Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Studies on the Low Temperature Fermentation

An attempt has been made to isolate the bacteria capable of accumulating amino acids during the growth at low temperature from various natural sources. A psychrophilic strain P 145 forming glutamic acid at 5°C was obtained and identified as a Brevibacterium sp. The bacterium grew in the range of 0° to 37°C and exhibited the optimum growth at 15°C. The bacterium was defined as a facultative psychrophile.

The strain strictly required methionine only at above 28°C; below this temperature it grew normally without the amino acid. When methionine was added thiamine and biotin stimulated the growth of this strain at 28°C.

With the Brevibacterium sp. P 145 isolated from soil, the effect of incubation temperature on the extracellular amino acid accumulation has been examined from cultural and enzymological points of view. The strain was found to accumulate l-glutamic acid up to 5.88 mg/ml and l-alanine 0.38 mg/ml at 5°C, whereas it formed 0.21 mg/ml of l-glutamic acid and 2.54 mg/ml of l-alanine at 28°C.

The accumulation of l-alanine in the medium at 28°C seemed to be related to the thiamine requirement of the strain. In the case of thiamine deficiency, l-alanine was the main product in the culture at 28°C. When the incubation temperature was abruptly shifted from 28° to 5°C or from 5° to 28°C, the amino acid accumulation was also changed to that of the final temperature. l-Alanine dehydrogenase existed even in the cells grown at 5°C but was not active at this low temperature. These results were in accord with the informations obtained from cultural experiments.     

Amperometric estimation of BOD by using living immobilized yeasts

Summary A microbial electrode consisting of immobilized living whole cells of yeasts, porous membrane and an oxygen electrode was prepared for continuous estimation of biochemical oxygen demand (BOD). Immobilized Trichosporon cutaneum was employed for the microbial electrode sensor for BOD. When a sample solution containing the equivalent amount of glucose and glutamic acid was injected into the sensor system, the current of the electrode decreased markedly with time until steady state was reached. The response time was within 18 min. A linear relationship was observed between the current decrease and the concentration below 41 mg l ^– of glucose and 41 mg l ^– glutamic acid (5-day BOD 60 mg l ^–). The current decrease was reproducible within ± 6% of the relative error when a sample solution containing 27 mg l ^– of glucose and 27 mg l ^– of glutamic acid (5-day BOD 40 mg l ^–) was employed. The microbial electrode sensor was applied to untreated waste waters from a fermentation factory. Good comparative results were obtained between BOD estimated by the microbial electrode and that determined by the conventional 5-day method (regression coefficient was 1.2). Furthermore, the effect of various compounds on BOD estimation was also examined. The current output of the microbial electrode sensor was almost constant for 17 d and 400 tests.     

Co‐utilization of acidified glycerol pretreated‐sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co‐utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30 g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6 g/L glucose from glycerol‐pretreated bagasse and 23.4 g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7 g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5 mg/L carotenoid as a by‐product. It is expected that microbial oil production can be significantly improved through process optimization.     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.     

Production of extracellular polysaccharides by a Rhizobium species from the root nodules of Melilotus alba

The Rhizobium sp., isolated from the root nodules of the leguminous fodder herb Melilotus alba, produced large amounts of extracellular polysaccharides (EPS) (963.5 μg/ml) in a yeast extract mannitol medium. Growth and EPS production started simultaneously, but EPS production reached its maximum during the stationary phase of growth of the bacteria, at 20 hours. EPS production was increased with all of the thirteen sugars tested. Different nitrogen sources, such as nitrates, glutamic acid, casamino acid and L-asparagine, increased the EPS production although it was inhibited by glycine, nitrite and ammonium salts. Among the vitamins and metal ions, only pyridoxal phosphate and ZnSO₄ promoted EPS production. Attempts were made to optimize the cultural requirements for growth and maximum EPS production. Maximum EPS production (1457.0 μg/ml) was obtained when the medium was supplemented with glucose (1%), pyridoxal phosphate (2 μ g/ml), ZnSO₄ × 7 H₂O (10 μg/ml) and glutamic acid (0.1%). Under these conditions, the production was increased by 254.3% compared to the control. The EPS contained arabinose, xylose and rhamnose monomers. The presence of arabinose and xylose in the EPS produced by a Rhizobium sp. was uncommon.     

Selection of hypercellulylytic derepressed mutants of Cellulomonas sp.

