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.     

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.     

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.     

Controlling filamentous fungi morphology with microparticles to enhanced β-mannanase production

β-mannanase was produced mainly by Aspergillus species and can degrade the β-1,4-mannose linkages of galactomannans. This study was undertaken to enhance mannanase production using talcum and aluminum oxide as the microparticles, which control cell morphology of recombinant Aspergillus sojae in glucose and carob extract medium. Both microparticles improved fungal growth in glucose and carob pod extract medium. Aluminum oxide (1 g/L) was the best agent for glucose medium which resulted in 514.0 U/ml. However, the highest mannanase activity was found as 568.7 U/ml with 5 g/L of talcum in carob extract medium. Increase in microparticle concentration resulted in decreasing the pellet size diameter. Furthermore, more than 10 g/L of talcum addition changed the filamentous fungi growth type from pellet to pellet/mycelium mixture. Results showed that right type and concentration of microparticle in fermentation media improved the mannanase activity and production rate by controlling the growth morphology.     

Rock phosphate solubilization by Aspergillus niger grown on sugar-beet waste medium

Solubilization of rock phosphate by Aspergillus niger was studied in solid-state fermentation on sugar-beet waste. This combination was selected after testing three agroindustrial waste materials, namely rice hulls, sugar-beet waste and alperujo. Sugar-beet waste was the best substrate for fungal growth with 69% mineralization, followed by rice hulls and alperujo. The fungus was successfully cultivated on sugar-beet waste supplemented with 3.0 g/l rock phosphate, acidifying the medium and thus decreasing the pH to 3–3.5. Solubilization of insoluble phosphate increased during the first half of the process, reaching a maximum of 292 g phosphate/ml, although a part of it was probably consumed by the mycelium.     

Fed-batch biotransformation of β-ionone by Aspergillus niger

Aspergillus niger IFO 8541 was found to be an efficient biocatalyst for the biotransformation of -ionone into hydroxy and oxo derivatives. The reaction had to be carried out with an inoculum made of about 4 × 10⁷ fresh spores/l and with a preliminary growth period giving at least 3 g/l biomass. The fungus developed in the form of pellets when cultivated as free mycelium; entrapment of the microorganism in calcium alginate beads was an efficient way to mimic this feature in an aerated, stirred bioreactor. The biotransformation was carried out using a fed-batch mode of operation involving sequential precursor addition. -Ionone stopped the fungal growth and was converted into metabolites only when the carbon source remained present in the medium; it was fully oxidized after sucrose exhaustion. These conditions allowed recovery of about 2.5 g/l aroma compounds after 230 h cultivation with a molar yield close to 100%.     

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.     

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.     

Evaluation and optimization of ethanol production from carob pod extract by <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> using response surface methodology

In this research, ethanol production from carob pod extract (extract) using Zymomonas mobilis with medium optimized by Plackett–Burman (P–B) and response surface methodologies (RSM) was studied. Z. mobilis was recognized as useful for ethanol production from carob pod extract. The effects of initial concentrations of sugar, peptone, and yeast extract as well as agitation rate (rpm), pH, and culture time in nonhydrolyzed carob pod extract were investigated. Significantly affecting variables (P = 0.05) in the model obtained from RSM studies were: weights of bacterial inoculum, initial sugar, peptone, and yeast extract. Acid hydrolysis was useful to complete conversion of sugars to glucose and fructose. Nonhydrolyzed extract showed higher ethanol yield and residual sugar compared with hydrolyzed extract. Ethanol produced (g g⁻¹ initial sugar, as the response) was not significantly different (P = 0.05) when Z. mobilis performance was compared in hydrolyzed and nonhydrolyzed extract. The maximum ethanol of 0.34 ± 0.02 g g⁻¹ initial sugar was obtained at 30°C, initial pH 5.2, and 80 rpm, using concentrations (g per 50 mL culture media) of: inoculum bacterial dry weight, 0.017; initial sugar, 5.78; peptone, 0.43; yeast extract, 0.43; and culture time of 36 h.     

Growth of Fusarium moniliforme on carob aqueous extract and nutritional evaluation of its biomass.

