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.     

Solid-state fermentation of carob pods byAspergillus niger for protein production: effect of particle size

Two strains ofAspergillus niger were cultured in solid-state fermentation system on carob pods ground from 1.25 to 8 mm diam. A particle size of 2.5 mm gave the highest protein content of the final product (20%, w/w) and 52% of the total soluble carbohydrates were utilized. The total tannin concentration of the carob pods decreased by 83% in 4 days of fermentation.T. Smail and O. Salhi are with the Laboratory of Microbiology, U.R.B.A.F., Institute of Biology, Tizi-Ouzou University, Algeria. J.S. Knapp is with the Department of Microbiology, The University of Leeds, Leeds LS2 9JT, UK;     

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.     

Effect of Water Extracts of Carob Pods, Tannic Acid, and Their Derivatives on the Morphology and Growth of Microorganisms

The effect of aqueous extracts of carob (Ceratonia siliqua) pods, gallotannic acid, gallic acid, and catechol on several microorganisms was studied. Carob pod extract and tannic acid showed a strong antimicrobial activity toward some cellulolytic bacteria. On the basis of tannin content, to which antimicrobial effect was related, carob pod extracts inhibited Cellvibrio fulvus and Clostridium cellulosolvens at 15 μg/ml, Sporocytophaga myxococcoides at 45 μg/ml, and Bacillus subtilis at 75 μg/ml. The inhibiting concentrations for tannic acid were found to be 12, 10, 45, and 30 μg/ml, respectively. Gallic acid and catechol were much less effective. Tannic acid and the tannin fraction of carob extract exerted both bacteriostatic and bactericidal effects on C. fulvus. Respiration of C. fulvus in the presence of bactericidal concentrations of tannic acid or tannin fraction of carob extract was inhibited less than 30%. A partial formation of “protoplasts” by C. fulvus was obtained after 2 hr of incubation in a growth medium to which 20% sucrose, 0.15% MgSO₄·7H₂O, and 10 to 50 μg/ml of tannic acid or 500μg/ml of penicillin, or both, had been added. Tannic acid and the tannin fraction of carob extract protected C. fulvus from metabolic lysis in sucrose solution. Although the growth of other microorganisms tested was only slightly affected, the morphology of some of them was drastically changed in the presence of subinhibitory concentrations of carob pod extracts of tannic acid. It is suggested that the site of action of tannins on sensitive microorganisms is primarily the cell envelope.     

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.     

Biology ofclausicella suturata [Dipt.: Tachinidae] a parasite ofEctomyelois ceratoniae [Lep.: Phycitidae]

The tachinid flyClausicella suturata Rondani is a solitary parasite ofEctomyelois ceratoniae (Zeller). It was grown under laboratory conditions (26±0.5°C, 55±5% R.H.) and the host-parasite relationships were studied. The different stages of the parasite were briefly described. The eggs are deposited on the carob (Ceratonia siliqua L.,Caesalpiniaceae) pods, near the openings of the host's tunnels and hatch immediately. The Ist instar maggot is directed by the web fibers of the host towards the caterpillar which is hidden in the carob pod. The parasites develop normally only in 4th and 5th instar caterpillars. The parasitized caterpillar spins its cocoon inside the carob pod before being killed by the parasite. The fully grown maggot leaves the host and pupates inside its cocoon. Adult longevity is dependent upon the presence of carbohydrate in the diet. Maximal male and female longevity (50% survival of 21 and 17.5 days respectively) was reached on a carbohydrate diet and water, at 26°C and 55% R.H. Newly emerged females readily mate with one or two-day old males. The average fecundity of a female is 202 eggs, deposited during 13.6 days, after an incubation period of 6.9 days. Superparasitism is common in nature but only one maggot is successful in completing its development.     

Kinetics of growth and chemical composition ofFusarium moniliforme cultivated on carob aqueous extract for microbial protein production

Summary The kinetics of growth and the chemical composition ofFusarium moniliforme cultivated on aqueous carob pod extract were investigated. The extract was adjusted to provide 0.5, 1.0, 2.0 and 4.0% carob sugars supplemented with inorganic salts at the ratio: carob sugar: NH₄H₂PO₄: MgSO₄.7H₂O=1:0.6:0.012. The extract contained 16 mg tannic acid (Folin-Dennis) per g of carob sugar.The phase of vigorous growth was exponential. Tannins were not observed to depress growth. The maximum value of 0.22 h^–1 for a specific growth rate corresponding to a generation time of 3.15 h was obtained when the fungus was cultivated on a 4% carob sugar medium. The dry mycelium produced per g of consumed carob sugar was then 0.515 g.The protein and purine content was affected by the composition of the growth medium. Protein values up to 37.7% true (Lowry) and 53.1% crude (NX6.25) of dry mycelium were recorded. Mean purine contents were 89 and 116 mol/g, corresponding to nucleic acid levels of 5.7 and 7.5% for mycelium grown on 0.5 and 4.0% carob sugar respectively.These findings linked with those previously reported regarding the good appearance and nutritional quality ofF. moniliforme (Drouliscos et al., 1976) make this fungus worthy of consideration for the production of protein.     

