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.     

Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate

Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH₄)₂SO₄, 0.09 % yeast extract, 0.05 % KH₂PO₄, and 0.05 % MgSO₄·7H₂O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.     

Optimization of Cultivating Conditions for Triterpenoids Production from Antrodia cinnmomea

The submerged cultivating conditions for triterpenoids production from Antrodia cinnamomea were optimized using uniform design method and the one-factor-at-a-time method was adopted to investigate the effect of plants oils and glucose supply on triterpenoids production and mycelia growth. Corn starch and culturing time were identified as more significant variables for triterpenoids production. The optimal conditions for triterpenoids production was 20.0 g/L corn starch, 20.0 g/L wheat bran, 1.85 g/L MgSO4, initial pH 3 and 16 days of cultivation. In addition, investigation of plant oils and glucose supply showed that 0.3 % (v/v) olive oil supply at the beginning of fermentation stimulated mycelia growth and significantly increased triterpenoids production; 0.2 % (w/v) glucose supplement at 10th day enhanced production of triterpenoids with slight effect on biomass, which is reported for the first time. The triterpenoids production experimentally obtained under the optimal conditions was 7.23 % (w/w). The uniform design method may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoids production from A. cinnamomea.     

Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes

Current study was focused on the development of a non-fastidious lactic acid producing strain having better growth rate, low pH tolerance and good productivity by genome shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and an amylase producing non-fastidious Bacillus amyloliquefaciens ATCC 23842. After the third cycle of the protoplast fusion, lactic acid production by few fusants was monitored and the best fusant was selected for further studies. Optimization of the important process parameters for lactic acid production was conducted using Plackett-Burman design and response surface methodology. Selected fusant could utilize the liquefied cassava bagasse starch directly with minimum nutrient supplementation for lactic acid production. During validation, 40g/L of lactic acid was obtained ( approximately 96% conversion of starch to lactic acid) by using fusant inoculum (3%, v/v) from 83g/L cassava bagasse (starch content 50% w/w) supplemented with yeast extract and peptone (0.2% each, w/v) and the buffering agent (2% CaCO(3), w/v).     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

Enzyme hydrolysis of cassava peels: treatment by amylolytic and cellulolytic enzymes

Cassava peels provide a cheap non-food biomass waste that can be hydrolyzed to simple sugars as a useful feedstock. Unlike most crop wastes, they have high starch content as well as lignocellulose. In this study, an enzymatic treatment of cassava peels by various concentrations of amylase and glucoamylase is considered. Steam explosion pre-treatments reduced rate and yield of hydrolysis. Milled peels suspended at 10% w/v yielded a maximum reducing sugar of 0.41?g (as glucose) per gram of peels. HPLC analysis showed that levels of soluble oligosaccharides remained low throughout. A pretreatment with amylase at 95?°C slightly increased rates although final yield was the same. Additional treatment with cellulolytic enzymes increases the total hydrolysis yield to 0.61?g (as glucose) per gram of peels representing 91% of the carbohydrate in cassava peels.     

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.

     

Ethanol fermentation of raw cassava starch with Rhizopus koji in a gas circulation type fermentor

Studies have been conducted in a gas circulation type fermentor in order to characterize the ethanol fermentation of uncooked cassava starch with Rhizopus koji. Results showed that ethanol concentration reached 13-14% (v/v) in 4-day broth, and the maximum productivity of ethanol was 2.3 g ethanol/L broth h. This productivity was about 50% compared to the productivity of a glucose-yeast system. Ethanol yield reached 83.5-72.3% of the theoretical yield for the cassava starch used. The fermentor used in the present work has been proven by experiment to be suitable for ethanol fermentation of the broth with solid substrate.     

