Production of 2,3-Butylene Glycol from Citrus Wastes. II. The Bacillus polymyxa Fermentation

The conditions required for production of levo 2,3-butylene glycol by Bacillus polymyxa from citrus molasses were studied. Starter cultures required acclimatization to the substrate prior to inoculation of the fermentation medium. Maximal production of butylene glycol with minimal residual sugar was obtained with a medium consisting of molasses, diluted to 20 degrees Brix, and 0.4% urea. Optimal environmental conditions included aeration at 0.11 volumes of air per volume of medium per minute, maintenance of pH at 6.0 to 6.2, a fermentation temperature of 30 C, and a stirring rate of 420 rev/min. The concentration of butylene glycol obtained in the fermentation beer ranged from 2.3 to 4.4%. The optical rotation of the glycol ranged from [alpha](D) (23 degrees ) = -1.01 degrees to -10.45 degrees . The variation in rotation was probably due to the presence of contaminating substances in the distillate.     

Biosurfactant production using molasses and whey under thermophilic conditions

Biosurfactant production was studied by Bacillus licheniformis K51, B. subtilis 20B, B. subtilis R1 and Bacillus strain HS3 using molasses or cheese whey as a sole source of nutrition at 45 degrees C. The isolates were able to grow and produce biosurfactant under shaking as well as static conditions. Maximum biosurfactant production was achieved with molasses at 5.0-7.0% (w/v). The biosurfactant retained its surface-active properties after incubation at 80 degrees C at a wide range of pH values and salt concentrations for nine days. Oil displacement experiments in sand pack columns with crude oil showed 25-33% recovery of residual oil.     

Preservation of crab or shrimp waste as silage for cattle

Five experiments were conducted to either ferment fresh shrimp or crab waste with molasses, molasses and bacterial inoculant, or to preserve it with salt. Experiment 1 was a 4 × 2 factorial arrangement. Crab waste was combined with 0, 5, 10, or 15% liquid molasses, and stored in mini-silos (15 l) with or without lids for 14 days. The addition of molasses slightly decreased pH and offensive odors; mini-silo temperatures without lids were higher than those with lids. Experiment 2 was a 5 × 2 factorial arrangement designed to enhance fermentation. Fresh shrimp waste was combined with 0, 10, 15, 20, or 25% dry molasses and 0 or 1.0 × 10⁸ colony forming bacteria/g inoculant and ensiled for six days. As the level of molasses increased, dry matter and lactic acid increased but, the pH, crude protein, ammonia acetic, butyric, and propionic acid concentrations decreased. Significant molasses by inoculant interactions occurred which were highly variable for each acid. Evidence of fermentation was supported by production of lactic acid at all levels of molasses. The pH decreased from 7.7 in the untreated waste to an average of 7.4 for the 10, 15 and 20% molasses treated wastes to 6.8 in the 25% molasses treated waste. The high pH was an indication that the waste may be unstable with longer storage (> 6 days). Therefore, in Experiment 3, designed as a 2 × 2 factorial arrangement, shrimp waste treated with 15 and 20% molasses, with or without inoculant was ensiled for 21 days to test stability. By day 21, shrimp waste had deteriorated as indicated by a mean pH of 7.5, low lactic acid, and high butyric acid concentration, an unacceptable odor, and the presence of mold on the surface of the samples.In Experiments 4 and 5, shrimp or crab waste was combined with salt at 0, 2.5, 5.0, 7.5, 10.0, and 12.5%. Increasing levels of salt decreased crude protein percent, ammonia concentration, and lactic and volatile fatty acids while increasing the pH and improving the acceptability of the odors in both the shrimp and crab wastes. Treatment of crustacean waste with 7.5% or greater salt was more effective at preserving crude protein and minimizing odor than either dry or liquid molasses.     

Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.     

Effect of controlled lactic acid bacterial fermentation on the microbiological and chemical qualities of Moroccan sardines (Sardina pilchardus)

Lactic acid bacteria (LAB) strains were assayed for the conservation of fresh sardine "Sardina pilchardus". Lactobacillus delbrueckii subsp. delbrueckii was used for inoculation of sardine fillets in a solution of NaCl (5%, w/w) and glucose (4%, w/w) concentration in water. Microbial counts including Standard Plate Count (SPC), LAB, yeasts, coliforms, Salmonella, staphylococci and Clostridium were followed during two weeks of storage at 30 degrees C. Determinations of chemical parameters including pH, dry matter, fat, ash, total nitrogen (NT), total volatile basic nitrogen (TVBN) and trimethylamine (TMA) were carried out under the same conditions. Chemical determinations showed a net pH decrease from an initial value of 6.05 in raw sardine fillets to 4.3 after 16 days of fermentation. Increases in TMA content and TBVN were observed. Microbiological control showed that LAB counts reached a level up to 3.10(9) cfu/g after 4 days of fermentation. After two weeks, fermented fish was free of coliforms and Salmonella. The inhibition of pathogenic microflora including staphylococci and Clostridium was also observed. The results indicated that controlled LAB fermentation could be used as a successful process for biopreservation of sardines produced in huge quantities in Morocco.     

