Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of L-lactic acid

Conversion of lignocellulose to lactic acid requires strains capable of fermenting sugar mixtures of glucose and xylose. Recombinant Escherichia coli strains were engineered to selectively produce L-lactic acid and then used to ferment sugar mixtures. Three of these strains were catabolite repression mutants (ptsG ⁻) that have the ability to simultaneously ferment glucose and xylose. The best results were obtained for ptsG ⁻ strain FBR19. FBR19 cultures had a yield of 0.77 (g lactic acid/g added sugar) when used to ferment a 100 g/l total equal mixture of glucose and xylose. The strain also consumed 75% of the xylose. In comparison, the ptsG ⁺ strains had yields of 0.47–0.48 g/g and consumed 18–22% of the xylose. FBR19 was subsequently used to ferment a variety of glucose (0–40 g/l) and xylose (40 g/l) mixtures. The lactic acid yields ranged from 0.74 to 1.00 g/g. Further experiments were conducted to discover the mechanism leading to the poor yields for ptsG ⁺ strains. Xylose isomerase (XI) activity, a marker for induction of xylose metabolism, was monitored for FBR19 and a ptsG ⁺ control during fermentations of a sugar mixture. Crude protein extracts prepared from FBR19 had 10–12 times the specific XI activity of comparable samples from ptsG ⁺ strains. Therefore, higher expression of xylose metabolic genes in the ptsG ⁻ strain may be responsible for superior conversion of xylose to product compared to the ptsG ⁺ fermentations. Received 14 December 2000/ Accepted in revised form 28 June 2002     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

Engineering industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for xylose fermentation and comparison for switchgrass conversion

Saccharomyces’ physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04–0.17 h⁻¹. Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17–0.31 g/l/h, with ethanol yields 0.18–0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13–17% more ethanol than the parental control strains because of their ability to ferment xylose.     

Glutamic acid production byArthrobacter globiformis

Two hundred and fiftyArthrobacter strains were tested in a basal salts-glucose medium for their ability to produce glutamic acid; 50 strains produced small amounts of glutamic acid and alanine, as well as traces of other amino acids. Five biotin-dependent strains produced extraordinarily large amounts of glutamic acid. One of these, which was identified asA. globiformis, was selected for further study. Glutamic acid was only produced by this organism at biotin levels suboptimal for growth; maximal production (0.45 moles of glutamic acid per mole of glucose consumed) occurred at a biotin level of 10^–5 µg/ml. Other factors which markedly influenced glutamic acid production were temperature, (NH₄)₂SO₄ concentration, and pH of the growth medium.The taxonomy of glutamic acid-producing bacteria and the correlation between biotin deficiency and glutamic acid production are discussed.     

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden–Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient␣anaerobic fermentation of this pentose. l-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under ‘academic’ conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.     

Acetone-butanol fermentation of xylose and sugar mixtures

Summary Three strains ofCl. acetobutylicum and one ofCl. butyricum have been tested for their ability to ferment xylose to butanol. ATCC 824 and NRRL 527 produced 0.28 g solvents/g xylose, while ATCC 8260 and NRRL 594 produced much butyric acid. In 2-stage fermentations in which ATCC 8260 or NRRL 594 acted upon xylose for 12 to 20 h, followed by NRRL 527 for a total of 3 days, yields of solvent were better, 0.32 g/g xylose. Upon fermenting a mixture of sugars simulating sulphite waste liquor 0.36 g solvents/g sugar were obtained. Sugar consumption in both cases was about 96%.     

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

Differentiation of Bacteroides from Sphaerophorus and Fusobacterium

This paper is concerned with new findings in the comparative study of the genera, Bacteroides, Sphaerophorus and Fusobacterium. Victoria blue 4R and barbiturates were found to be effective in differentiating the genera by their discriminative inhibition of the growth. A new character, glutamic acid decarboxylation, was found in Bacteroides but neither in Sphaerophorus nor in Fusobacterium. The procedure for this test was presented. In the classification of newly isolated fecal strains this test and other differential features were compared. An improved method for the threonine deamination test was reported. The use of these characters and tests would be a step in the improvement of the present incomplete classification of gram-negative anaerobic non-sporulating rods.     

