Optimization of single cell protein production from cassava starch (Rhizopus oligosporus)

The fermentation pattern of cassava starch utilization was investigated at 37°C using Rhizopus oligosporus UQM 145 F and eight different media. Depending on the medium used, the addition of zinc or zinc plus iron to a combination of calcium plus manganese switches the fermentation from glucose accumulation to biomass (single cell protein) production. Complete starch hydrolyzation was obtained in both cases, with a complete glucose utilization resulting in 24 g biomass containing 30% true protein per 100 g cassava starch (= 7.45 g SCP/100 g substrate) in 24 hours. In the case of glucose accumulation, biomass was kept low and 15.5 g/l glucose representing 57.3% of starch supplied were obtained in 36 hours. R. oligosporus UQM 145 F grows well between 30° and 45°C. At 45°C and pH 5.0, 7.0 g SCP/100 g substrate were obtained, which rose to 8.6 g if cassava starch is replaced by ground cassava tuber.     

Optimization of cassava starch conversion to glucose byRhizopus oligosporus

Summary Incubation temperature, inoculum size, initial pH and pH control play a major role in cassava starch to glucose conversion byRhizopus oligosporus. Maximal glucose production was obtained after 45 to 48 h fermentation at 45°C, pH control at 4.0, 5% cassava starch, agitation rate of 300 rev./min. and aeration rate of 85 ml/min. Under these conditions, starch hydrolysis was 99.4% with a starch-to-glucose conversion efficiency of 91.6% and a final yield of 35.2 g/l glucose with a biomass yield of only 2.8 g/100 g cassava starch.

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Optimisation de la conversion de l'amidon de manioc en glucose par Rhizopus oligosporus

Résumé La température d'incubation, la taille de l'inoculum, le pH initial et le contrôle du pH jouent un rôle majeur dans la conversion de l'amidon de manioc en glucose parRhizopus oligosporus. On obtient la production maximum de glucose après 45–48 h de fermentation à 45°C, avec un contrôle de pH à 4.0, 5% d'amidon de manioc, une vitesse d'agitation de 300 tpm et une vitesse d'aération de 85 ml/min. Dans ces conditions, l'hydrolyse de l'amidon atteint 99.4% avec une efficacité de conversion de l'amidon en glucose de 91.6% et un rendement final de 35.2 g de glucose par litre pour un rendement en biomasse de 2.8 g seulement par 100 g d'amidon de manioc.

     

Cassava starch fermentation pattern ofRhizopus oligosporus

Summary Rhizopus oligosporus UQM 145F was grown on cassava starch medium without a preliminary treatment of starch. The fermentation pattern ofR. oligosporus is shown to be influenced significantly by the medium composition, pH of the growth culture and the size of the spore inoculum. By varying the carbon, nitrogen and mineral salt concentrations, it was found that either glucose accumulated due to excess hydrolysis of starch or single cell protein production became dominant. In the case of the carbon source, increases in the concentration of starch led to higher biomass values, but lower rates of protein production. These observations led to a new formulated medium, resulting in an 82.9% cassava starch utilization and a true protein yield of 5.32 g/100 g initial substrate. The response ofR. oligosporus to inorganic compounds such as (NH₄)₂SO₄, urea, NaCl and MgSO₄ indicates an interesting fermentation control system.

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Resumen Rhizopus oligosporus UQM 145F se hizo crecer en un medio a base de almidón de casava sin que hubiera pasado por un tratamiento previo con almidón. Se demostró que el patrón de fermentación seguido porRh.oligosporus estaba significativamente influenciado por: la composición del medio, su pH y el tamaño del inóculo. Al variar las concentraciones de carbono, nitrógeno y sales minerales se observó que predominaba la acumulación de glucosa o bien la producción de proteínas unicelulares. Un incremento en la fuente de carbono aumentó la biomasa pero disminuyó el ritmo de producción de proteínas. Estas observaciones permitieron elaborar un nuevo medio de cultivo que resultó en la utilización del 82.9% del almidón de casava con un rendimiento en proteínas de 5.32 g/100 g de sustrato inicial. La respuesta deRh. oligosporus a compuestos inorgánicos como sulfato amónico, urea, cloruro sódico y sulfato magnésico indican la existencia de un sistema de control de la fermentación muy interesante.

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Résumé On a fait croîtreRhizopus oligosporus UQM 145 F sur un milieu à base d'amidon de manioc sans traitement préalable de l'amidon. Le profil de fermentation parR. oligosporus apparaît être influencé de manière significative par la composition du milieu, le pH de la culture en croissance et la taille de l'inoculum de spores. En variant les concentrations en carbone, en azote et en sels minéraux, on remarque la prédominance tantôt de la protéine unicellulaire, tantôt de l'accumulation de glucose. Un accroissement en concentration conduit à des valeurs plus élevées de la biomasse mais en une moindre production de protéines. Ces observations ont abouti à une nouvelle formulation du milieu avec pour résultat une utilisation à 82.9% de l'amidon de manioc et un rendement vrai en protéines de 5.32 g/100 g de substrat initial. La réponse deR. oligosporus aux composés inorganiques comme le sulfate d'ammonium, l'uréi, le chlorure de sodium et le sulfate de magnésium sont indicateurs d'un système très intéressant de contrôle de la fermentation.

