A genetically engineered strain of Saccharopolyspora erythraea that produces 6,12-dideoxyerythromycin A as the major fermentation product

The erythromycin producer, Saccharopolyspora erythraea ER720, was genetically engineered to produce 6,12-dideoxyerythromycin A, a novel erythromycin derivative, as the major macrolide in the fermentation broth. Inspection of the biosynthetic pathway for erythromycin would suggest that production of this compound could be achieved simply through the disruption of two genes, that encoding the erythromycin C-6 hydroxylase (eryF ) and that encoding the erythromycin C-12 hydroxylase (eryK ). The double mutant, however, was found to produce a mixture of 6,12-dideoxyerythromycin A and the precursor, 6-deoxyerythromycin D. Complete conversion to the desired product (to the limit of detection by TLC) was achieved by inserting an additional copy of the eryG gene, encoding the erythromycin 3′′-O-methyltransferase and driven by the ermE* promoter, into the S. erythraea chromosome. Received: 6 October 1997 / Received revision: 27 January 1998 / Accepted: 24 February 1998     

Sophorose lipid fermentation with differentiated substrate supply for growth and production phases

Sophorose lipid production by Candida bombicola is a two-step process where sophorose lipids are mainly produced after a first stage of growth, ending because of nitrogen limitation. The influence of the following parameters was individually studied for both the stages of growth and of product formation with respect to final sophorose lipid production performance: pH, temperature and carbon source. Glucose and rapeseed ethyl esters were supplied individually or as a dual carbon source. The lipidic substrate was added by continuous feeding. It was found that supplying both carbon sources during the production step was crucial for obtaining a high production performance ranging from 250 g l⁻¹ to 300 g l⁻¹ or more. Controlling the feeding of rapeseed ethyl esters to avoid inhibition by fatty acids was essential for a successful scale-up of the fermentation on the industrial scale. The conditions of substrate feeding markedly affected the composition of the mixture of sophorose lipids produced, namely the extent of acetylation of the sophorose moieties and distribution of the acidic and lactonic forms. The results suggest that the physiological role of sophorose lipid production is related to the regulation of energy metabolism. Received: 26 June 1996 / Received revision: 12 December 1996 / Accepted: 15 December 1996     

Biotechnology of succinic acid production and markets for derived industrial products

Succinic acid, derived from fermentation of agricultural carbohydrates, has a specialty chemical market in industries producing food and pharmaceutical products, surfactants and detergents, green solvents and biodegradable plastics, and ingredients to stimulate animal and plant growth. As a carbon-intermediate chemical, fermentation-derived succinate has the potential to supply over 2.7 × 10⁸ kg industrial products/year including: 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, adipic acid, n-methylpyrrolidone and linear aliphatic esters. Succinate yields as high as 110 g/l have been achieved from glucose by the newly discovered rumen organism Actinobacillus succinogenes. Succinate fermentation is a novel process because the greenhouse gas CO₂ is fixed into succinate during glucose fermentation. New developments in end-product recovery technology, including water-splitting electrodialysis and liquid/liquid extraction have lowered the cost of succinic acid production to U.S. $ 0.55/kg at the 75 000 tonne/year level and to $ 2.20/kg at the 5000 tonne/year level. Research directions aimed at further improving the succinate fermentation economics are discussed. Received: 27 October 1998 / Received revision: 22 January 1999 / Accepted: 22 January 1999     

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

  When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO₂ manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO₂ levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO₂ at a low level during the initial phase of the fermentation. However, both the timing of the pO₂ decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates. Received: 29 February 1996 / Received revision: 11 July 1996 / Accepted: 15 July 1996     

The effect of heat-shocking on batch fermentation by Clostridium beijerinckii NRRL B592

 In spite of the large-scale industrial use of the acetone-butanol fermentation process earlier this century (until 1983 in South Africa), very little has been published on the inoculum preparation techniques required for successful fermentation using these bacteria. In particular, heat-shocking has often been referred to as “useful” but no quantitative data are available. Data presented in this paper demonstrate and quantify the effect of heat-shocking on batch fermentation yields using one organism capable of this fermentation. Received: 27 August 1999 / Received revision: 23 December 1999 / Accepted: 5 January 2000     

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut^s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut⁺ clones. The mixed-feeding/Mut^s strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l. Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997     

Development of a low-cost fermentation medium for ethanol production from biomass