Fusarium moniliforme was cultured semicontinuously on a carob medium in a 14-liter fermentor (8.5-liter working volume). The growth medium provided 2.4% carob sugar, 0.72% NH4H2PO4, and 0.03% MgSO4-7H2O. The biomass harvest was 8.8 g/liter per day. Ninety percent of the sugars were consumed, and the pH dropped from 5.9 to about 3.7. The crude protein (N X 6.25) of the spray-dried mycelium was 380 g/kg, 300 g/kg for the true protein (Lowry), and 4.8 g/kg for the (Folin-Denis) tannic acid. The mycelium was evaluated nutritionally with the weanling rat as experimental animal. The protein efficiency ratio and net protein utilization values for the unsupplemented mycelium were 1.15 and 0.42, respectively, and for the mycelium supplemented with DL-methionine (5 g/kg) they were 2.31 and 0.72, respectively. No growth depression was observed in the experimental rats, and on dissection of the carcasses the internal organs were found to be normal.     

Growth of Fusarium moniliforme on carob aqueous extract and nutritional evaluation of its biomass.

Fusarium moniliforme was cultured semicontinuously on a carob medium in a 14-liter fermentor (8.5-liter working volume). The growth medium provided 2.4% carob sugar, 0.72% NH4H2PO4, and 0.03% MgSO4-7H2O. The biomass harvest was 8.8 g/liter per day. Ninety percent of the sugars were consumed, and the pH dropped from 5.9 to about 3.7. The crude protein (N X 6.25) of the spray-dried mycelium was 380 g/kg, 300 g/kg for the true protein (Lowry), and 4.8 g/kg for the (Folin-Denis) tannic acid. The mycelium was evaluated nutritionally with the weanling rat as experimental animal. The protein efficiency ratio and net protein utilization values for the unsupplemented mycelium were 1.15 and 0.42, respectively, and for the mycelium supplemented with DL-methionine (5 g/kg) they were 2.31 and 0.72, respectively. No growth depression was observed in the experimental rats, and on dissection of the carcasses the internal organs were found to be normal.     

Lactic acid production by batch fermentation of whey permeate: A mathematical model

Summary The batch fermentation of whey permeate to lactic acid was improved by supplementing the broth with enzyme-hydrolyzed whey protein. A mathematical model based on laboratory results predicts to a 99% confidence limit the kinetics of this fermentation. Cell growth, acid production and protein and sugar use rates are defined in quantifiable terms related to the state of cell metabolism. The model shows that the constants of the Leudeking-Piret model are not true constants, but must vary with the medium composition, and especially the peptide average molecular weight. The kinetic mechanism on which the model is based also is presented.Nomenclature K _i lactic acid inhibition constant (g/l) - K _pr protein saturation constant during cell growth (g/l) - K _pr protein saturation constant during maintenance (g/l) - K _s lactose saturation constant (g/l) - [LA] lactic acid concentration (g/l) - [PR] protein concentration (g/l) - [S] lactose concentration (g/l) - t time (h) - [X] cell mass concentration (g/l) - , fermentation constants of Leudeking and Piret - specific growth rate (l/h) - Y _{g, LA/S} acid yield during cell growth (g acid/g sugar) - Y _{m, LA/S} acid yield during maintenance (g acid/g sugar) - Y _x/pr yield (g cells/g protein) - specific sugar use rate during cell growth (g sugar/h·g cell) - specific sugar use rate during maintenance (g sugar/h·cell)     

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.     

Microorganisms for degrading simmondsin and related cyanogenic toxins in jojoba

Summary Four microorganisms that metabolize simmondsin (S) and related cyanogenic toxins from jojoba (Simmondsia chinensis) were isolated by enrichment: Pseudallescheria boydii, a fungus which specifically degrades simmondsin ferulate but not S; Fusarium moniliforme; Flavobacterium aurantiacum; and Pseudomonas maltophilia. The latter three organisms grow on S as a sole carbon and nitrogen source in culture media, but only F. moniliforme attacks S in the complete jojoba meal. Combinations of the four microorganisms at two temperatures, and with free air or limited air exchange for up to 20 days, were tested on jojoba meal to determine an optimum detoxification method. Degradation of toxins was most rapid and complete when Pseudallescheria boydii and Fusarium moniliforme together were incubated on jojoba meal at 25°C with free air exchange for 20 days. Mice were fed fermented meals at 0, 5, 10 and 20% substitution levels to determine detoxification and nutritional quality. Average daily gains during rapid growth of weanling (1–3 weeks) and mature (4–8 weeks) mice did not differ significantly from controls for mice on all diets containing fermented meal. Diets containing fungally detoxified jojoba meal were more efficient for maintaenance of mature weight than jojoba meal detoxified with enzymes naturally present in the meal. Meal can be detoxified by ensilage for 20 days at 80% water content. Detoxification is attributed to as yet unidentified enzymes inherent in the jojoba seed.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentionedOffprint requests to: Thomas P. Abbott     