Summary Mutants from Cellulomonas sp.IIbc were obtained combined treatment of UV light and N-methyl-N-nitrosoguanidine. T The selection criterion for the screening of catabolite-repression-resistant mutants was based on the formation of clear zones around the bacterial colonies in medium containing 0.5% Walseth cellulose and 0.5% glucose. Mutants produced not only clear zones in significantly lower times than the parent strain, but also exhibited higher specific growth rates and cellulolytic activity when grown on bagasse pith. The cellulase-derepressed character of the mutants was demonstrated by the presence of cellulolytic activity in cultures grown in the presence of high levels of glucose. These results raise the possibility of enhancing the productivity of bacterial degradation of lignocellulosic substrates for single cell protein production. Offprint requests to: F. Alea     

Biosynthesis and Hydrolysis of Poly(γ-glutamic acid) from Bacillus subtilis IF03335

Poly(γ-glutamic acid) (PGA) production in Bacillus subtilis IF03335 was studied. When citric acid as a carbon source was added to a glutamic acid medium containing L-glutamic acid and ammonium sulfate, a large amount of pure PGA was produced. On the other hand, when glucose was added to the glutamic acid medium, a by-product was produced, which seemed to be a polysaccharide. Moreover, the mode of hydrolysis was investigated with PGA in aqueous solutions at 80, 100, and 120°C by monitoring the time-dependent changes in the molecular weights. Hydrolytic degradation of PGA was found to proceed through a random chain scission.     

Single cell protein from bagasse pith by a mixed bacterial culture

A spontaneous association of Cellulomomas sp. with another bacterial strain was studied for its capabilities for single cell protein (SCP) production from bagasse pith. The associated strain was identified as Pseudomonas sp. and further characterized for its physiological properties. The effect of the initial proportions of both strains, the way of propagation, and the effect of pH on the growth of the mixed culture on bagasse pith was studied. Separate propagation of both strains before the fermentation step (“controlled mixed culture”), a range of proportions Cellulomonas-Pseudomonas from 4:1 to 100: 1, and pH 7.0, were found to be the most appropriate conditions of growth. A mutualistic symbiotic relationship was demonstrated to take place between both strains during the mixed growth on bagasse pith, the Cellulomonas supplying the carbon source (glucose produced from bagasse degradation) to the Pseudomonas, and the latter producing the vitamin supplements necessary for the Cellulomonas growth, allowing the growth of the mixed culture in a minimal medium, without any growth factor supplement. Fed-batch cultivation of the mixed culture on this substrate was successful, giving rise to high biomass production (19.4 g/l), thus increasing the productivity of the system. Due to its improved productivity, high biomass production, inexpensiveness of the culture medium, (without any vitamin supplement), and good stability, this culture presents economical advantages and constitutes an attractive choice for lignocellulosic substrate utilization.     

Neuronal and glial enzyme studies in cell culture

Summary Newborn BALB/c mouse brain was cultured as disaggregated cells after serial trypsin dissociations. The ontogeny of the cultures was followed by assays of cell number, deoxyribonucleic acid, and protein content and by the activities of three enzymes considered to be markers of neuronal differentiation. Aliquots of the freshly dissociated cells were assayed for choline acetylase, acetylcholinesterase, and glutamic acid decarboxylase activities and compared with intact brain. The percentages of recovery of activities, expressed as¹⁴C product formed per mg of protein per 10 min, at pH 6.8 and 37°C, were 37% for choline acetylase, 54% for acetylcholinesterase, and 24% for glutamic acid decarboxylase. The remainder of the freshly dissociated cells were placed into culture; enzyme assays were performed as the cells multiplied and then when the cultures became static. Choline acetylase activity increased as the cells rapidly divided, and glutamic acid decarboxylase activity increased only after the cultures became confluent. Under the culture conditions, acetylcholinesterase was not induced, despite active synthesis of acetylcholine. Neuroblastoma clone N18, C1300 cell line, was grown in cell culture, and the activity of acetylcholinesterase was measured as the cells multiplied and came to confluency. The specific activity of mouse neuroblastoma acetylcholinesterase increased 25-fold when the rate of cell division was restricted. The rate of cell division could be regulated by adjusting the serum concentration. By removing fetal calf serum during the growth period, cell division ceased, and acetylcholinesterase activity was significantly and rapidly induced. Choline-O-acetyltransferase specific activity was measured in rapidly dividing and in static cultures. Its specific activity was highest in nondividing cultures, compared to cultures containing actively dividing cells (6-fold), and the specific activity of thymidylate synthetase was increased 2.5-fold in actively dividing cultures, compared to static cultures. Glioblastoma cells obtained from the rat astrocytoma, clone C6, were grown in culture, and glucose metabolism was measured in control cultures, and in cultures containing norepinephrine (0.017 mg per ml). Norepinephrine produced a 50% inhibition in the incorporation ofd-[¹⁴C]glucose. Cells incubated for 2 hr in the presence ofd-[¹⁴C]glucose, washed and then incubated in control medium or in medium containing norepinephrine, resulted in the release of greater than 50% of radioactive metabolites in the norepinephrine treated plates. Norepinephrine caused a 50% increase in¹⁴CO₂ production in glioblastoma cells incubated withd-[1-¹⁴C]glucose. Norepinephrine, under similar conditions, did not affect the metabolism of glucose in clone C46, C1300 mouse neuroblastoma cells. Portions of this work were supported by a research grant (6-444946-58605) from the American Cancer Society.     