Nutrient cost is an important aspect in the fermentation of biomass to ethanol. With a goal of developing a cost-effective fermentation medium, several industrially available nutrient sources were evaluated for their effectiveness in the simultaneous saccharification and fermentation of pretreated poplar with Saccharomyces cerevisiae D₅A. These studies showed that a low-cost medium containing 0.3% corn steep liquor and 2.5 mM MgSO₄ · 7H₂O was similar in performance to a nutrient-rich medium. Besides its low cost, this alternative medium consists of components that are available on a commercial scale, thereby making it industrially relevant. Received: 14 August 1996 / Received revision: 7 January 1997 / Accepted: 24 January 1997     

Application of agro-industrial by-products for riboflavin production by Eremothecium ashbyii NRRL 1363

Riboflavin production is significantly determined by the type and initial concentration of the carbon and nitrogen sources and also by other flavinogenic stimulants. Using an optimum carbon and nitrogen concentration, an industrial fermentation medium has been designed with molasses as the carbon source and peanut seed cake as the nitrogen source. In addition the stimulatory effect of some of the low-cost agro-industrial by-products on riboflavin yield was investigated. Received: 10 March 1996 / Received revision: 25 June 1996 / Accepted: 14 July 1996     

Investigation of poly ( β-L-malic acid) production by strains of Aureobasidium pullulans

 Eight strains of the genus Aureobasidium obtained from culture collections were tested for their capability to produce poly(β-L-malic acid) (PMA). Four of the tested strains showed positive results. The most productive strain, A. pullulans CBS 591.75, was used to study the production of PMA in stirred-tank reactors. It was found that PMA was mainly produced in the late exponential phase, and the production related positively to glucose consumption. At the beginning of the fermentation the pH increased from 4.0 to about 7.0; subsequently the pH decreased and remained stable at around 3.0–3.5 for several days. Temperatures higher than 25^°C were detrimental to PMA production and cell growth. PMA production and cell growth at 20^°C and 25^°C exhibited no significant differences. PMA production and cell growth were studied under pH-controlled fermentation (at pH 2.0, 4.0, 5.5). The highest PMA production occurred at pH 4.0. PMA production was reduced at pH 2.0 although quite reasonable cell growth occurred at this pH value. Under optimized conditions 9.8 g PMA/l was produced during 9 days of fermentation in the stirred-tank reactors with an overall yield of 0.11 g PMA/g glucose. A procedure for the isolation of PMA and its separation from the other components of the fermentation broth was developed. The isolated PMA was characterized by ¹H and ¹³C-NMR spectroscopy as well as by infrared absorption spectroscopy. Gel-permeation chromatography revealed a relative molecular mass of approximately 3000–5000 by comparison with polyethylene glycol standards. Received: 13 February 1996/Received revision: 25 April 1996/Accepted: 1 May 1996     

Production of methoxyphenol-type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp.

In our screening program for microorganisms that are able to metabolize eugenol, the main component of the essential oil of the clove tree Syzigium aromaticum (sy. Eugenia cariophyllus), we found a new Pseudomonas sp. that produces several substituted methoxyphenols when eugenol is fed to the culture. A taxonomic characterization of this new organism has been performed. Examples of the biotransformation products, produced in high amounts, were vanillic acid with 3.25 g/l within 99 h, ferulic acid with 5.8 g/l within 75 h and coniferyl alcohol with 3.22 g/l within 47.5 h. By changing the culture conditions the ratio of the different metabolites could be varied. Based on these results a scheme for the degradation of eugenol by this strain has been established. Received: 1 April 1996 / Received revision: 24 June 1996 / Accepted: 1 July 1996     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Application of response-surface methodology to evaluate the optimum environmental conditions for the enhanced production of surfactin

Response-surface methodology was applied to determine the effect of the fermentation process conditions, namely pH, temperature, rates of agitation and aeration, on surfactin production. The effects of the mutual interactions between these parameters were extensively studied to optimize the process conditions for the maximum production of surfactin. With a view to simultaneously reducing the number of experiments and obtaining the mutual interactions between the variables required for achieving the optimal experimental conditions, a 2⁴ full-factorial central composite design followed by multi-stage Monte-Carlo optimization was employed for experimental design and analysis of the results. The optimum process conditions for the enhanced production of surfactin were as follows: pH = 6.755, temperature = 37.4 °C, agitation = 140 rpm and aeration = 0.75 vvm. Relative surfactin concentrations were denoted by the reciprocal of the critical micelle concentrations. Received: 25 October 1996 / Accepted: 15 November 1996     

Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Streptoverticillium mobaraense