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.     

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.     

Seed size variability: from carob to carats

The seeds of various plants were used as weights because their mass reputedly varies so little. Carob (Ceratonia siliqua), which has given its name to the carat, is particularly famous in this regard. But are carob seeds unusually constant in weight and, if not, how did the myth arise? The variability of seeds sampled from a collection of carob trees (CV=23%) was close to the average of 63 species reviewed from the literature (CV=25%). However, in a perception experiment observers could discriminate differences in carob seed weight of around 5% by eye demonstrating the potential for humans to greatly reduce natural variation. Interestingly, the variability of pre-metrication carat weight standards is also around 5% suggesting that human rather than natural selection gave rise to the carob myth.     

Yeasts associated with algarrobo trees (Prosopis spp.) in northwest Argentina: A preliminary report

Summary Yeasts were isolated from exudates from trees at three sites in northwest Argentina, two between the towns of Amaicha del Valle and Cafayate and one in the Quebrada de Cafayate, a deep river valley north of Cafayate. The majority of the yeasts were identified asCandida famata andRhodotorula graminis, though isolates of other species ofRhodotorula, Candida boidinii, Pichia membranaefaciens, and occasional isolates of other species were obtained. None of the species was the same as those isolated in Crete, from pods of the carob (European algarrobo). Of 96 cultures investigated, 26 utilized methanol as sole carbon source. The frequency of isolation of methylotrophic yeasts from this habitat may prove to be of considerable scientific and technological interest.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Inhibitors from carob (Ceratonia siliqua L.)

Summary Inhibitory extracts of carob and abscisic acid (ABA) were compared and found to behave differently in three types of tests. The carob inhibitors remained at the origin upon thin-layer chromatography in two different solvent systems while a cis-trans mixture of ABA had Rf's of 2.5 and 3.5 in the first system (chloroform:acetic acid, 95:5), and 3.5 and 4.5 in the second system (benzene:acetic acid:water, 8:3:5). When ABA and carob extract were mixed and then chromatographed, the ABA had the same Rf values as ABA chromatographed alone.Assays utilizing light-grown, dwarf peas showed that a weight ratio of 1000: 1 ABA:gibberellic acid (GA₃) was necessary to inhibit GA₃-induced growth by 50% while carob fraction C is inhibitory to GA₃ at a ratio of 17:1. The amount of ABA which inhibited 50% of the growth induced by 0.05 g GA₃ reduced the endogenous growth of both dwarf and non-dwarf pea seedlings; in contrast, concentrations of carob extract up to 100 times greater than the amount necessary for 50% inhibition of the growth response caused by 0.05 g GA₃ did not affect endogenous growth.Only very small amounts of inhibitory activity from carob extract were transferred from water to chloroform at a pH (2.0) at which most of the ABA was transferred.     

Incorporation of galactomannans in the diet of newly weaned piglets: Effect on bacteriological and some morphological characteristics of the small intestine

In search of substances replacing antibiotics as growth promoters for farm animals, non-digestible oligosaccharides (NDO) or non-starch polysaccharides (NSP) have been proposed as possible alternatives. In this context, the influence of galactomannans on bacteriological and morphological aspects of the gastrointestinal tract in weanling pigs was investigated. Four groups of five newly weaned piglets received one of the following diets: control feed (C), C supplemented with guar gum (1%), C supplemented with locust bean gum (1%) and C supplemented with 10% of carob tree seeds meal as source of locust bean gum. The animals were euthanized after 11?–?12 days and digesta were sampled in stomach, jejunum (proximal and distal) and caecum, while mucosal scrapings and ring shaped tissue samples were taken of proximal and distal jejunum. On these samples bacteriological, biochemical and morphological determinations were carried out. Total count of bacteria in digesta and mucosal scrapings was not influenced by the different diets, with the exception of the proximal jejunum where a small decrease (0.5 log₁₀ CFU) was noted with the guar gum and carob tree seeds diet. The number of E. coli increased by feeding both gums and carob tree seeds. With the latter diet, higher counts of streptococci were observed. In agreement with the lower concentration of lactic acid in jejunal contents, guar gum decreased the number of lactobacilli. Locust bean gum decreased the molar proportion of acetate in caecal contents while butyrate and valerate were augmented. Feeding the carob tree seeds resulted in shorter villi and a lower villus height/crypt depth ratio in the jejunum mucosa, which was an indication for a faster renewal rate of the epithelium. Both locust bean gum feeds significantly lowered the mitotic index in the crypts of the small intestine. Only with the carob tree seeds diet, viscosity of jejunal contents was increased. In conclusion, the effects of the addition of 1% of pure guar gum or locust bean gum were inconsistent and not very outspoken, whereas 10% of carob tree seeds meal in the diet resulted in influences on intestinal characteristics at the bacteriological and morphological level.     

