Influence of bioreactor design on exopolysaccharide production byAureobasidium pullulans

Summary Exopolysaccharide yields byAureobasidium pullulans were affected by the bioreactor configuration in batch culture. However the yields obtained in the stirred-tank and airlift vessels were also dependent on the nitrogen source used, and independent of cell morphology.     

A two stage continuous process for the production of thermogelable curdlan-type exopolysaccharide

Summary A laboratory-scale, two-stage continuous process for the production of curdlan-type exopolysaccharide has been operated in steady-state for 500hr. Two continuous flow, constant volume fermenters are connected in series. A stable, curdlan-producing strain of Alcaligenes faecalis var myxogenes ATCC 31749 is grown aerobically in a nitrogen-limited chemostat operating near D_max at 0.24 hr^–1. The effluent is introduced directly into a second larger constant volume fermenter which is being simultaneously fed a glucose solution at a fixed rate. Under sub-optimal conditions associated with curdlan production, the observed Qp was 0.05 g curdlan/g cell/hr. At a biomass level of 4 g/L in the second stage, curdlan was present at 10 g DW/L and the volumetric productivity was 0.2 g/g cell/hr. The substrate (glucose) conversion efficiency was 42%. The process is described in patents applied for on behalf of George Weston Ltd. (Toronto, Canada).     

Glycollate production and excretion byAlcaligenes eutrophus

Autotrophic cultures of the facultative chemolithotrophAlcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO₂ to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic conditions.Extracts from autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction ofd0ribulose 1,5-diphosphate carboxylase under the same conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - HPMS 2-pyridyl-hydroxymethane sulphonie acid - RuDP d-ribulose 1,5-diphosphate     

Effects of molecular weight and stereoregularity on biodegradation of poly(vinyl alcohol) byAlcaligenes faecalis

Summary The biodegradability of poly(vinyl alcohol) (PVA) was analyzed with respect to its molecular weight and stereoregularity using the isolated PVA-assimilating microbial strain,Alcaligenes faecalis KK314. The biodegradability of PVA was influenced by its stereoregularity, and the isotactic moiety was preferentially biodegraded. However, there is no difference in the biodegradability based on the molecular weight of PVA being larger than the octamer.     

Xylitol production in a bubble column bioreactor: Influence of the aeration rate and immobilized system concentration

This work evaluated the xylitol production from sugarcane bagasse hemicellulosic hydrolysate in a bubble column bioreactor using cells of the yeast Candida guilliermondii immobilized in calcium-alginate. The fermentation runs were performed according to a 2² full factorial design with three replicates at the center point in order to determine the effect of the variables: aeration rate (0.66–1.33 vvm) and immobilized system concentration (20–40% v/v), on the efficiency of xylose-to-xylitol conversion and on the xylitol volumetric productivity. The results indicated a significant influence of both variables on xylitol production. The highest conversion efficiency (41%) was attained using 1.33 vvm aeration rate and 40% immobilized system. Under these conditions, the volumetric productivity was 0.21 g l⁻¹ h⁻¹.     

Influence of nutritional conditions on the mycelial growth and exopolysaccharide production in Paecilomyces sinclairii

AIMS: The objective of the study was to optimize the submerged culture conditions for the production of exopolysaccharide from Paecilomyces sinclairii. METHODS AND RESULTS: The optimal temperature and initial pH for exopolysaccharide production by Paecilomyces sinclairii in shake flask culture were found to be 30 degrees C and 6.0, respectively. Sucrose (60 g l(-1)) and corn steep powder (10 g l(-1)) were the most suitable carbon and nitrogen source for exopolysaccharide production. CONCLUSIONS: Under optimal culture medium, the maximum exopolysaccharide concentration in a 5-l stirred-tank fermenter indicated 7.4 g l(-1), which was approximately three times higher than that in basal medium. The maximum specific growth rates (micro max) and yield coefficient (Y(P/S)) in the optimal culture medium was 0.16 h(-1) and 0.19, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimal culture conditions reported in this article can be widely applied to the processes for submerged cultures of other mushrooms.     