In the later stages of a batch fermentation for microbial transglutaminase production by Streptoverticillium mobaraense the availability of a nitrogen source accessible to the microorganism becomes critical. Fed-batch fermentation is investigated with the aim of avoiding this substrate limitation. When peptone is used as a nitrogen source in the feed, no significant improvement of growth and transglutaminase production is observed. This is probably due to crosslinking of the nitrogen source by the transglutaminase produced. Using an inorganic nitrogen source alone does not give satisfactory growth and production. A fed-batch fermentation method has thus been developed to deal with this problem. In the batch phase of the fermentation, an initial medium containing peptone, designed on the basis of the stoichiometric requirements of the microorganism, is used to ensure optimal growth. In the feeding phase, ammonium sulphate is used instead to avoid the crosslinking effect. The feed composition, mainly the amount of nitrogen and carbon source, is also based on the stoichiometric requirements of the organism, taking into account the replacement of peptone by ammonium sulphate. By using this fed-batch fermentation technique, cell-mass dry weight and transglutaminase production could be increased by 33% and 80% respectively, compared to those in a batch fermentation. Received: 10 July 1997 / Received revision: 24 October 1997 / Accepted: 24 October 1997     

Process development for high-level secretory production of carboxypeptidase Y by Saccharomyces cerevisiae

In order to develop a production process for carboxypeptidase Y (CPY, yeast vacuolar protease) secreted by Saccharomyces cerevisiae KS58-2D, medium composition, culture conditions, and expression systems were investigated. We found that the addition of histidine to thiamine-free medium, in which CPY production was almost negligible, raised the intracellular thiamine level, resulting in the increase of CPY production. On the basis of the choice of an expression system that uses an inducible GAL10 promoter, reassessment of histidine concentration in the medium, and optimization of the pH level during cultivation (pH 6.5), active CPY was secreted in a quantity of over 400 mg/l, which was more than tenfold that higher than that previously reported. The process developed could be easily scaled-up to industrial-scale fermentation. Received: 16 January 1998 / Received revision: 16 February 1998 / Accepted: 27 February 1998     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Bioconversion of 2-cyanopyrazine to 5-hydroxypyrazine-2-carboxylic acid with Agrobacterium sp. DSM 6336

5-Hydroxypyrazine-2-carboxylic acid, a versatile building block for the synthesis of new antituberculous agents, was prepared by whole-cell biotransformation from 2-cyanopyrazine via pyrazinecarboxylic acid using Agrobacterium sp. DSM 6336. By developing a fermentation process for this two-enzyme-step bioconversion, a product concentration of 286 mM (40 g/l) was obtained. After the isolation method had been optimized the total yield was 80%. Received: 28 February 1997 / Received revision: 28 April 1997 / Accepted: 4 May 1997     

High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium

The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome of Klebsiella planticola with high transposition frequency. A Bacillus hybrid β-glucanase located on an RSF1010-derived plasmid was mobilized from E.coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments it was shown that the production of β-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter used for the expression of the kil gene the secretion of β-glucanase into the medium started at the transition from the exponential to the stationary phase, as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins. Received: 22 July 1998 / Received revision: 25 November 1998 / Accepted: 29 November 1998     

Critical factors in chitin production by fermentation of shrimp biowaste

Factors affecting Lactobacillus fermentation of shrimp waste for chitin and protein liquor production were determined. The objective of the fermentation is medium conditioning by Lactobacillus through production of proteases and lowering of the pH. The efficiency was tested by conducting fermentation of biowaste in 1-l beakers with or without pH adjustment using different acids. Addition of 5% glucose to the biowaste supported the growth of lactic acid bacteria and led to better fermentation. Among four acids tested to control pH at the start and during fermentation, acetic acid and citric acid proved to be the most effective. In biowaste fermented with 6.7% L. plantarum inoculum, 5% glucose, and pH 6.0 adjusted with acetic acid, 75% deproteination and 86% demineralization was achieved. Replacement of acetic acid by citric acid gave 88% deproteination and 90% demineralization. The fermentation carried out in the presence of acetic acid resulted in a protein fraction that smelled good and a clean chitin fraction. Received: 4 April 2000 / Received revision: 9 June 2000 / Accepted: 9 June 2000     

Continuous production of ethanol using yeast cells immobilized in preformed cellulose beads

 Saccharomyces cerevisiae cells were immobilized on preformed cellulose beads by adsorption. The fermentation capacity of the immobilized yeast cells was found to be practically independent of the hydrogen ion concentration between pH 3.1 and 6.25. The fermentation capacity was maximal at 30 °C. The immobilized yeast cells were used for continuous production of ethanol in a fluidized-bead reactor. The average values characteristic for the process were an ethanol concentration of 41.9±0.1 g l^-1, a fermentation efficiency of 82.9±2.1% and a volumetric productivity of 3.94±0.52 g l^-1 h^-1. Received: 9 October 1995/Accepted: 22 April 1996     

Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks

The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/l) than in conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution. In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size. Received: 13 December 1996 / Received revision: 10 April 1997 / Accepted: 27 April 1997