Viricidal effects of Lactobacillus and yeast fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40 degrees C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30 degrees C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20 degrees C but was inactivated more rapidly at 30 and 40 degrees C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20 degrees C and day 6 at 30 degrees C. The porcine picornavirus was inactivated by day 7 at 30 degrees C but survived the entire test period at 20 degrees C. Frog virus 3 was inactivated by day 3 at 20 degrees C and day 2 at 30 degrees C.     

Protein enrichment of potato processing waste through yeast fermentation

Potato starch obtained from waste waters of chips manufacturing was used as a fermentation substrate for yeast protein enrichment. Among 18 yeast strains, 6 strains were screened according to their biomass yield and protein content after fermentation for 16 h at 30 degrees C in an aerated glucose-based liquid media (4.5 Ls). Using concentrated media (25% solids) made from potato starch pre-hydrolyzed with malt flour and batch-fermented for 20 h at 26 degrees C under aerobic conditions, Candida utilis ATCC 9256 was the most efficient protein-forming strain. Scaled-up at the 100 Ls level, the aerobic batch process was improved under fed-batch conditions with molasses supplementation. After drying, fermented starch contained 11-12% protein, including 7-8% yeast protein.     

Clostridium botulinum type A growth and toxin production in media and process cheese spread

We found that Clostridium botulinum type A grew well and produced toxin in media with a water activity (a(w)) of 0.972 or 0.965 and a pH of 5.7, but no growth or toxin production was observed at or below an a(w) of 0.949 during incubation at 30 degrees C for 52 to 59 days. a(w) and pH values of media were adjusted to those of cheese spreads commercially produced. Solutes used to adjust a(w) included combinations of NaCl, cheese whey powder, emulsifying salt, sodium tripolyphosphate, and glycerol. In agreement with results obtained for media, toxin was produced in samples of cheese spread (a(w), 0.970; pH, 5.7) at 30 to 70 days of incubation at 30 degrees C.     

Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation

Effects of medium components and culture conditions on biomass and lipid production of Trichosporon fermentans were studied. The optimal nitrogen source, carbon source and C/N molar ratio were peptone, glucose and 163, respectively. The favorable initial pH of the medium and temperature were 6.5 and 25 degrees C. Under the optimized conditions, a biomass of 28.1 g/l and a lipid content of 62.4% could be achieved after culture for 7 days, which were much higher than the original values (19.4 g/l and 50.8%) and the results reported by other groups. T. fermentans could grow well in pretreated waste molasses and a lipid yield of 12.8 g/l could be achieved with waste molasses of 15% total sugar concentration (w/v) at pH 6.0, representing the best result with oleaginous microorganisms on agro-industrial residues. Addition of various sugars to the pretreated molasses could efficiently enhance the accumulation of lipid and the lipid content reached as high as above 50%. Similar to vegetable oils, the lipid mainly contains palmitic acid, stearic acid, oleic acid and linoleic acid and the unsaturated fatty acids amount to about 64% of the total fatty acids. The microbial oil with an acid value of 5.6 mg KOH/g was transesterified to biodiesel by base catalysis after removal of free fatty acids and a high methyl ester yield of 92% was obtained.     

Presence of nitrosable mutagen precursors in cooked meat and fish

Broiled chicken, pork, mutton, beef and sun-dried sardine were found to yield direct-acting mutagenicity after nitrite treatment. When 50% methanol extracts of cooked foods were treated with 50 mM nitrite at pH 3 for 1 h at 37 degrees C, they induced 3800-17,900 revertants of Salmonella typhimurium TA100 and 15,000-43,600 revertants of TA98 per g. In contrast, raw meat and uncooked sun-dried sardine showed little or no mutagenicity after nitrite treatment. Treatment of broiled chicken with 0.5-3 mM nitrite, which is a physiologically feasible concentration in the human stomach under some conditions, induced direct-acting mutagenicity. When broiled chicken was treated with 1 mM nitrite at pH 3 for 1 h at 37 degrees C, its mutagenicities on TA100 and TA98 without S9 mix were 7100 and 5400 revertants/g, respectively.     

