Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides

A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes. Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998     

Effect of cations, pH and sulfate content on the viscosity and emulsifying activity of the Halomonas eurihalina exopolysaccharide

The effects of monovalent and divalent cations on the rheological behavior of Halomonas eurihalina exopolysaccharide (EPS) were studied. Sodium, potassium, magnesium and calcium were added and the relative abilities to increase viscosity were as follows: KCl > NaCl > MgCl₂ > CaCl₂. The highest viscosity value was measured in acidic 10⁻⁴ M KCl, in which a gel formed. A loss of sulfate content seemed to correlate with the increase of viscosity. H. eurihalina produced EPS in all growth media. Addition of hydrophobic substrates to culture media produced changes in chemical composition and emulsifying activity of the EPS. Xylene was the most effectively emulsified substance and the EPS produced on tetradecane and on corn oil the most active emulsifier. Received 25 July 1997/ Accepted in revised form 30 January 1998     

Comparison of industrial yeast strains for fermentation of spent sulphite pulping liquor fortified with wood hydrolysate

Ethanol production from spent sulphite pulping liquor (SSL) was compared for four different yeasts. A second strain of S. cerevisiae as well as a 2-deoxyglucose-resistant strain formed through protoplast fusions between S. uvarum and S. diastaticus produced up to 27% more ethanol from SSL fortified with hydrolysis sugars than was produced by S. cerevisiae. The incremental improvement in ethanol yield appeared to vary with the degree of fortification, ranging from 5.8% for unfortified SSL, to 27% for the highest level of fortification tested. Decreasing fermentation rates were observed for SSL fortified with glucose, mannose and galactose, respectively. Sugar uptake rates in SSL fortified with glucose, galactose and mannose were 6.8, 2.8 and 2.0 g L⁻¹ h⁻¹, respectively. However, when these sugars were fermented along with a glucose cosubstrate, the rate at which the combined glucose/mannose medium was fermented was nearly identical to that of the glucose control. Received 18 April 1996/ Accepted in revised form 27 August 1996     

Comparison of exopolysaccharide production by strains of Lactobacillus rhamnosus and Lactobacillus paracasei grown in chemically defined medium and milk

Exopolysaccharide (EPS) production was compared among three strains of lactobacilli. Lactobacillus rhamnosus strain 9595M can be classified among the highest EPS-producing strains of lactic acid bacteria reported to date with a maximum EPS production of 1275 mg L⁻¹. Under controlled pH, no significant differences in the quantity of EPS produced could be detected between carbon source (glucose or lactose) or fermentation temperature (32 or 37°C). In milk, strains ATCC 9595M and R produced more than 280 mg L⁻¹ EPS whereas strain Type V produced less than 80 mg L⁻¹ EPS. Journal of Industrial Microbiology & Biotechnology (2000) 24, 251–255. Received 10 September 1999/ Accepted in revised form 22 December 1999     

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     

The adverse effect of nitrogen limitation and excess-cellobiose on Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 cultures that were cellobiose-limited converted cellobiose to succinate and acetate, produced little glucose or cellotriose, maintained an intracellular ATP concentration of 4.1 mM and a membrane potential of 140 mV for 24 h, did not lyse at a rapid rate once they had reached stationary phase, and had a most probable number of viable cells that was greater than 10⁶/ml. When the cellobiose concentration was increased 6-fold (5 mM to 30 mM), ammonia was depleted and the cultures left 10 mM cellobiose. Cultures provided with excess cellobiose produced succinate and acetate while they were growing, but there was little increase in fermentation acids after the ammonia was depleted and growth ceased. The stationary-phase, cellobiose-excess cultures had a lysis rate that was 7-fold faster than that of the cellobiose-limited cultures, and the most probable number was only 3.3 × 10³ cells/ml. The stationary-phase, cellobiose-excess cultures had 2.5 times as much cellular polysaccharide as the cellobiose-limited cultures, but the intracellular ATP and membrane potential were very low (0.1 mM and 40 mV respectively). Methylglyoxal, a potentially toxic end-product of carbohydrate fermentation, could not be detected, and fresh inocula grew rapidly in spent medium that was supplemented with additional ammonia. Stationary-phase, cellobiose-excess cultures converted cellobiose to glucose and cellotriose, but the apparent K _m of cellotriose formation was 15-fold lower than the K _m of glucose production (0.7 mM compared to 10 mM). Received: 26 June 1997 / Received revision: 12 August 1997 / Accepted: 29 August 1997     