The effect of lignocellulose on lignocellulolytic activity of Pleurotus pulmonarius in submerged culture

Summary The possible effects of lignocellulose substrate on lignocellulolytic activity of the fungus Pleurotus pulmonarius was studied in submerged culture. This study included fungal growth rates, nitrogen and carbon consumption, and enzymatic activity rates, with and without added cotton-wheat straw (CWS) mixtures. Addition of CWS to the media caused increased consumption of glucose and NH _inf4 ^sup+ by the fungal mycelium, induced carboxymethylcellulase (CMC-ase), increased poly-B decolourization, and enhanced the activity of laccase tenfold, while -glucosidase activity was also enhanced: its first peak was higher and second peak earlier. Lignin peroxidase, however, was not detected. These results give some indication that the lignocellulolytic activity of P. pulmonarius in liquid culture is enhanced by the presence of lignocellulosic substrates such as CWS.Offprint requests to: D. Levanon     

Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

AIMS: To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium. METHODS AND RESULTS: The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22-25 degrees C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1.1 day(-1), reaching a cell density of 23.0 g dw l(-1) in 7-9 days. Exopolysaccharides accumulated in the liquid culture to about 0.3 g l(-1) glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11.7-microg) and carbohydrate (654.6-microg) but much higher contents of polysaccharide (244.2 mg vs 129.5 mg), adenosine (1116.8-microg vs 264.6 microg) and cordycepin (65.7 microg vs 20.8 microg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour growth in mice. CONCLUSIONS: The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated by liquid fermentation. The mycelium biomass contained the major bioactive compounds of C. sinensis, and the mycelium extract had significant antitumour activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The Cs-HK1 fungus may be a new and promising medicinal fungus and an effective and economical substitute of the wild C. sinensis for health care.     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

Semi-defined Medium for in vitro Cultivation of the Fastidious Insect Pathogenic Fungus Cordyceps unilateralis

Cordyceps unilateralis is a fastidious fungal pathogen affecting ants. Up to now, only the complex and expensive Grace’s insect cell culture medium has been used for in vitro cultivation (as blastospores and mycelium) of this fungus. To obtain an inexpensive and less complicated medium, the effects of carbon and nitrogen sources, salt solution and carbon-to-nitrogen (C:N) ratio on the growth of this fungus were examined. Glucose was the most important factor for blastospore formation, and yeast extract could be used as a nitrogen source for blastospore formation and mycelial growth. A suitable C:N ratio (glucose: yeast extract) was 33.3:1. As a result, a new semi-defined medium was achieved, composed of 26.68 g L⁻¹ glucose, 3.3 g L⁻¹ yeast extract and salt solution. This medium supported blastospore formation and mycelial growth of all tested C. unilateralis isolates.     

β-Mannanase production and kinetic modeling from carob extract by using recombinant Aspergillus sojae

The main objectives of this study were to optimize β-mannanase fermentation conditions by using Response Surface Methodology (RSM) and to model kinetically using the kinetic models. Based on the results, the optimum fermentation conditions were found to be initial sugar concentration of 10°Bx, whey concentration of 0.75% [w/v], and inoculum size of 8% (v/v). Under optimized conditions, β-mannanase activity (P), sugar consumed (ΔS), maximum β-mannanase production rate (Q_P), and sugar utilization yield (SUY) were 687.89 U/mL, 47.38 g/L, 118.54 U mL^–1 day^–1, and 69.73%, respectively. Kinetic models were employed to describe the optimum β-mannanase fermentation process. The kinetic analysis of β-mannanase fermentation showed that β-mannanase fermentation is growth associated because the α value (U/mgX) is approximately 330-fold higher than the β value (U/mgX·hr). Nevertheless, maintenance value (Z) was lower than γ value, thus showing that Aspergillus niger mainly utilizes the sugars for β-mannanase production and fungal growth. Consequently, carob extract and whey powder could be used to be cost-effective carbon and organic nitrogen sources, respectively. It was clearly indicated that the suggested kinetic models can successfully describe the fungal growth, β-mannanase production, and substrate consumption.