411 bp) and studied in terms of its ability for producing polyhydroxybutyrate (PHB) by implementing different fermentation configurations on formulated media. The isolated strain was able to produce PHB up to 59 and 60% of its dry cell weight during bioreactor experiments employing glucose and glycerol as carbon source, respectively. The produced biopolymer was characterized and identified by using carbon-13 nuclear magnetic resonance (¹³C-NMR) and Fourier transform infrared (FTIR) techniques. In spite of the sporulation phenomenon existing in Bacillus strains, obtained results demonstrate that the new isolated strain has the potential of accumulating high levels of intracellular PHB. Supported by these experimental results and by those reported by other authors, the last section of this paper gives an outlook of future research topics on PHB and polyhydroxyalkanoate (PHA) copolymers production by Gram-positive bacteria. The importance of combining bioprocessing/biorefinering concepts with bioreactor optimization approaches is stressed and analyzed based on current PHAs research trends.     

Production of Short Side Chain-Poly[Hydroxyalkanoate] by a Newly Isolated Ralstonia Pickettii Strain

In order to obtain better bacterial species or strains for production of short side chain-poly[hydroxyalkanoate](ssc-PHA) from cheap carbon sources, a bioprospecting programme was performed in a subtropical rainforest soil. From 398 bacterial isolates, one produced high amounts of ssc-PHA when grown on sugarcane molasses or sucrose as detected by spectrophotometric scanning and gas chromatography coupled to mass spectrometry. Also, the GC—MS analysis indicated that the polymer was composed basically of poly[3-hydroxybutyrate](PHB). Phylogenetic studies using 16S rDNA analysis showed that the isolated bacterium belonged to the Ralstonia pickettii species and had a high identity/similarity with 16S rDNA obtained from total DNA of uncultured strains of soils and with unidentified bacteria at species level. The new strain was named R. pickettii 61A6. Spectrofluorometric analysis showed that the best rates of ssc-PHA accumulation within the cells occurred in 10%(w/v) sucrose and in 5%(w/v) sugarcane molasses at the stationary phase, with a yield of 231 and 357 mg/l of ssc-PHA per g dry cell weight, respectively.     

Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium

Fourteen yeast strains were screened for production of 2-phenylethanol from l-phenylalanine with molasses as carbon source. Up to 1 g 2-phenylethanol l^–1 was obtained. Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l^–1 at 35 °C. The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.     

Desulfotomaculum geothermicum sp. nov., a thermophilic,fatty acid-degrading,sulfate-reducing bacterium isolated with H2 from geothermal ground water

A strictly anaerobic, thermophilic, fatty acids-degrading, sporulating sulfate-reducing bacterium was isolated from geothermal ground water. The organism stained Gram-negative and formed gas vacuoles during sporulation. Lactate, ethanol, fructose and saturated fatty acids up to C₁₈ served as electron donors and carbon sources with sulfate as external electron acceptor. Benzoate was not used. Stoichiometric measurements revealed a complete oxidation of part of butyrate although growth with acetate as only electron donor was not observed. The rest of butyrate was oxidized to acetate. The strain grew chemolithoautotrophically with hydrogen plus sulfate as energy source and carbon dioxide as carbon source without requirement of additional organic carbon like acetate. The strain contained a c-type cytochrome and presumably a sulfite reductase P582. Optimum temperature, pH and NaCl concentration for growth were 54°C, pH 7.3–7.5 and 25 to 35 g NaCl/l. The G+C content of DNA was 50.4 mol %. Strain BSD is proposed as a new species of the spore-forming sulfate-reducing genus Desulfotomaculum, D. geothermicum.     

Evaluation of agro-food byproducts for gluconic acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ORS-4.410

Certain cost-effective carbohydrate sources in crude as well as after purification were utilized as the sole sources of carbon for gluconic acid production using Aspergillus niger ORS-4.410 under submerged fermentation. Crude grape must (GM) and banana-must (BM) resulted into significant levels of gluconic acid production i.e. 62.6 and 54.6 g/l, respectively. The purification of grape and banana-must led to a 20–21% increase in gluconic acid yield. Molasses as such did not favour gluconate production (12.0 g/l) but a significant increase in production (60.3 g/l) was observed following hexacyanoferrate (HCF) treatment of the molasses. Rectified grape must (RGM) appeared to be best suitable substrate which after 144 h resulted in 73.2 g of gluconic acid/l with 80.6% yield followed by the yield obtained from the rectified banana must (RBM) (72.4%) and treated cane molasses (TM) (61.3%). Abundant growth of mould A. niger ORS-4.410 was observed with crude grape (0.131 g/l/h) and banana must (0.132 g/l/h).     

Determination of poly-β-hydroxybutyrate (PHB) production by some <Emphasis Type="Italic">Bacillus</Emphasis> spp.