Production of Acetylacetoin by Bacterial Fermentation

Bacillus sp. YUF-4 did not produce acetylacetoin with general culture media, such as bouillon medium containing glucose or acetoin. When diacetyl was added to a medium cultured for 18–20 h in the presence of glucose or acetoin, AAC was produced as culture continued. AAC was assayed by GLC with a Carbowax 20M capillary column. The AAC produced was purified by several steps: the final HPLC using a Shodex E411 column was effective. The yield of AAC was 346 mg per liter of the medium (7.5% recovery) and the purity was 97%. AAC was identified by ¹H-NMR and ¹³C-NMR.     

Cyanuric acid biodegradation by a mixed bacterial culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. in a packed bed biofilm reactor

Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L⁻¹), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L⁻¹) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.     

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.     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Metal ion accumulation by immobilised cells of Brevibacterium sp

This paper explores the use of an experimental system based on polyacrylamide-entrapped cells of Brevibacterium sp strain PBZ for the removal of metal ions from solutions. Experiments were performed in columns filled with the immobilised cells and challenged with influents containing 20 mg L⁻¹ of lead and 10 mg L⁻¹ of cadmium. The cells were able to accumulate lead (about 40 mg g⁻¹ dry biomass) and, to a lesser extent, cadmium (about 13 mg g⁻¹ dry biomass) from solutions. In the presence of 0.4 g L⁻¹ of glucose, the cells removed up to 53% of lead. Lead competed with cadmium for attachment to the binding sites when a solution containing both the metals was applied. Lead removal occurred by a combination of fast physico-chemical adsorption and prolonged low rate accumulation mediated by cell metabolism. The biosorptive capacity of the cells was sensitive to pH. Desorption of the metal with EDTA restored the binding capability of the cells. Received 07 July 1997/ Accepted in revised form 26 November 1997     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

Emulsification and utilization of high-speed diesel by a <Emphasis Type="Italic">Brevibacterium </Emphasis>species isolated from hydraulic oil

Summary A contaminant of hydraulic oil has been isolated and identified using conventional microbiological and biochemical techniques. The PCR amplification and sequencing of 16S-rDNA indicated it to be a Brevibacterium sp. closely related to B. casei strain DSM 20657, based on sequence homology (98%). In view of being an oil contaminant, its ability of high speed diesel (HSD) emulsification and utilization have been studied and compared with two strains of Pseudomonas putida (MTCC2445 and Biotype-A MTCC 1274) and Pseudomonas fluorescens (MTCC670). After enrichment in minimal medium containing HSD as sole source of carbon Brevibacterium sp. (Met-1) was grown in Bushnell Haas broth containing 0.4% HSD (w/v). Bacterial growth at 28 °C, oil utilization, emulsification and surface active properties were determined after 5 days of incubation and compared with type cultures. All bacteria were found to grow well in the presence of HSD at the tested concentration. However, better utilization of HSD was observed in the Brevibacterium Met-1 isolate and Pseudomonas putida (MTCC 2445).     

Eicosapentaenoic and docosahexaenoic acids production by and okara-utilizing potential of thraustochytrids

Nine thraustochytrid strains isolated from subtropical mangroves were screened for their eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production potential in a glucose yeast extract medium. Their ability to utilize okara (soymilk residue) for growth and EPA and DHA production was also evaluated. EPA yield was low in most strains, while DHA level was high on glucose yeast extract medium, producing 28.1–41.1% of total fatty acids, for all strains, with the exception of Ulkenia sp. KF13. The DHA yield of Schizochytrium mangrovei strains ranged from 747.7 to 2778.9 mg/l after 52 h of fermentation at 25°C. All strains utilized okara as a substrate for growth, but DHA yield was lower when compared with fermentation in a glucose yeast extract medium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 199–202. Received 11 December 2000/ Accepted in revised form 29 June 2001     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

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.