Production of exopolysaccharide by Pseudomonas sp. ATCC 31461 (Pseudomonas elodea ) using whey as fermentation substrate

An improved strain of Pseudomonas sp. ATCC 31461 (Pseudomonas elodea), capable of producing broth viscosities of 11 000 and 4700 mPa s (cP) when grown in enriched whey permeate and enriched sweet whey broths respectively, was isolated. The isolation was by serial transfers of the parent on lactose-rich and sweet whey broths. Maximum viscosities and biopolymer production were observed in 25% (v/v) whey concentration. In whey concentrations of 50% (v/v) or greater, residual glucose was detected in the broth and biopolymer production was low. This strain is capable of totally utilising the lactose in up to 50% (v/v) whey in 64 h. Enzyme activities suggest that the transport of lactose in P. elodea is by the permease system as opposed to the phosphotransferase system. The location of β-galactosidase is mainly intracellular. The improved strain is able to utilise lactose better than the parent and produce 1.6 times more intracellular β-galactosidase activity compared to the parent. Received: 3 May 1996 / Received revision: 8 August 1996 / Accepted: 10 August 1996     

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     

Formation of benzaldehyde by Pseudomonas putida ATCC 12633

Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal. Received: 16 March 1998 / Received revision: 20 May 1998 / Accepted: 21 May 1998     

Fermented whey – an inexpensive feed source for a laboratory-scale selenium-bioremediation reactor system inoculated with Thauera selenatis

It is critical that an inexpensive electron- donor/carbon-source be found for selenium bioremedia-tion using the selenate-respiring bacterium, Thauera selenatis. Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in whey to large amounts of acetate. Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous culture at a very slow dilution rate (D = 0.05 h⁻¹). The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation reactor system, inoculated with T. selenatis, treating selenium-contaminated drainage water was also demonstrated. Selenium oxyanions and nitrate were reduced by 98%. Received: 30 October 1998 / Received revision: 26 January 1999 / Accepted: 5 February 1999     

Production of sophorolipids from whey

Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1′,4′′-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFT^min 39 mN m⁻¹, CMC 130 mg l⁻¹), water solubilities (2–3 g l⁻¹) and low cytotoxicities (LC₅₀ 300–700 mg l⁻¹). In contrast, purified sophorolipids were more surface active (SFT^min 36 mN m⁻¹, CMC 10 mg l⁻¹), less water soluble (max. 70 mg l⁻¹) and showed stronger cytotoxic effects (LC₅₀ 15 mg l⁻¹). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect. Received: 23 February 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

Sophorolipid Production with High Yields on Whey Concentrate and Rapeseed Oil without Consumption of Lactose

Sophorolipids were produced by single-step batch cultivation of Candida bombicola ATCC 22214 on deproteinized whey concentrate and repeated feed of rapeseed oil. A mild sterilization method for whey was developed. High yields of 280 g dry sophorolipids l^–1 were obtained from deproteinized whey concentrate containing 100 g lactose l^–1 and 300 g rapeseed oil l^–1. Surprisingly, the whey lactose was not consumed by the organism. Growth only on the oil was assumed and a high lipase activity of 24 U per g cell dry weight resulted.     

Production of xanthan gum and ice-nucleating material from whey by Xanthomonas campestris pv. translucens

Xanthomonas campestris pv. translucens IFO13599 could produce xanthan gum (18.5 mg/100 mg, lactose) with lactose as the growth substrate in spite of a low level of β-galactosidase. This productivity corresponded to one-fifth that with glucose. This strain could also produce ice-nucleating material having an ice-nucleating temperature, T ₅₀, of −2.8 °C with xanthan gum in the culture broth. We found that this strain produced both materials in whey medium from which the insoluble components had been removed. The production of xanthan with ice-nucleating material reached a maximum after cultivation for 168 h under optimum conditions. Furthermore, the xanthan obtained had a low viscosity because of its variant structure revealed, by TLC and HPLC analyses, to be lacking pyruvic acid. Furthermore, we concluded that this mixture had considerable potential as a regeneratic agent, when compared to other regeneratic agents such as carboxymethylcellulose. Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222

Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1% v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO₃ with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature (15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days. Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Production of sophorolipids in high concentration from deproteinized whey and rapeseed oil in a two stage fed batch process using Candida bombicola ATCC 22214 and Cryptococcus curvatus ATCC 20509

High concentrations of 422 g sophorolipids l–1 were produced using a two-stage cultivation process: first deproteinized whey concentrate (DWC) containing 110 g lactose l–1 was used for cultivation of the yeast Cryptococcus curvatus ATCC 20509, resulting in 34 g dry weight l–1, 20 g single-cell oil l–1 and reducing the chemical oxygen demand (COD) from 159 g l–1 to 35 g oxygen l–1. Afterwards cells were disrupted by passing the cell suspension directly through a high pressure laboratory homogeniser. After autoclavation, the resulting crude cell extract containing the single-cell oil served as substrate for growth of Candida bombicola ATCC 22214 and for sophorolipid production in a second stage. When the single-cell oil was consumed, repeated feeding of 400 g rapeseed oil l–1 was started increasing the yield of sophorolipids to 422 g l–1. A simple technique for product isolation, sedimentation, could be used to harvest the crude sophorolipids. © Rapid Science Ltd. 1998     