In this study, 29 strains of the genus Bacillus were isolated from different soil samples which were taken from grasslands of Ankara, Turkey and were identified as B. brevis, B. sphaericus, B. cereus, B. megaterium, B. circulans, B. subtilis, B. licheniformis and B. coagulans. Two strains, B. sphaericus ATCC 14577 and B. subtilis ATCC 6633 were also included in this study. Poly-β-hydroxybutyrate (PHB) production by these strains was determined by the spectrophotometric method, and it was found that PHB production ranged from 1.06–41.67% (w/v) depending on the dry cell weight. The highest PHB production and productivity percentage was found in B. brevis M6 (41.67% w/v).     

Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens

Summary Poly--hydroxybutyric acid (PHB) and similar bacterial polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of methanol, one of the cheapest noble substrates available, may help to reduce the cost of producing such bioplastics. As a first step, a culture collection of 118 putative methylotrophic microorganisms was obtained from various soil samples without any laboratory enrichment step to favour culture diversity. The most promising culture was selected based on rapidity of growth and PHB accumulation and later identified as Methylobacterium extorquens. This isolate was obtained from soml contaminated regularly with used oil products for some 40 years. Concentrations of methanol greater than 8 g/l affected growth significantly and the methanol concentration was optimal at 1.7 g/l. PHB concentrations averaged 25–30% (w/v) of dry weight under non-optimized conditions. Controlling methanol concentration, using an open-loop configuration, led to biomass levels of 9–10 g/l containing 30–33% PHB while preventing methanol accumulation. The new isolate was also able to produce the co-polymer PHB/poly--hydroxyvalerate (PHV) using the mixture methanol + valerate. The PHV-to-PHB ratio was about 0.2 at the end of the fermentation. An average molecular mass varying between 2 and 3 × 10⁵ Da was obtained for three PHB samples using two different measurement methods.Publication number NRCC No. 33672 Offprint requests to: D. Groleau     

Engineering <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> for poly-3-hydroxybutyrate production

Strains of Yarrowia lipolytica were engineered to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway. The genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase were cloned and inserted into the chromosome of Y. lipolytica. In shake flasks, the engineered strain accumulated PHB to 1.50 and 3.84% of cell dry weight in complex medium supplemented with glucose and acetate as carbon source, respectively. In fed-batch fermentation using acetate as sole carbon source, 7.35 g/l PHB (10.2% of cell dry weight) was produced. Selection of Y. lipolytica as host for PHB synthesis was motivated by the fact that this organism is a good lipids producer, which suggests robust acetyl-CoA supply also the precursor of the PHB pathway. Acetic acid could be supplied by gas fermentation, anaerobic digestion, and other low-cost supply route.     

Effect of simple and complex carbon sources,low temperature culture and complex carbon feeding policies on poly-3-hydroxybutyric acid (PHB) content and molecular weight (Mw) from Azotobacter chroococcum 6B

The molecular weight (M _w) of poly-3-hydroxybutyrate (PHB), produced by shake-flask culture of Azotobacter chroococcum showed little variation with increasing glucose concentration as carbon source (being in the range of 400–500 kDa), while M _w increased from 300–400 to 640 kDa when grown with increasing concentration of sugar cane molasses. Molecular weight increased nearly 30% from 48 to 72 h culture time when 5% molasses as carbon source was used, while with glucose the highest M _w was reached at 48 h. Under fermentor cultivation A. chroococcum produced PHB with a relatively high M _w of 1590 kDa at 53 h culture time when grown in modified Burk's medium with glucose as carbon source at an initial C/N ratio (molar basis) of 69 under fermentor cultivation. A batch glucose-grown ammonium-limited fermentor culture was repeatedly fed with sugar cane molasses (initial C/N ratio 69) and it was observed that PHB content curve decreased at a slower rate than in the fed-batch culture in which glucose and sucrose were not consumed in the culture medium after the feed.     

Degradation of polycyclic aromatic hydrocarbons and long chain alkanes at 6070 °C by Thermus and Bacillus spp

Although polycyclic aromatic hydrocarbons (PAH) and alkanesare biodegradable at ambient temperature, in some cases low bioavailabilities are thereason for slow biodegradation. Considerably higher mass transfer rates and PAH solubilities and hence bioavailabilities can be obtained at higher temperatures. Mixed and pure cultures of aerobic, extreme thermophilic microorganisms (Bacillus spp., Thermus sp.) were used to degrade PAH compounds and PAH/alkane mixtures at 65 °C. The microorganismsused grew on hydrocarbons as sole carbon and energy source. Optimal growthtemperatures were in the range of 60–70 °C at pH values of 6–7. The conversion of PAH with 3–5 rings (acenaphthene, fluoranthene, pyrene, benzo[e]pyrene) was demonstrated. Efficient PAH biodegradation required a second, degradable liquid phase. Thermus brockii Hamburg metabolized up to 40 mg (l h)^-1 pyrene and 1000 mg(1 h)^-1 hexadecane at 70 °C. Specific growth rates of 0.43 h^-1 were measured for this strain with hexadecane/pyrene mixtures as the sole carbon and energy source in a 2-liter stirred bioreactor. About 0.7 g cell dry weight were formed from 1 g hydrocarbon. The experiments demonstrate the feasibility and efficiency of extreme thermophilic PAH and alkane biodegradation.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.