Solid-state fermentation of carob pods for ethanol production

The production of ethanol from carob pods by Saccharomyces cerevisiae in solid-state fermentation was investigated. The maximal ethanol concentration (160±3 g/kg dry pods), ethanol productivity (6.7 ± 0.2 g/kg per hour), ethanol yield (40 ± 1.8%), biomass concentration (7.5 ± 0.4 x 10⁸ cells/g carob pulp) and fermentation efficiency (80 ± 2%) were obtained at an inoculum amount of 3%, a particle size of 0.5 mm, a moisture level of 70%, a pH of 4.5 and a temperature of 30°C. Under the same fermentation conditions both sterilized and non-sterilized carob pods pulp gave the same maximum ethanol concentration.     

EVALUATION OF BIOETHANOL PRODUCTION FROM CAROB PODS BY Zymomonas mobilis AND Saccharomyces cerevisiae IN SOLID SUBMERGED FERMENTATION

Bioethanol production from carob pods has attracted many researchers due to its high sugar content. Both Zymomonas mobilis and Saccharomyces cerevisiae have been used previously for this purpose in submerged and solid-state fermentation. Since extraction of sugars from the carob pod particles is a costly process, solid-state and solid submerged fermentations, which do not require the sugar extraction step, may be economical processes for bioethanol production. The aim of this study is to evaluate the bioethanol production in solid submerged fermentation from carob pods. The maximum ethanol production of 0.42 g g^?1 initial sugar was obtained for Z. mobilis at 30°C, initial pH 5.3, and inoculum size of 5% v/v, 9 g carob powder per 50 mL of culture media, agitation rate 0 rpm, and fermentation time of 40 hr. The maximum ethanol production for S. cerevisiae was 0.40 g g^?1 initial sugar under the same condition. The results obtained in this research are comparable to those of Z. mobilis and S. cerevisiae performance in other culture mediums from various agricultural sources. Accordingly, solid submerged fermentation has a potential to be an economical process for bioethanol production from carob pods.     

Improvement of Carob Pods as Feed by Solid-substrate Fermentation

Chopped carob pods were autoclaved in an ammonium salt solution. The solution was absorbed by the pods during autoclaving. The material was inoculated with either Rhizopus oligosporus or Monascus ruber and incubated for 3 d at 30 °C. The growing mycelium penetrated the particles to form a cake-like structure containing ca. 7% protein. The fermentation product contained 73–83% of the original carob sugars, whereas the amount of tannins was considerably lower than in the pods.     

Study of docosahexaenoic acid production by the heterotrophic microalga <Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis> CCMP 316 using carob pulp as a promising carbon source

In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation product.     

Kinetics of sugars consumption and ethanol inhibition in carob pulp fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in batch and fed-batch cultures

The waste materials from the carob processing industry are a potential resource for second-generation bioethanol production. These by-products are small carob kibbles with a high content of soluble sugars (45–50%). Batch and fed-batch Saccharomyces cerevisiae fermentations of high density sugar from carob pods were analyzed in terms of the kinetics of sugars consumption and ethanol inhibition. In all the batch runs, 90–95% of the total sugar was consumed and transformed into ethanol with a yield close to the theoretical maximum (0.47–0.50 g/g), and a final ethanol concentration of 100–110 g/l. In fed-batch runs, fresh carob extract was added when glucose had been consumed. This addition and the subsequent decrease of ethanol concentrations by dilution increased the final ethanol production up to 130 g/l. It seems that invertase activity and yeast tolerance to ethanol are the main factors to be controlled in carob fermentations. The efficiency of highly concentrated carob fermentation makes it a very promising process for use in a second-generation ethanol biorefinery.     