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.     

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.     

Natural enemies of the carob mothEctomyelois ceratoniae (Zeller)

The presence of natural enemies of the carob moth was investigated on carob trees, acacias and citrus trees in the various regions of Israel. The highest number of parasite species was found on carob trees, while only a few were observed on acacias and a single one on citrus. A list is given of the natural enemies of the carob moth.     

Pod development of groundnut (Arachis hypogaea L.) in solution culture

Normal pods (containing seed) of groundnut (Arachis hypogaea L.) (cv. TMV-2) were successfully raised in darkened, aerated, nutrient solution, but not in the light. The onset of podding was evident 7 to 8 d after gynophores were submerged in the darkened nutrient solution. An examination of pods and submerged portions of gynophore surfaces by scanning electron microscopy showed the presence of two distinctly different protuberances: unicellular root-hair-like structures that first developed from epidermal cells of the gynophores and developing pods; and branched septate hairs that developed later from cells below the epidermal layer. The septate hairs became visible only after the epidermal and associated unicellular structures had been shed by the expanding gynophore and pods. Omission of Mn and Mg from the podding environment increased pod and seed weight, whilst omission of Zn reduced pod and seed weight.     

Sucrose syrup from carob pod

Summary The ripe carob pod (pericarp) is rich in water-soluble sugars, mainly sucrose (63% on total sugars). Sucrose crystallization from aqueous carob extract is prevented by its reducing sugar content. The selective consumption of these sugars by mixed culture ofRhizopus oligosporus andSaccharomyces rouxii gives a sucrose syrup suitable for several uses e.g. sucrose extraction.     

Incorporation of galactomannans in the diet of newly weaned piglets: effect on bacteriological and some morphological characteristics of the small intestine

In search of substances replacing antibiotics as growth promoters for farm animals, non-digestible oligosaccharides (NDO) or non-starch polysaccharides (NSP) have been proposed as possible alternatives. In this context, the influence of galactomannans on bacteriological and morphological aspects of the gastrointestinal tract in weanling pigs was investigated. Four groups of five newly weaned piglets received one of the following diets: control feed (C), C supplemented with guar gum (1%), C supplemented with locust bean gum (1%) and C supplemented with 10% of carob tree seeds meal as source of locust bean gum. The animals were euthanized after 11-12 days and digesta were sampled in stomach, jejunum (proximal and distal) and caecum, while mucosal scrapings and ring shaped tissue samples were taken of proximal and distal jejunum. On these samples bacteriological, biochemical and morphological determinations were carried out. Total count of bacteria in digesta and mucosal scrapings was not influenced by the different diets, with the exception of the proximal jejunum where a small decrease (0.5 log10 CFU) was noted with the guar gum and carob tree seeds diet. The number of E. coli increased by feeding both gums and carob tree seeds. With the latter diet, higher counts of streptococci were observed. In agreement with the lower concentration of lactic acid in jejunal contents, guar gum decreased the number of lactobacilli. Locust bean gum decreased the molar proportion of acetate in caecal contents while butyrate and valerate were augmented. Feeding the carob tree seeds resulted in shorter villi and a lower villus height/crypt depth ratio in the jejunum mucosa, which was an indication for a faster renewal rate of the epithelium. Both locust bean gum feeds significantly lowered the mitotic index in the crypts of the small intestine. Only with the carob tree seeds diet, viscosity of jejunal contents was increased. In conclusion, the effects of the addition of 1% of pure guar gum or locust bean gum were inconsistent and not very outspoken, whereas 10% of carob tree seeds meal in the diet resulted in influences on intestinal characteristics at the bacteriological and morphological level.