The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis

Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 ± 0.12 h⁻¹) than on glucose (1.67 ± 0.12 h⁻¹), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less␣intracellular fructose 1,6-bisphosphate (Fru1,6P ₂) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Fru1,6P ₂. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more than 1.2 h⁻¹) of S.␣bovis. Received: 3 June 1997 / Received revision: 10 September 1997 / Accepted: 6 January 1998     

Effects of culture conditions on β-poly(l-malate) production by Physarum polycephalum

Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO₃. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO₃, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C. Received: 12 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l⁻¹ exopolysaccharides with glucose and 30 mg l⁻¹ with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M _r) of 1.7 × 10⁶ and 4 × 10⁴ in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M _r of 4 × 10⁴. The high-M _r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M _r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M _r fractions appeared to be dependent on the carbohydrate source, whereas the low-M _r fractions were produced more continuously. Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997     

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation

 The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation. Received: 6 October 1999 / Accepted: 18 November 1999     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized first, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose. Received: 5 May 1998 / Received revision: 3 August 1998 / Accepted: 8 August 1998     

Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass

A Saccharomyces-cerevisiae-based simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass is limited to an operating temperature of about 37 °C, and even a small increase in temperature can have a deleterious effect. This points to a need for a more thermotolerant yeast. To this end, S. cerevisiae D₅A and a thermotolerant yeast, Candida acidothermophilum, were tested at 37 °C, 40 °C, and 42 °C using dilute-acid-pretreated poplar as substrate. At 40 °C, C. acidothermophilum produced 80% of the theoretical ethanol yield, which was higher than the yield from S.cerevisiae D₅A at either 37 °C or 40 °C. At 42 °C, C. acidothermophilum showed a slight drop in performance. On the basis of preliminary estimates, SSF with C. acidothermophilum at 40 °C can reduce cellulase costs by about 16%. Proportionately greater savings can be realized at higher temperatures if such a high-temperature SSF is feasible. This demonstrates the advantage of using thermophilic or thermotolerant yeasts. Received: 20 February 1997 / Received revision: 24 June 1997 / Accepted: 4 July 1997     

Characterization of salt-tolerant mutant for enhancement of l-threonine production in Escherichia coli

 Escherichia coli strain HS3, metabolically engineered to have Met^–, AHV^r, Ile^L and AEC^r characteristics, produced 58.0 g/l of l-threonine, but it was neither salt-tolerant nor osmotolerant; and the growth and threonine production of the strain were severely inhibited both by the addition of NaCl with a concentration higher than 2% and by the presence of glucose with a concentration higher than 10%. Therefore, salt-tolerant mutants were isolated. The salt-tolerant mutants, HS454 and HS528 which were derived from strain HS3, were both tolerant to salt (2%) and hyperproductive. The growth and l-threonine production by the mutant strain HS454 were almost unaffected by a glucose concentration lower than 10%, but gradually reduced with increasing glucose concentration, up to 15%. However, the mutant strain HS528 showed slightly enhanced growth and l-threonine production with increasing glucose concentration, up to 10–12.5%. Strains HS454 and HS528 produced 69.8 g/l and 74.0 g/l of l-threonine, respectively in a 5-l jar fermentor. Received: 21 January 2000 / Received revision: 31 March 2000 / Accepted: 5 May 2000     

Enhanced production of penicillin V acylase from Streptomyces lavendulae

At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G. Received: 22 March 1999 / Received revision: 16 June 1999 / Accepted: 20 June 1999     

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     

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     

Characterization of polygalacturonase from the brown-rot fungus Postia placenta

 Two extracellular isoenzymes of polygalacturonase, isolated from the brown-rot fungus Postia placenta, were purified 342-fold by Mono S cation-exchange chromatography. The temperature optimum ranged from 25 ^°C to 37 ^°C, and the pH optimum ranged from 3.2 to 3.9. Apparent pI values of the isoenzymes (3.2 and 3.4) were lower than any previously reported. The estimated molecular mass from a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (PAGE) was 34 kDa. Isoenzymes of polygalacturonase in native PAGE and isoelectric focusing gels were identified by substrate/ agar overlays (zymograms). Comparison of viscosity reduction rates with release of reducing sugars indicated that the enzyme from P. placenta is endo-acting. The objective of this study was to isolate polygalacturonase from the brown-rot fungus P. placenta and characterize the properties of the enzyme. Received: 31 October 1995/Received revision: 12 February 1996/Accepted: 4 March 1996     

Production of 2,3-butanediol by newly isolated Enterobacter cloacae

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of l-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars). Received: 17 December 1998 / Revision received: 9 March 1999 / Accepted: 20 March 1999