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 °C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium. Received: 11 April 1997 / Received revision: 19 June 1997 / Accepted: 23 June 1997     

Influence of pulp density and bioreactor design on microbial desulphurization of coal

Summary Pyrite was microbiologically removed by Thiobacillus ferrooxidans in pure and mixed cultures from German bituminous coal at 10% pulp density with maximum pyrite oxidation rate of 350 mg pyritic S/l per day. However, at pulp densities above 20% bacterial growth and consequently pyrite oxidation were completely prevented both in a conventional airlift reactor and in a stirred-tank reactor. Modifying the airlift reactor by adapting a conical bottom part, bacterial growth and pyrite oxidation could be achieved even at 30% pulp density, resulting in a pyrite removal of more than 90% at a pyrite oxidation rate of 230 mg pyritic S/l per day.Dedicated to Prof. Dr. H. Jüntgen on the occasion of his 60th birthday     

Influence of culture conditions on exopolysaccharide production by Lactobacillus rhamnosus strain C83

The influence of culture conditions on growth and exopolysaccharide (EPS) production by Lactobacillus rhamnosus strain C83 was investigated using fermentor batch cultures. A temperature shift (from 37 to 25 °C) at the beginning of the exponential growth phase (0–5 h) enhanced EPS production. Furthermore, an optimal environmental pH of 6·2–7·2 improved the performance of strain C83 and partially anaerobic culture conditions (pO₂ from 0 to 10%) triggered EPS over-production by the cells. The sugar composition of this polymer was independent of culture conditions, such as carbon source, medium composition, temperature, pH, pO₂ and growth phases. In all cases, galactose and glucose were the principal components.     

Continuous production of poly(3-hydroxybutyrate-co-3-hyhroxyvalerate) byAlcaligenes eutrophus

Summary Copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced in a continuous culture ofAlcaligenes eutrophus with 17.5 gl ^–1 of fructose and 2.5gl ^–1 of pentanoic acid in the feed. The P(3HB-co-3HV) productivity was maximal at a dilution rate of 0.17h^–1 and yielded 0.31gl ^–1h^–1 under an ammonium-limited condition. The 3HV content in copolymers increased from 11 to 79 mol% as the dilution rate of cells was increased from 0.06 to 0.32h^–1.     

Effect of ammonium supplementation on production of poly-B-hydroxybutyric acid byAlcaligenes eutrophus in batch culture

Summary Alcaligenes eutrophus H16 showed some degree of oxygen sensitivity, having the fastest specific growth rate at a dissolved oxygen tension of 60% air saturation. Growth and PHB accumulation kinetics were studied and an accelerated rate of PHB synthesis was obtained when a low concentration of NH₄Cl was supplied during the PHB synthesis stage.     

Influence of mist intervals and aeration rate on growth and second metabolite production of Pseudostellaria heterophylla adventitious roots in a siphon-mist bioreactor

Plant adventitious root culture in bioreactors is a promising alternative for the efficient production of medicinal herbs. Adventitious roots of Pseudostellaria heterophylla were induced from callus and then cultivated in a siphon-mist bioreactor. An orthogonal test established that the optimal medium for adventitious root induction was MS medium supplemented with 1.0 mg/L naphthaleneacetic acid and 2.0 mg/L 3-indolybutyric acid. Under these conditions, the average root number was more than 14 on each 1.0 cm diameter callus and the rooting rate reached 100%. The bioreactor was equipped with an integral siphon-spraying device designed to automatically supply the liquid medium. The operation parameters of the bioreactor were assessed by varying the mist interval and the aeration velocity. The mist interval was negatively related to average growth rate of the adventitious roots and positively related to saponin and polysaccharide content. A relatively high aeration rate was necessary to achieve the maximum biomass production, but the secondary metabolite production was not enhanced by increasing the aeration velocity.     