Isolation of thermotolerant ethanologenic yeasts and use of selected strains in industrial scale fermentation in an Egyptian distillery

An enrichment and isolation program for new ethanol-producing thermotolerant yeasts as well as a screening program of some known thermotolerant strains resulted in the selection of several strains capable of growth at 40-43 degrees C. Among these strains four grew and fermented sugar cane molasses at 43 degrees C under batch conditions with sugar-conversion efficiencies >94% and ethanol concentrations 6.8-8.0% (w/v). The two best-performing strains, a Saccharomyces cerevisiae F111 and a Kluyveromyces marxianus WR12 were used in eight 87.5 m(3) fermentation runs (four using each strain) for industrial ethanol production in an Egyptian distillery using sugar cane molasses. Mean ethanol production was 7.7% and 7.4% (w/v), respectively, with an added advantage of cooling elimination during fermentation and higher ethanol yields compared to the distillery's S. cerevisiae SIIC (ATCC 24855) strain in use. The isolate S. cerevisiae F111 was subsequently adopted by the distillery for regular production with significant economical gains and water conservation.     

Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses

Fresh, defrosted and delignified brewer's spent grains (BSG) were used as yeast supports for alcoholic fermentation of molasses. Glucose solution (12%) with and without nutrients was used for cell immobilization on fresh BSG, without nutrients for cell immobilization on defrosted and with nutrients for cell immobilization on delignified BSG. Repeated fermentation batches were performed by the immobilized biocatalysts in molasses of 7, 10 and 12 initial Baume density without additional nutrients at 30 and 20 degrees C. Defrosted BSG immobilized biocatalyst was used only for repeated fermentation batches of 7 initial Baume density of molasses without nutrients at 30 and 20 degrees C. After immobilization, the immobilized microorganism population was at 10(9) cells/g support for all immobilized biocatalysts. Fresh BSG immobilized biocatalyst without additional nutrients for yeast immobilization resulted in higher fermentation rates, lower final Baume densities and higher ethanol productivities in molasses fermentation at 7, 10 and 12 initial degrees Be densities than the other above biocatalysts. Adaptation of defrosted BSG immobilized biocatalyst in the molasses fermentation system was observed from batch to batch approaching kinetic parameters reported in fresh BSG immobilized biocatalyst. The results of this study concerning the use of fresh or defrosted BSG as yeast supports could be promising for scale-up operation.     

Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11

An alkalophilic, environmental micro-organism, Bacillus sp. BG-11, has been isolated and characterized. It produced 76 U ml-1 of chitinase in liquid batch fermentation after 72 h of incubation at 50 degrees C using chitin-enriched medium. The molecular weight of purified chitinase was estimated to be 41 kDa by SDS-PAGE. The pH and temperature optima of chitinase immobilized on chitosan and calcium alginate were 8.5 and 50 degrees C, respectively, which were same as that of free enzyme. The pH and thermostability of immobilized chitinase were enhanced significantly. The chitinase immobilized on chitosan was stable between pH 5.0 and 10.0, and the half-life of chitosan-immobilized enzyme at 70, 80 and 90 degrees C was 90, 70 and 60 min, respectively. The end-products formed during the enzyme-substrate reaction were identified by 13C-NMR, and N-acetyl-D-glucosamine was found to be the major end-product. GlcNAc (GlcNAc)2 and (GlcNAc)3 inhibited the chitinase activity by 32, 25 and 18%, respectively, at a concentration of 10 mmol l-1. The shelf-life of chitinase (retained 100% activity) at 4 degrees C was 8 weeks in the presence of either sodium azide (100 microgram ml-1), sodium metabisulphite (0.1% w/v) or KCl (15% w/v). The enzyme was resistant to the action of proteases and allosamidin.     

Growth of the salt-tolerant yeast Zygosaccharomyces rouxii in microtiter plates: effects of NaCl,pH and temperature on growth and fusel alcohol production from branched-chain amino acids

Zygosaccharomyces rouxii, a salt-tolerant yeast isolated from the soy sauce process, produces fusel alcohols (isoamyl alcohol, active amyl alcohol and isobutyl alcohol) from branched-chain amino acids (leucine, isoleucine and valine, respectively) via the Ehrlich pathway. Using a high-throughput screening approach in microtiter plates, we have studied the effects of pH, temperature and salt concentration on growth of Z. rouxii and formation of fusel alcohols from branched-chain amino acids. Application of minor variations in pH (range 3-7) and NaCl concentrations (range 0-20%) per microtiter plate well allowed a rapid and detailed evaluation of fermentation conditions for optimal growth and metabolite production. Conditions yielding the highest cell densities were not optimal for fusel alcohol production. Maximal fusel alcohol production occurred at low pH (3.0-4.0) and low NaCl concentrations (0-4%) at 25 degrees C. At pH 4.0-6.0 and 0-18% NaCl, considerable amounts of alpha-keto acids, the deaminated products from the branched-chain amino acids, accumulated extracellularly. The highest cell densities were obtained in plates incubated at 30 degrees C. The results obtained under various incubation conditions with (deep-well) microtiter plates were validated in Erlenmeyer shake-flask cultures.     