Optimization of fermentation conditions for ethanol production from whey

Summary Optimal conditions for ethanol production in 7% whey solutions by the yeast Candida pseudotropicalis ATCC 8619 included initial pH of 4.57 and 30°C. Complete fermentation of the available lactose took place without supplementary nutrients; additions of nitrogen or phosphorus salts, yeast extract or corn steep liquor resulted in increased yeast production and lower ethanol yields. A positive correlation was observed between increases in yeast inocula and lactose utilization and ethanol production rates; 8.35 g/l of ethanol was obtained within 22 h by using yeast inoculum of 13.9 g/l. No differences in fermentation rates or ethanol yields were observed when whole or deproteinized whey solutions were used. Concentrated whey permeates, obtained after removal of the valuable proteins from whey, can be effectively fermented for ethanol production.     

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens were systematically studied for the production of succinic acid from whey. Addition of 2.5 g l⁻¹ yeast extract and 2.5 g l⁻¹ polypeptone per 10 g l⁻¹ whey was most effective for succinic acid production from both treated and nontreated whey. When 20 g l⁻¹ nontreated whey and 7 g l⁻¹ glucose were used as cosubstrates, the yield and productivity of succinic acid reached at the end of fermentation were 95% and 0.46 g (l h)⁻¹, respectively. These values were higher than those obtained using nontreated whey alone [93% and 0.24 g (l h)⁻¹ for 20 g l⁻¹ whey]. Continuous fermentation of A. succiniciproducens at an optimal dilution rate resulted in the production of succinic acid with high productivity [1.35 g (l h)⁻¹], high conversion yield (93%), and higher ratio of succinic acid to acetic acid (5.1:1) from nontreated whey. Received: 23 July 1999 / Received revision: 17 November 1999 / Accepted: 24 December 1999     

Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate

 Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, q _P, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with K _S at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, q _S,max, varied between 4.32±0.02 g lactose g cells^-1 h^-1 and 4.89 ±0.02 g lactose g cells^-1 h^-1. The initial specific lactose uptake rate for non-growing cells, q _S,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells. Received: 4 October 1995/Received last revision: 23 April 1996/Accepted: 29 April 1996     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Construction of a flocculent Saccharomyces cerevisiae fermenting lactose

A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for β-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting yeast strains. Moreover, the flocculating capacity of the strain used in this work gives the process several advantages. On the one hand, it allows for operation in a continuous mode at high cell concentration, thus increasing the system's overall productivity; on the other hand, the biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs. Received: 2 October 1998 / Received revision: 15 January 1999 / Accepted: 17 January 1999     

Development of markers for product formation and cell cycle in batch cultivation of Clostridium acetobutylicum ATCC 824

Experiments were performed to identify relationships between morphological and physiological events during batch fermentation of Clostridium acetobutylicum ATCC 824. Differing fermentation conditions were obtained by manipulation of the culture pH value during the process. The bacterium showed marked changes in morphology during its cultivation, similar to those previously observed for other strains. However, although the acidogenic phase was characterized by the presence of rod-shaped cells, and the solventogenic phase by clostridial forms, there was no simple relationship between the proportion of clostridial forms present and the ratio of solvents to acids. Nevertheless, the shift from acidogenesis to solventogenesis was always coincident with the presence of some clostridial forms and with the accumulation of granulose within the cells. In addition, the “solvent shift” was associated with major changes in the cellular protein pattern, as analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Hence, the potential solventogenic ability of any particular culture may be recognised by its morphological appearance and/or its cellular protein pattern. Received: 19 September 1997 / Received revision: 9 February 1998 / Accepted: 9 February 1998     

Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture

The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h⁻¹. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production. Received: 9 January 1997 / Received revision: 5 March 1997 / Accepted: 13 April 1997     

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     

Effects of yeast extract on the production and the quality of the exopolysaccharide, zooglan, produced by Zoogloea ramigera 115SLR

Although many studies have examined the influence of culture conditions on the production and composition of polysaccharides, little is known about the factors influencing the quality of exopolysaccharides (EPS). In this work we studied the effect of yeast extract on the production, composition and molecular weight of the EPS zooglan produced by Zoogloea ramigera 115SLR. This bacterium was grown on a new completely defined synthetic medium and on a medium containing yeast extract. Growth and polysaccharide production performances were comparable on the two media with a glucose to exopolysaccharide conversion yield of 35% (g/g). The polysaccharides produced on these two media have an identical composition but a different molecular weight and molecular weight distribution. The yeast extract medium leads to a more homogeneous polysaccharide solution. Received: 12 June 1998 / Received revision: 19 September 1998 / Accepted: 11 October 1998