Carbon and nitrogen source effects on basidiomycetes exopolysaccharide production

The capability to synthesize the extracellular polysaccharide (EPS) is widespread among eight mushroom species which accumulated 0.6–2.2 g/1 of EPS in submerged cultivation. Glucose, maltose, and mannitol were the most appropriate carbon sources for biomass and EPS production. Organic nitrogen sources appeared to be the most suitable nitrogen sources for biomass and EPS accumulation. The cultivation process in shake flasks was successfully reproduced in a laboratory fermentor with enhanced EPS production. The highest yield of EPS (3.8–4.0 g/1) was achieved in cultivation of Agaricus nevoi and Inonotus levis.     

pH-control modes in a 5-L stirred-tank bioreactor for cell biomass and exopolysaccharide production by Tremella fuciformis spore

The effect of pH-control modes on cell growth and exopolysaccharide production by Tremella fuciformis was evaluated in a 5-L bioreactor. The results show that the maximal dry cell weight (DCW) and exopolysaccharide production were 23.57 and 4.48 g L⁻¹ in pH-stat fermentation, where the maximal specific growth rate (μ_max) and specific production rate of exopolysaccharide (P_P/X) were 1.03 and 0.24 d⁻¹, respectively; under pH-shift cultivation, the maximal DCW and exopolysaccharide production were 30.57 and 3.90 g L⁻¹, where the μ_max and P_P/X were 1.21 and 0.06 d⁻¹. Unlike batch fermentation, maximal DCW and exopolysaccharide production merely reached 15.04 and 2.0 g L⁻¹, where the μ_max and P_P/X were 0.86 and 0.05 d⁻¹, respectively. These results suggest that a pH-stat strategy is a more efficient way of performing the fermentation process to increase exopolysaccharide production. Furthermore, this research has also proved that the three-stage pH-control mode is effective for cell growth.     

Intraspecific variability and exopolysaccharide production inAureobasidium pullulans

Forty seven strains of the black yeasts,Aureobasidium pullulans andHormonema dematioides, and the type strain ofHormonema macrosporum were examined using PCR-ribotyping and universally primed PCR with subsequent hybridization. Four groups (populations) were distinguished withinA. pullulans with PCR-ribotyping, which largely coincided with UP-PCR/hybridization groups. The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied. Five strains identified asHormonema dematioides on the basis of physiological and morphological data formed a group recognizable with PCR-ribotyping and UP-PCR/hybridization, which also includedH. macrosporum. Aureobasidium pullulans is characterized by the absence of RsaI restriction sites in rDNA amplified with primers 5.8S-R and LR7, whileHormonema species possessed several bands after RsaI digestion. For analysis of distance between populations, PCR-ribotyping with AluI and MspI is sufficient. Strains ofA. pullulans produce exopolysaccharides in liquid media with different nitrogen sources, while the strains ofHormonema synthesize minor amounts of polysaccharides in media with peptone. Populations ofA. pullulans differ slightly from each other in their optimal, medium-dependent production of polysaccharides.     

Effect of oxygen supply and dilution rate on the production of 2,3-butanediol in continuous bioreactor by Klebsiella pneumoniae

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae (NRRL B199) from glucose have been studied in a continuous bioreactor. The effect of oxygen supply rate and dilution rate on the product output rate and yield of 2,3-butanediol were investigated. For a feed glucose concentration of 100 g l⁻¹, the optimum oxygen transfer rate is between 25.0–35.0 mmol l⁻¹ h⁻¹. Under these conditions, maximum product concentration obtained was 35 g l⁻¹ at a dilution rate of 0.1 h⁻¹ and the maximum product output rate obtained was 4.25 g l⁻¹ h⁻¹. The product yield based on the substrate utilized approached the theoretical value (50%) at low values of oxygen transfer rate but decreased with increasing oxygen transfer rate.     

High rate treatment of terephthalic acid production wastewater in a two-stage anaerobic bioreactor