Solid-substrate fermentation ofKloeckera apiculata andCandida utilis on apple pomace to produce an improved stock-feed

Apple pomace was used in solid-substrate fermentation with the yeastsKloeckera apiculata orCandida utilis Y15. A total crude protein content of 7.5% (w/w) was achieved after 72 h for each yeast. The concentration of essential amino acids in the modified apple pomace was more than twice that in the control, enhancing its nutritive value as a stock-feed supplement. The fermentation of pomace as described can be used to reprocess this waste material into a useful value-added product for the agricultural sector.H. Rahmat was and R.A. Hodge, G.J. Manderson and P.L. Yu are with the Biotechnology Group, Department of Process and Environmental Technology, Massey University, Palmerston North, New Zealand; H. Rahmat is now with the Department of Chemical Engineering, Queen's University of Belfast, Belfast BT9 5AG, Northern Ireland, UK.     

Mathematical modeling of ethanol production in solid-state fermentation based on solid medium’ dry weight variation

In this work, mathematical modeling of ethanol production in solid-state fermentation (SSF) has been done based on the variation in the dry weight of solid medium. This method was previously used for mathematical modeling of enzyme production; however, the model should be modified to predict the production of a volatile compound like ethanol. The experimental results of bioethanol production from the mixture of carob pods and wheat bran by Zymomonas mobilis in SSF were used for the model validation. Exponential and logistic kinetic models were used for modeling the growth of microorganism. In both cases, the model predictions matched well with the experimental results during the exponential growth phase, indicating the good ability of solid medium weight variation method for modeling a volatile product formation in solid-state fermentation. In addition, using logistic model, better predictions were obtained.     

Inulinase production and mathematical modeling from carob extract by using Aspergillus niger

The main objectives of the study were to produce inulinase from carob extract by Aspergillus niger A42 (ATCC 204447) and to model the inulinase fermentation in the optimum carob extract-based medium. In the study, carob extract was used as a novel and renewable carbon source in the production of A. niger inulinase. For medium optimization, eight different variables including initial sugar concentration (°Bx), (NH₄)₂HPO₄, MgSO₄.7H₂O, KH₂PO₄, NH₄NO₃, yeast extract, peptone, and ZnSO₄.7H₂O were employed. After fermentations, optimum medium composition contained 1% yeast extract in 5°Bx carob extract. As a result of the fermentation, the maximum inulinase activity, maximum invertase-type activity, I/S ratio, maximum inulinase- and invertase-type activity rates, maximum sugar consumption rate, and sugar utilization yield were 1507.03 U/ml, 1552.86 U/ml, 0.97, 175.82 and 323.76 U/ml/day, 13.26 g/L/day, and 98.52%, respectively. Regarding mathematical modeling, the actual inulinase production and sugar consumption data were successfully predicted by Baranyi and Cone models based on the model evaluation and validation results and the predicted kinetic values, respectively. Consequently, this was the first report in which carob extract was used in the production of inulinase as a carbon source. Additionally, the best-selected models can serve as universal equations in modeling the inulinase production and sugar consumption in shake flask fermentation with carob extract medium.     

Influence of culture conditions on the production of milk-clotting enzyme from Rhizomucor

The ratio of milk-clotting activity to proteolytic activity (MC/PA) was used as an index to determine the quality of milk-clotting enzyme. Solid-state fermentation on wheat bran for 5 days at room temperature gave optimal production for enzyme by Rhizomucor miehei and R. pusillus. A ratio of wheat bran to moisture of 1:0.6 (w/v) gave best results. Adding skim milk powder to the media of R. miehei did not improve the MC/PA ratio but 4% (w/w) of the powder did improve the ratio with R. pusillus. Co-cultivation of R. miehei with R. pusillus did not change the MC/PA ratio.The authors are with the Department of Biotechnogy, Bharathiar University. Coimbatore-641 046 India     