Slaughterhouse by-products preserved by Lactobacillus plantarum fermentation as feed for mink and foxes

Lactic acid fermentation was evaluated as a method to preserve abattoir waste for use in fur animal diets. The method used involved grinding, acidifying to pH 5–5.2 by formic acid and propionic acid, addition of 6% molasses as a carbohydrate source and a starter culture of Lactobacillus plantarum. Fermentation was completed after 2–3 days at 25°C. The final pH of the fermented product was 3.8–4.1.

Storage experiments revealed satisfactory stability. The fermented products could be kept for weeks at room temperature (20°C) and for months in a cold room (4°C).

The amino acid composition of the 4 types of abattoir waste tested was not significantly changed by fermentation. Digestibility studies with mink revealed slight, but significant (P < 0.05), effects of fermentation. The digestibility of cystine and threonine was reduced and that of glycine and proline increased.

Two types of fermented abattoir waste were investigated in 2 long-term feeding experiments with mink and blue foxes. In the mink trial, diets with 10 or 20% fermented abattoir waste supported normal reproduction, kit mortality and body growth. In one experiment, there was a significant reduction in mink kit body weights with 20%, but not with 10% fermented abattoir waste in the diet. In the fox trial, litter sizes and kit viability tended to improve with 20% fermented abattoir waste, while kit body weights were unaffected. It is concluded that fermentation could be an acceptable process for the preservation of abattoir waste intended for the feeding of fur animals.     

Effect of incubation atmosphere and temperature on isolation of Campylobacter jejuni from human stools

To determine the optimal conditions for isolation of Campylobacter jejuni from human fecal specimens, we compared incubation atmospheres that contained about 5, 10, and 15% oxygen with the 17% oxygen produced in candle jars and also compared incubation temperatures of 37 and 42 degrees C. At 42 degrees C, C. jejuni was isolated from all 16 specimens; however, colony sizes were larger when plates were incubated in 5 and 10% oxygen than in the other two atmospheres. At 37 degrees C some positive cultures were missed in 15% oxygen and in the candle jar. The largest colony sizes were obtained in 5% oxygen. For each atmospheric condition tested, the colonies were larger at 42 than at 37 degrees C. When incubation is done at 42 degrees C, use of a candle jar is adequate; however, at 37 degrees C candle jars should not be used for isolation of C. jejuni from human feces.     

Production of β-carotene from deproteinized waste whey filtrate using Mucor azygosporus MTCC 414 in submerged fermentation

The cheese whey, a by-product of dairy industry proved to be an attractive substrate for production of β-carotene. The β-carotene production from Mucor azygosporus MTCC 414 by using deproteinized waste whey filtrate under submerged fermentation was investigated. Various fermentation variables, such as lactose content in whey, initial pH, production temperature, incubation time, and carbon and nitrogen sources played significant role on β-carotene production. Maximum β-carotene production (385 μg/g dcw) was obtained with the whey (pH 5.5) containing 3.5% (w/v) lactose supplemented with soluble starch at (1.0%, w/v) at 30°C after a 5 days incubation. Moreover, unlike other microorganisms which utilize pre-hydrolyzed lactose, this Mucor azygosporus MTCC 414 was found to be capable of utilizing unhydrolyzed lactose present in the whey.     

Fermentation of molasses by Zymomonas mobilis: effects of temperature and sugar concentration on ethanol production

Fermentations utilizing strains of Zymomonas mobilis, in place of the traditional yeasts, have been proposed due their ethanol yields being close to theoretical. Ethanol production from sugar cane molasses was analyzed under different culture conditions using Z. mobilis in batch fermentation. The total reducing sugars (TRS) concentrations in the molasses, temperature, agitation and culture time effects were studied simultaneously through factorial design. The best conditions for ethanol production were 200 g L(-1) of total reducing sugars in the molasses, temperature of 30 degrees C and static culture and time of fermentation of 48 h, achieving 55.8 g L(-1). The pH of the medium was kept constant during the experiments, showing that molasses presents a buffering effect.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.