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.     

Kinetic analysis on formation of poly(3-hydroxybutyrate) from acetic acid by Ralstonia eutropha under chemically defined conditions

Batch cultures of Ralstonia eutropha in chemically defined media with acetic acid (HAc) as the sole carbon source were conducted to investigate acetate utilization, formation of poly(3-hydroxybutyrate) (PHB) and growth of active biomass (ABM) under different carbon to nitrogen (C/N) weight ratios. The specific acetate utilization rate based on ABM approached 0.16 g/g ABM h⁻¹, which was not affected very much by the extracellular HAc concentration from 1 to 5 g/l, but was affected by the C/N weight ratio. A low C/N ratio or high nitrogen supply sped up the specific acetate utilization rate to produce more ABM and less PHB. A high HAc concentration (>6 g/l), however, depressed acetate utilization as well as the ABM growth and PHB formation. A high cell mass concentration enhanced the tolerance of R. eutropha to the toxicity of HAc at pH 7 to 8.5. The viscosity-average molecular size of PHB generally increased first and then declined in batch cultures. Larger PHB molecules and less PHB per ABM were produced at a low C/N ratio with enough nutrient nitrogen than those under a high C/N ratio with less nutrient nitrogen available. Journal of Industrial Microbiology & Biotechnology (2001) 26, 121–126. Received 06 June 2000/ Accepted in revised form 21 October 2000     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

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.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P _adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.     

High production of Poly-β-hydroxybutyrate (PHB) from Methylobacterium organophilum under potassium limitation

Summary Mass production of Poly(-hydroxybutyrate) (PHB) from Methylobacterium organophilum under potassium-limited condition was carried out using a microcomputeraided automatic fed-batch culture system. The concentration of methanol was kept within the range of 2 – 3 g/l which did not show any inhibitory effect on cell growth. The PHB accumulation was stimulated when potassium concentration in the culture broth fell below 25 mg/l. After 70 hours of cultivation, the concentrations of cell mass and PHB were obtained to be 250 g/l and 130 g/l, respectively, which corresponded to a volumetric productivity of 1.8 – 2.0 g-PHB/1-hr. PHB contents ranged from 52% to 56% of dry cell weight with a yield factor (Y_P/S) of 0.19 g-PHB/g-methanol.     

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.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Polymer production by two newly isolated extremely halophilic archaea: application of a novel corrosion-resistant bioreactor

A novel corrosion-resistant bioreactor composed of polyetherether ketone (PEEK), tech glass and silicium nitrite ceramics was constructed and applied for the cultivation of two newly isolated, extremely halophilic archaea producing poly(γ-glutamic acid) (PGA), or poly(β-hydroxy butyric acid) (PHB), respectively. These bacteria were isolated from hypersaline soil close to Aswan (Egypt). The isolate strain 40, which is related to the genus Natrialba, produced large amounts of PGA when cultivated on solid medium. Culture conditions were optimised applying the corrosion-resistant bioreactor. PGA production was dependent on NaCl concentration and occurred about at 20% (w/v) NaCl in the medium. A maximum cell density of about 1.6 g cell dry matter/l was obtained when the bioreactor was stirred and aerated in a batch fermentation process using proteose-peptone medium. The supernatant was monitored with respect to PGA formation, and after 90 h a maximum of 470 mg/l culture volume was detected by HPLC analysis. Culture conditions were optimized for the isolate 56, which accumulated PHB as intracellular granules. Batch fermentations in the stirred and aerated bioreactor applying acetate and n-butyric acid as carbon sources led to cell density of 2.28 g cell dry matter/l and a maximum PHB accumulation contributing to about 53% of cellular dry weight. About 4.6 g PHB were isolated from 10.6 g dried cells of strain 56, which exhibited a weight average molar mass of 2.3 × 10⁵ g mol⁻¹ and a polydispersity of about 1.4. Received: 3 December 1999 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

Poly-3-hydroxybutyrate production byAzotobacter chroococcum

Thirty-seven soil isolates and mutants ofAzotobacter chroococcum tested for poly-3-hydroxybutyrate (PHB) production using Sudan black B staining method were found to be positive. One mutant showed a higher number of PHB-producing cells and maximum number of granules per cell. Using 2% glucose and 15 mmol/L ammonium acetate, PHB production was found to be maximum at 36 and 48 h of growth under submerged cultivation and under stationary cultivation, respectively. PHB production was found to be higher on sucrose and commercial sugar (as carbon sources) as compared to glucose and mannitol. As commercial sugar is cheaper than sucrose it was selected as carbon source for PHB production, that being found to be maximum at 1% concentration. Inorganic nitrogen sources seemed to have no stimulatory effect on the production of PHB. However, ammonium acetate (15 mmol/L) was found to be best for PHB production. Peptone (0.2 %) gave a better yield of PHB under both growth conditions. Using all optimized conditions, PHB production was studied in ten selected strains. Two of them were found to be best PHB producers under both growth conditions, one producing 621 and 740 μg/g dry mass under submerged cultivation and under stationary cultivation, respectively, while the second one produced 589 and 733 μg/g.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Production of poly-β-hydroxybutyrate (PHB) by <Emphasis Type="Italic">Alcaligenes latus</Emphasis> from maple sap