Frementation of biowaste to H2 by Bacillus licheniformis

Completely damaged wheat grains, unfit for human consumption, were fermented to H₂ by Bacillus licheniformis strain JK1. Batch-culture fermentation of wheat slurries [6% (w/v) total solids and 5.8% (w/v) organic solids (OS)] evolved 225, 205 and 203 l of biogas-H, a mixture of H₂, CO₂ and H₂S, per kg OS at pH 6, 7 and 8, respectively. H₂ constituted 25% to 41% of the total biogas-H evolved. In single-stage continuous culture, H₂ generated/kg OS reduced was 70 l at pH 6 and 74 l at pH 7 and 8.V.C. Kalia, S.R. Jain, and A. Kumar are and A.P. Joshi was with the Centre for Biochemical Technology, Mall Road, University Campus, Delhi-110007, India; A.P. Joshi is now with the Chemical Engineering Division, National Chemical Laboratory, Pune-410008, India.     

Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation

Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l^–1 h^–1 and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h^–1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.     

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

Fusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr^?1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr^?1 were obtained at μ = 0.205 hr^?1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr^?1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr^?1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr^?1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr^?1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr^?1 in order to maximize protein production.     

Effect of growth rate on the amino acid composition of Fusarium moniliforme cultivated continuously for fungal protein production.

Fusarium moniliforme was shown to be a promising fungus for microbial protein production. ^1?3The fungus grows well on the aqueous extracts of carob pods, a low value agricultural product well known in the Mediterranean areas. In a previous paper²quantitative data were presented on the production of fungal protein by growing F. moniliforme on a carob aqueous extract in a continuous culture. In the present paper the amino acid profile of the biomass was determined and the resulting essential amino acid index was calculated.     

Quantification of the geocarposphere and rhizosphere effect of peanut (Arachis hypogaea L.)

Roots and pods of field-grown peanut (groundnut) (Arachis hypogaea L.) were sampled at the R3, R5, and R7 developmental stages and examined in comparison to root- and pod-free soil for microbial population densities to assess the geocarposphere and rhizosphere effects. G/ S (no. geocarposphere microorganisms/no. soil microorganisms) and R/S (no. rhizosphere microorganisms/no. soil microorganisms) ratios were calculated for total fungi,Asperigillus flavus, spore-forming bacilli, coryneform bacteria, fluorescent pseudomonads, and total bacteria isolated on low- and high-nutrient media. A clear geocarposphere effect was evidenced by increased population densities of bacteria and fungi associated with developing pods compared to soil. G/S and R/S ratios were generally greater than 1.0 for all groups of microorganisms except bacilli. G/S ratios were greater for total bacteria than for total fungi at two of the three sample times, suggesting that bacteria were stimulated more than fungi in the zone around developing pods. In contrast, R/S ratios, were higher for total fungi than for total bacteria at two of three sample times. The preferential association of fungi and bacteria with early developmental stages of the pod indicates that some microorganisms are particularly well adapted for colonization of the peanut geocarposphere. These microorganisms are logical candidates for evaluation as biological control candiates forA. flavus.     

Natural fermentation of soybean (glycine max) with added salt for ‘soy-daddawa’ production

Microbial growth, pH and titratable acidity decreased with increasing NaCl concentration during the fermentation of soybeans. NaCl at 1% (w/w) in the fermenting mash improved the organoleptic quality of soy-daddawa.The author is with the Department of Microbiology, Obafemi Awolowo University, lle-lfe, Nigeria.     

Processing and characteristics of soybean-fortified gari

Two cassava cultivars were fermented for 3 days with the incorporation of soybean residue or full-fat flour at 25% (w/w) to produce gari. Unfortified gari was superior in taste (P<0.05) to the soybean-fortified gari, though consumers still accepted the latter. Fortified samples from two of the three procedures used, in which soybean was added at different stages of the fermentation, were rated similarly (P>0.05). The water activity of soybean-fortified gari was <0.7, the swelling capacity was 2.5 to 3.3, pH was 5.1±0.2 and total cyanide was 0.8 to 1.3 mg/100 g. The crude protein content of soybean-fortified gari was 8 to 12% (w/w), much higher than the 1 to 2% (w/w) of the unfortified gari. Soybean-fortified gari is recommended for use in areas where cassava is consumed and protein intake is inadequate.The authors are with the Biology Department, The Polytechnic, Ibadan, Nigeria.     