     

A one-step process for the production of single-cell protein and amyloglucosidase

Summary A new Rhizopus species was isolated from traditional Indonesian food, tempeh. The newly isolated species was similar in its morphological characteristics to Rhizopus oligosporus UQM 145F, but grew faster on potato-dextrose agar as well as in submerged culture. The new isolate was found to convert ground cassava tuber directly into single cell protein without pretreatment due to its high amyloglucosidase formation.From 100 g ground tuber, a dry biomass of 33.75 g containing 26.48% true protein together with 60 ml of highly active amyloglucosidase (282 units) was obtained in 12 h. The amyloglucosidase was recovered by ultrafiltration, releasing 26.226 millimol glucose/l/min from soluble starch. The crude enzyme exhibited a pH optimum between 4.6 and 5.0, a temperature optimum between 55 and 60° C and an apparent Km of 3.125 g/l. High substrate concentrations and ammonium sulphate are inhibitory to the enzyme.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

A model substrate for solid-state fermentation

Summary A model substrate consisting of cassava starch embedded in kappa-carrageenan was used to mimic the growth ofRhizopus oligosporus on cassava tubers. Growth on the model substrate was similar to that during solid-state fermentation of the actual cassava. However, protein production and starch utilization were slower on the model substrate.     

Preparation and evaluation of dehydrated mycological media from West African raw materials

Twelve culture media powders prepared from local raw materials were evaluated for their mycological utility using Nigrospora oryzae as the initial test organism. Radial growth of N. oryzae was greater (P < 0.05) on media from pigeon pea, soybean and ripe plantain than on Oxoid potato-dextrose agar (PDA) control. Two other media supported radial growth comparable to potato-dextrose agar. More conidia were produced on the ripe plantain medium (44/microscope field) than on PDA (3.4/microscope field). In an additional test, Fusarium oxysporum f. sp. cubense, Aspergillus niger, Curvularia lunata and Choanephora cucurbitarum grew and sporulated well on selected media. Local media were inexpensive and easy to produce but tended to be characterised by reduced clarity and presence of particles after autoclaving and jelling.     

Degradation of stachyose,raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of -galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean -protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the -galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.     

Culture conditions for yellow pigment formation byMonascus sp. KB 10 grown on cassava medium

An isolate ofMonascus, from a commercial, fermented soybean curd (sufu) was grown on a cassava medium. With medium at an initial pH of 7.0 an orange-red pigmentation was produced but with an initial pH below 4, a light golden pigment was obtained. A medium containing, w/v, 3% cassava starch, 0.4% peptone and 0.1% glutamic acid, with an initial pH of 2.5 was optimal for the production of this yellow pigment, which had a single maximum absorption spectrum at 330 nm. The spectroscopic characterization of the purified yellow pigments demonstrated a monascin-ankaflavin-monascorubrin skeleton.B. Yongsmith, W. Tabloka and W. Yongmanitchai are with the Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. R. Bavavoda is with the Department of Botanical Pharmacy, Chulalongkorn University, Bangkok, Thailand.     

The effect of urea on growth of moulds and biomass yield in solid-state cultivation

Urea, added at 2 to 20 mg/g in solid bran medium supporting growth of Aspergillus oryzae and Rhizopus oligosporus, led to a higher concentration of NH₄ ⁺ and pH but a decrease in biomass production.The authors are with the Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 61000 Ljubljana, Slovenia.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Production of a raw starch saccharifying amylase byBacillus alvei grown on different agricultural substrates

Maximum activity of the amylase ofBacillus alvei was attained after growth of the organism on sorghum starch. Rice, corn, yam, cassava and potato starch gave high enzyme activities as did soluble starch. Glucose, maltose and glycerol were less effective. Optimum conditions for both growth and enzyme production were pH 6.8 at 40°C.     

Improvement of growth ofRhizopus oligosporus on a model solid substrate

Summary Metal ions and cassava extracts stimulated growth ofRhizopus oligosporus on a model solid substrate. A large improvement in protein content was obtained by simultaneously increasing the nitrogen content and decreasing the particle size of the substrate. No improvement occurred when these conditions were applied to cassava, however, because of the sticky consistency of the cassava after gelatinization.     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.     

Desulfotomaculum geothermicum sp. nov., a thermophilic,fatty acid-degrading,sulfate-reducing bacterium isolated with H2 from geothermal ground water

A strictly anaerobic, thermophilic, fatty acids-degrading, sporulating sulfate-reducing bacterium was isolated from geothermal ground water. The organism stained Gram-negative and formed gas vacuoles during sporulation. Lactate, ethanol, fructose and saturated fatty acids up to C₁₈ served as electron donors and carbon sources with sulfate as external electron acceptor. Benzoate was not used. Stoichiometric measurements revealed a complete oxidation of part of butyrate although growth with acetate as only electron donor was not observed. The rest of butyrate was oxidized to acetate. The strain grew chemolithoautotrophically with hydrogen plus sulfate as energy source and carbon dioxide as carbon source without requirement of additional organic carbon like acetate. The strain contained a c-type cytochrome and presumably a sulfite reductase P582. Optimum temperature, pH and NaCl concentration for growth were 54°C, pH 7.3–7.5 and 25 to 35 g NaCl/l. The G+C content of DNA was 50.4 mol %. Strain BSD is proposed as a new species of the spore-forming sulfate-reducing genus Desulfotomaculum, D. geothermicum.     

Efficient and Direct Fermentation of Starch to Ethanol by Sake Yeast Strains Displaying Fungal Glucoamylases

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999     

Oats tempeh

Oats was used as a substrate in tempeh fermentation. The time needed to obtain sufficient mold growth was at least 30 hours at 31°C (Rhizopus oligosporus NRRL 2710). pH was decreasing during the first 32 hours of incubation reaching pH = 5.30. Fermentation of oats led to an increase in water soluble nitrogen, but it did not change protein nitrogen content. R. oligosporus proteinases of optimum pH = 5.50 are postulated to play an important role in oats tempeh fermentation. When a mixture of oats and soybean (1:1) was used, mold growth was faster and the cake tougher. Mixing cereals with legumes to produce good tempeh is recommended.