The feasibility was studied of anaerobic treatment of wastewater generated during purified terephthalic acid (PTA) production in two-stage upflow anaerobic sludge blanket (UASB) reactor system. The artificial influent of the system contained the main organic substrates of PTA-wastewater: acetate, benzoate, and terephthalate. Three parallel operated reactors were used for the second stage, and seeded with a suspended terephthalate degrading culture, with and without additional methanogenic granular sludge (two different types). The first stage UASB-reactor was seeded with methanogenic granular sludge. Reactors were operated at 37 degrees C and pH 7. During the first 300 days of operation a clear distinction between the biomass grown in both reactor stages was obtained. In the first stage, acetate and benzoate were degraded at a volumetric loading rate of 40 g-COD/L . day at a COD-removal efficiency of 95% within the first 25 days of operation. No degradation of terephthalate was obtained in the first stage during the first 300 days of operation despite operation of the reactor at a decreased volumetric loading rate with acetate and benzoate of 9 g-COD/L . day from day 150. Batch incubation of biomass from the reactor with terephthalate showed that the lag-phase prior to terephthalate degradation remained largely unchanged, indicating that no net growth of terephthalate degrading biomass occurred in the first stage reactor. From day 300, however, terephthalate degradation was observed in the first stage, and the biomass in this reactor could successfully be enriched with terephthalate degrading biomass, resulting in terephthalate removal capacities of 15 g-COD/L . day. Even though no single reason could be identified why (suddenly) terephthalate degradation was obtained after such a long period of operation, it is suggested that the solid retention time as well the prevailing reactor concentrations acetate and benzoate may have played an important role. From day 1 of operation, terephthalate was degraded in the second stage. In presence of methanogenic granular biomass, high terephthalate removal capacities were obtained in these reactors (15 g-COD/L . day) after approximately 125 days of operation. From the results obtained it is concluded that terephthalate degradation is the bottleneck during anaerobic treatment of PTA-wastewater. Pre-removal of acetate and benzoate in staged bioreactor reduces the lag-phase prior to terephthalate degradation in latter stages, and enables high rate treatment of PTA-wastewater.     

Studies on the production of extracellular protease by Alcaligenes faecalis

Alcaligenes faecalis produced extracellular protease when incubated in media containing protein substrates. Enzyme production was found to be influenced by various culture conditions. Enzyme production was growth-associated, expressed linearity with growth and reached a maximum at the end of the growth phase. Carbohydrates and inorganic nitrogen sources could not be utilized by the bacterium for its growth, and organic nitrogen appeared to be a primary determinant in protease production. Enzyme production reached its maximum level of 171.2 U/ml when the culture was incubated at 30 °C at pH 8.0. Ca²⁺ and Mg²⁺ enhanced the enzyme production. The crude enzyme powder was stable at high alkaline pH and stable upto 6 months at the storage temperature of 0–4 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antioxidant property and production of exopolysaccharide from Armillaria mellea in submerged cultures: Effect of culture aeration rate

Effects of culture aeration rate on production and antioxidant property of exopolysaccharide (EPS) by Armillaria mellea were investigated in a 5‐L stirred‐tank bioreactor where an optimal biomass aeration rate of 1.2 vvm with 0.22 g/g cell yield and 0.6 vvm EPS formation rate with 7.66 mg/g product yield were achieved. A two‐stage aeration process to maximize the biomass and EPS productions proceeded with a 1.55‐fold enhancement (from 4.28 to 6.65 g/L) in biomass formation and a 2.68‐fold enhancement (from 86.9 to 233.2 mg/L) in the EPS production, as compared with those from the aeration rate of 0.3 vvm. The molecular weights of EPS in cultures of different aeration rates are closely correlated with their protein/polysaccharide ratios (R²=0.830) and EC₅₀ (EC₅₀, the effective concentration where the antioxidant property is 50%) values in antioxidant activity (R²=0.960), reducing power (R²=0.894) and chelating ability (R²=0.954). EPS from the two‐stage aeration rate culture shows a strong antioxidant property by the conjugated diene method, reducing power and chelating ability on ions. Therefore, we present results to regulate and to optimize A. mellea cultures to efficiently produce biomass and EPS. The fermented EPS has the potential to be used as for antioxidant‐related functional foods and pharmaceutical industries.     

Estimation of Chinese hamster ovary cell density in packed-bed bioreactor by lactate production rate

A method is described for estimating recombinant Chinese hamster ovary (rCHO) cell density in a packed-bed bioreactor by lactate production rate. The lactate production rate, which depended on both the cell numbers and cell growth rate, was modeled by segregating the cell population into two parts: one growing at a maximum specific growth rate and another non-growing. The individual cell in each part had the same lactate production rate. The established rate equation of lactate production matched the experimental data reasonably well and could be used to estimate the cell growth in the batch culture with microcarriers. Furthermore, in the perfusion culture of rCHO cells in a packed-bed bioreactor, the final cell density, 1.3×10¹⁰ cells l^–1, estimated by lactate production rate, was comparable to the direct sample counting of 1.2×10¹⁰ cells l^–1, showing that lactate production rate method would be useful in tracing the cell growth in packed-bed bioreactors.