Tourmaline ceramic balls stimulate growth and metabolism of three fermentation microorganisms

Effects of tourmaline ceramic balls on growth and metabolism of Saccharomyces cerevisiae, Lactobacillus acidophilus and Aspergillus oryzae were studied. Treatments with 3, 6, 9 or 12 g of tourmaline ceramic balls in a 50 ml culture showed significant stimulation of the growth of the three microorganisms. In optimal treatments with 12 g of tourmaline balls, the growth of S. cerevisiae, L. acidophilus, and A. oryzae was increased by 34, 32 and 10%, respectively. After 72 h fermentation of S. cerevisiae, total carbohydrate content in the culture medium was decreased by 65% and ethanol production was increased by 150%. Total carbohydrate content was decreased by 80% and the pH value was decreased by 0.3, as a result of organic acid production in the medium of L. acidophilus after 72 h fermentation. In the case of A. oryzae, enzyme activities of protease and amylase were increased by 90 and 31%, respectively, after 96 h fermentation. Results indicated that tourmaline stimulates initiation of growth in the early lag stage and increases production of metabolites at a later stage of fermentation. The strong stimulatory effect of tourmaline on growth, utilization of substrates and production of metabolites in the three microorganisms suggests a potential application in the fermentation industry.     

Production and Chemical Characterization of Antifungal Metabolites From Micromonosporasp. M39 Isolated From Mangrove Rhizosphere Soil

Micromonospora sp. M39 was selected as a producer of antifungal substances. Cell mass and antifungal activity of the organism was dependent on the carbon and nitrogen sources used. A combination of glucose, starch and glycerol as carbon sources at 1%(w/v) and corn steep powder as a nitrogen source at 0.25%(w/v) concentration in basal medium gave a maximum growth of 17.2 PCV/50 ml and in vitro an antifungal activity of 19.0 mm against the rice blast pathogen Pyricularia oryzaeMPO 293. In the selected production medium, optimum antifungal activity of the crude extract of the strain M39 was on day 16 of fermentation at 28 ± 2 °C. The crude extract of the strain M39 was analyzed and characterized by HPLC-DAD-UV–visible spectra. The peaks with a retention time of 4.88 min had a u.v. spectrum identical to that of 2,3-dihydroxybenzoic acid. The peak with a retention time of 6.08 min was confirmed by an automated spectral library search as phenylacetic acid. Two other peaks in the chromatogram were identified as cervinomycin A1 with a retention time at 12.78 min and cervinomycin A2 with a retention time at 13.46 min.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

虎掌菌菌丝富硒培养的研究

为生产合适的硒源提供一种思路,以菌丝体生物量、含硒量、还原糖、氨态氮和蛋白质为指标,采用四因素三水平正交设计法优化虎掌菌的富硒发酵条件,探讨不同浓度的硒对虎掌菌固体培养菌丝生长和液体培养产生的生物组分的影响。结果表明,高浓度的硒抑制虎掌菌菌丝体生长;正交试验选择不同的衡量指标,由极差分析得出各因素的影响程度大小,结合证实试验得到以还原糖为指标的最优组合为葡萄糖6%(质量分数)、酵母浸膏3%(质量分数)、KH_2PO_4 0.1%(质量分数)、Na_2SeO_3 0.6 mmol/L,其菌丝体生物量和氨态氮含量较高。与对照相比,加硒后虎掌菌发酵液中氨态氮、还原糖和总糖含量显著增加(P0.05);当硒浓度为0.5 mmol/L时,氨态氮、还原糖和总糖含量均达到最高值。菌丝体生物量和可溶性蛋白质分别在硒浓度为0.2 mmol/L和0.4 mmol/L时达到最大值。虎掌菌富硒培养后,发酵液的营养成分含量会发生变化。