In vitro fermentation of prebiotics by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> CFR 2194: selectivity,viability and effect of metabolites on β-glucuronidase activity

Prebiotic Fructooligosaccharides (FOS) escape metabolism in upper GI tract undergo microbial metabolism in colon and thereby influence the nature, type and number of intestinal microbiota to improve host’s health. The present study focuses on the ability of Lactobacillus plantarum CFR 2194 to utilize FOS as a selective carbon and energy source. The effect of fermentative metabolites of L. plantarum on the β-glucuronidase was also investigated. A total of 16 strains of lactobacilli were assessed for their ability to ferment oligosaccharides. L. plantarum CFR 2194, an isolate from kanjika was found to utilize FOS effectively. Lactic acid was the main metabolic end product, followed by acetic acid, butyric acid, formic acid and ethanol. The inhibitory effects of these metabolites have been confirmed through the reduction of β-glucuronidase activity. L. plantarum when co-cultured with β-glucuronidase producing E. coli, in a basal media containing FOS as an energy source, could inhibit the growth of the pathogen during the course of fermentation. The results showed that L. plantarum CFR 2194 has the ability to utilize the prebiotic FOS as a selective carbon and energy source. The organism could inhibit the growth of the pathogen which produces β-glucuronidase and lowered its activity by the metabolites of FOS which indicates the probable use of L. plantarum through dietary intervention in combating colon carcinogenesis.     

Thermophilic anaerobic bacteria which ferment hemicellulose: characterization of organisms and identification of plasmids

Seven thermophilic anaerobic bacteria which ferment xylan were isolated from natural geothermal features in the western United States. Typically, these strains were Gram-negative non-sporeforming rods with an unusual double-layered cell wall which resembled that observed in Thermobacteroides acetoethylicus. The strains differed from known thermophilic anaerobes in their ability to utilize a very wide variety of carbohydrates and in their ability to grow in a chemically-defined medium and/or at pH 3.5. Four of the strains contained cryptic plasmids of 1.2 or 1.5×10⁶ daltons. The taxonomic characteristics of the strains are discussed in terms of their relatedness to those of Thermoanaerobium, Thermoanaerobacter, and Thermobacteroides species.Contribution No. 3222 of the Central Research and Development Department     

Changes of Curdlan Biosynthesis and Nitrogenous Compounds Utilization Characterized in <Emphasis Type="Italic">ntrC</Emphasis> Mutant of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

The regulatory function of global regulator NtrC on curdlan biosynthesis and nitrogen consumption under nitrogen-limited condition in Agrobacterium sp. ATCC 31749 was investigated. The ntrC mutant of Agrobacterium sp. was constructed by homologous recombination. The ability to utilize NH₄Cl and KNO₃ was impaired in the mutant. Other nitrogenous compounds, such as glutamic acid and glutamine, were utilized normally. Curdlan production capability was impaired severely in the mutant. Curdlan production was 5-fold lower than the wild type strain in batch fermentation with NH₄Cl as the sole nitrogen source. However, up to 6.5 g l⁻¹ of a newly found alkali-insoluble biopolymer was produced by the ntrC mutant when glutamic acid was used as nitrogen source. The new biopolymer had glycosidic bond and hydroxyl group but no β-configuration absorption peak on IR spectrum was found as different from curdlan. In addition, the mutant exhibited a rapid morphological change from the dot to rod form. These results deduced that the global regulator NtrC was involved in curdlan and other biopolymer biosynthesis in Agrobacterium sp. ATCC 31749 in response to nitrogen-limited condition.     

Changes in cell volume,growth and respiration rate in response to hyperosmotic stress of NaCl,sucrose and glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum

Responses to hyperosmotic shock in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032 were studied in batch and continuous culture. The strains were chosen because they are used in commercial production of lysine and glutamic acid. Both strains, as well as the wild type of B. lactofermentum, were able to grow at high osmotic stress, at least 3 osmol/kg, from NaCl, sucrose, glutamic acid or lysine. The specific growth rate decreased in a nearly linear fashion with increasing stress. However, low stress from glutamic acid stimulated growth, especially in the wild type of B. lactofermentum. Both cell and cytoplasmic volume decreased spontaneously after hyperosmotic shock and no plasmolysis was observed. Addition of betaine stimulated the subsequent increase in the volumes. The volumes decreased linearly with increasing stress, except at low glutamic acid stress, which caused a volume increase. The respiration rate, measured as CO₂ evolution, decreased immediately after shock, but increased again and stabilized within 2 h. Betaine stimulated the respiration recovery rate.     