Maple sap, an abundant natural product especially in Canada, is rich in sucrose and thus may represent an ideal renewable feedstock for the production of a wide variety of value-added products. In the present study, maple sap or sucrose was employed as a carbon source to Alcaligenes latus for the production of poly-β-hydroxybutyrate (PHB). In shake flasks, the biomass obtained from both the sap and sucrose were 4.4 ± 0.5 and 2.9 ± 0.3 g/L, and the PHB contents were 77.6 ± 1.5 and 74.1 ± 2.0%, respectively. Subsequent batch fermentation (10 L sap) resulted in the formation of 4.2 ± 0.3 g/L biomass and a PHB content of 77.0 ± 2.6%. The number average molecular weights of the PHB produced by A. latus from maple sap and pure sucrose media were 300 ± 66 × 10³ and 313 ± 104 × 10³ g/mol, respectively. Near-infrared, ¹H magnetic resonance imaging (MRI), and ¹³C-MRI spectra of the microbially produced PHB completely matched those obtained with a reference material of poly[(R)-3-hydroxybutyric acid]. The polymer was found to be optically active with [α]²⁵ _D equaled to −7.87 in chloroform. The melting point (177.0°C) and enthalpy of fusion (77.2 J/g) of the polymer were also in line with those reported, i.e., 177°C and 81 J/g, respectively.     

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A _w=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A _w in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 271–275. Received 05 July 2000/ Accepted in revised form 27 January 2001     

Growth-associated production of poly(hydroxybutyric acid) by Azotobacter beijerinckii from organic nitrogen substrates

Poly(hydroxybutyric acid) (PHB) was produced by a selectant of Azotobacter beijerinckii in media containing only organic nitrogen sources such as N substrates. The chosen compounds were casein peptone, yeast extract, casamino acids and urea, each combined with carbon substrates glucose or sucrose. The PHB was synthesized under growth-associated conditions. The concentrations amounted to more than 50% of cell dry mass on casein peptone/glucose as well as urea/glucose medium within 45 h fermentation time. Corresponding to these yields, productivities of about 0.8 g PHB l⁻¹ h⁻¹ were discovered. The highest values increased to 1.06 g PHB l⁻¹ h⁻¹ on casein peptone/glucose medium and 1.1 g PHB l⁻¹ h⁻¹ on yeast extract/glucose medium after a period of 20 h. It was found that oxygen limitation was essential for successful product formation, as demonstrated earlier. These data from basic research may support further investigations into the use of technical proteins from renewable sources as substrates for PHB production by a strain of A. beijerinckii. Received: 3 June 1997 / Received revision: 29 August 1997 / Accepted: 15 September 1997     

The influence of poly-β-hydroxybutyrate accumulation on cell volume and buoyant density in alcaligenes eutrophus

Accumulation of poly--hydroxybutyrate (PHB) was studied in Alcaligenes eutrophus strain N9A. Under nitrogen limitation and heterotrophic conditions, the cells accumulated PHB at a rate of 50 fg cell^-1 h^-1. Volume increased from 1.208 to 3.808 m³ and buoyant density from 1.110 to 1.145 pg m^-3 with an increase in PHB from 0 up to 1.699 pg cell^-1. Volume was found to change linearly with PHB content. The changes were due to increases in cell width and not in cell length. PHB explained 93% of the changes in cellular volume. The relationship between density and PHB was hyperbolic. PHB explained 96% of the changes in density. When a mutant strain unable to accumulate PHB was analyzed together with the wild type, the PHB-less mutant and the wild type showed densities of 1.100 pg m^-3 and 1.120 pg m^-3, respectively, in gradients of 65% Percoll. In sucrose gradients, nevertheless, the results were reversed. This discrepancy was explained by the high osmolarity of sucrose which gives artificial results. Thus, we conclude that Percoll is a more suitable medium than sucrose to measure the density of live bacterial cells.Abbreviation PHB poly--hydroxybutyrate     

Growth and formation of poly (hydroxybutyric acid) by Methylobacterium rhodesianum at methanol concentrations of above 25 g/I

Methylobacterium rhodesianum MB 126 was cultivated using extended cultures without outflow. The feeding regime was based on the pH-regulated synchronous dosages of ammonia, methanol, phosphatc and trace elements according to supposed stoichiometric relations. The acidity of the culture medium was kept constant at pH 6.8, whereas the dissolved oxygen concentration was adjusted at 80% of saturation by autoregulation of the stirrer speed. However, besides testing technical conditions, two types of fermentations were discovered which are described in this paper. Firstly, although at the beginning of the bioprocesses the impeller speed increased up to 2,000 rpm, a decrease of dissolved oxygen down to zero was unavoidable. Secondly, methanol was accumulated temporarily up to 44 g/l and 26 g/l at 23 h of fermentation time and without inhibition of growth at least up to 30 g/l or PHB production. During this accumulation of the carbon substrate, exponential growth phases were detected showing growth rates of μ = 0.20/h and 0.21/h. But then, phases of retarded growth followed, whereas the methanol disappeared either continuously or after a steady level. In the course of a 54-h fermentation period, the synthesized PHB amounted to a content of above 50% of cell dry mass. From this data, a volumetric productivity of 0.4 g PHB/lxh was estimated. Moreover, the growth related yield coefficients were calculated to Y_X/MeOH = 0.21 and Y_X/MeOH = 0.14, whereas the product related yield coefficients amounted to Y_PHB/MeOH = 0.12 and Y_PHB/MeOH = 0,09. Since the shift down of growth rates as well as the production of PHB agreed in time with partial oxygen limitation (40% oxygen saturation), the competition observed between the tricarboxylic acid cycle and PHB synthesis was discussed. Summarizing the results, it was concluded that the frequently described inhibitory effect of methanol of above 2 g/l seems to be rather an effect of experimentally chosen conditions than of a general physiological phenomenon. Therefore, it could be demonstrated that the toxicity of methanol could be overcome if it was not dosed at different times but simultaneously with other medium components.