BIOTYPING CONFIRMS A NEARLY CLONAL POPULATION STRUCTURE IN ESCHERICHIA COLI

A. reference collection of 72 natural isolates of Escherichia coli (the ECOR collection) has been examined with respect to eight metabolic capabilities (biotype characters) plus motility and resistance or sensitivity to five common antibiotics. Data from biotype characters were analyzed by means of unweighted pair-group cluster analysis, and the genetic variation among the strains defines three major clusters of strains with substantial variation within each cluster but greater genetic similarity of strains within a cluster than between clusters. These clusters define an infraspecific population structure in E. coli, which reflects the predominantly clonal mode of reproduction in this organism. The clusters identified by the biotype characters are in good agreement with those resulting from an analysis of 11 enzyme polymorphisms (allozymes) among the strains, and these are in good agreement with the infraspecific structure detected by factor analysis of allozyme data. The clusters of strains also differ in several genetic characteristics that are independent of those used in making the classification.     

Lactic acid bacteria in Hamei and Marcha of North East India

Hamei and Marcha are mixed dough inocula used as starters for preparation of various indigenous alcoholic beverages in Manipur and Sikkim in India, respectively. These starters are traditionally prepared from rice with wild herbs and spices. Samples of Hamei and Marcha, collected from Manipur and Sikkim, respectively, were analysed for lactic acid bacterial composition. The population of lactic acid bacteria (LAB) was 6.9 and 7.1 Log cfu/g in Hamei and Marcha, respectively. On the basis of phenotypic and genotypic characters, LAB strains isolated from Hamei and Marcha were identified as Pediococcus pentosaceus, Lactobacillus plantarum and Lactobacillus brevis. Technological properties of LAB such as antimicrobial properties, effect on acidification, ability to produce biogenic amines and ethanol, degree of hydrophobicity and enzymatic activities were also performed. Pediococcus pentosaceus HS: B1, isolated from Hamei, was found to produce bacteriocin. None of the strains produced biogenic amines. LAB strains showed a strong acidifying ability and they also produced a wide spectrum of enzymes.     

Variation in the fermentative pattern of some Saccharomyces species

Summary The acquisition of the ability to ferment galactose, maltose, sucrose, raffinose and melibiose by yeast strains of the genus Saccharomyces was investigated. During cultivation in selective media 11 strains belonging to 5 species gained the ability to ferment one or several of these sugars. De-adaptation was not usually observed after cultivation in glucose medium, indicating that the saltants are stable in this medium.     

Comparative use of amino acids by three auxotypes of Neisseria gonorrhoeae

Auxotypes of Neisseria gonorrhoeae are usually distinguishable by their particular requirements for growth; these requirements often include amino acids. It is possible that strains needing particular substrates to grow can be distinguished not merely by their growth requirements but also by their metabolism of these particular substrates. In this work amino acid utilization and oxidation studies were performed enabling prototype, pro- and thia-strains to be distinguished. The metabolism study also underlined the importance of proline as an energy source and pointed to the probability of distinct relationships with the metabolism of the key amino acids, glutamic and aspartic acids, for the three auxotypes.It is proposed that the specific amino acid required by the naturally occurring auxotype serves as an energy source at the site of infection and has important implications with respect to particular auxotypes at various sites.     

Fermentation of Fructooligosaccharides by Lactic Acid Bacteria and Bifidobacteria

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.     

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     

Dekkera anomala sp. nov., the teleomorph of Brettanomyces anomalus,recovered from spoiled soft drinks

A new ascogenous yeast species, Dekkera anomala, is described. The strains studied were isolated from spoiled soft drinks. This species differs morphologically and physiologically from the species presently accepted in the genus Dekkera by the formation of blastese and the ability to ferment lactose.