一株厌氧嗜热杆菌生长及发酵特性的初步研究

论文对筛选并鉴定为Thermoanaerobacterium saccharolyticum菌株的生长、底物利用情况、产物生成以及酒精耐受性进行了研究。结果表明,该菌在以甘露糖、葡萄糖和木糖为碳源时生长较好,同时能够较好的利用木聚糖和木薯淀粉;最适底物浓度为15g/L;不同的葡萄糖:木糖比例对其生长无显著影响;能耐受的培养基最高初始酒精浓度为3%(V/V)。在5g/L的木聚糖、木糖和木薯淀粉培养基中发酵60h后,产物主要有乙醇、乳酸和乙酸,乙醇产量分别为0.824、0.867和0.916g/L。     

Preparation of Koji from corn hulls for alcoholic fermentation without cooking

Corn hulls, the outer peel covering the corn grain, were used for preparation of koji, which was applied to alcoholic fermentation without cooking of raw strachy materials. Corn hull koji had lower saccharifying power, α-amylase, CMCase and xylanase than wheat bran koji, but higher protease and pectinase activities, Its alcoholic fermentations of cassava starch and sweet potato were also superior to those of wheat bran koji: corn hull koji gave 10.3% (v/v) of alcohol with 93% yield from 20 g of cassava starch, while wheat bran koji gave 9.4% (v/v) of alcohol with 90.4% yeild; and corn hull koji gave 9.1% (v/v) of alcohol with 92.6% yielded from 50 g of sweet potato, while wheatbran koji gave 8.1 (v/v) of alcohol with 88.6% yield.     

虹鳟幼鱼对不同淀粉的利用

试验旨在考察不同淀粉种类对虹鳟(Oncorhynchus mykiss)幼鱼生长性能、血清生化指标、肝脏糖代谢酶、肝脏组织学和摄食后血糖含量的影响。选用木薯淀粉、小麦淀粉、豌豆淀粉和玉米淀粉为淀粉源,设置相同的淀粉水平(15%),配制4组等氮等脂饲料,饲喂初始体重(7.7±0.1) g的虹鳟56d。结果表明,木薯淀粉组增重率最高(1049.3%),饲料系数最低(0.83)。各组脏体比、肝体比、全鱼和肝脏常规组成无显著差异(P>0.05);豌豆淀粉组肝糖原含量显著低于其他组(P<0.05)。各组血清甘油三酯、总胆固醇、谷草转氨酶和谷丙转氨酶无显著差异(P>0.05)。木薯淀粉组和小麦淀粉组肝脏己糖激酶活性显著高于豌豆淀粉组和玉米淀粉组(P<0.05),玉米淀粉组肝脏磷酸果糖激酶活性显著低于木薯淀粉组和小麦淀粉组(P<0.05)。摄食后,各组的血糖含量均在7h达到最高,木薯淀粉组在摄食后1—7h内的血糖含量显著高于其余3组(P<0.05)。各组的肝脏组织形态无显著差异。综上,木薯淀粉较其他3种淀粉更适宜作为虹鳟饲料的淀粉源。     

Heterologous protein secretion by Aspergillus niger growing in submerged culture as dispersed or aggregated mycelia

The secreted production of a heterologous enzyme, hen egg-white lysozyme, by Aspergillus niger was studied in shake flasks containing media of different initial viscosities. Raising the viscosity of the medium by addition of polyvinylpyrrolidone (PVP) brought about a transition in the form of growth from aggregated mycelia (pellets) to dispersed mycelia. The specific yield of lysozyme in cultures containing an initial concentration of 5% (w/v) starch was 8 mg lysozyme/g dry weight. Addition of 2% (w/v) PVP to the medium resulted in a specific yield of 14 mg lysozyme/g dry weight.     

Ethanol production from cassava and sago starch using Zymomonas mobilis

Summary Cassava and sago starch were evaluated for their feasibilities as substrates for ethanol production using Zymomonas mobilis ZM4 strain. Before fermentation, the starch materials were pretreated employing two commercial enzymes, Termamyl (thermostable -amylase) and AMG (amyloglucosidase). Using 2 l/g of Termamyl and 4 l/g of AMG, effective conversion of both cassava and sago starch into glucose was found with substrate concentration up to 30%(w/v) dry substances. Fermentation study performed using these starch hydrolysates as substrates resulted in ethanol yield at an average of 0.48g/g by Z. Mobilis ZM4.     

Effects of cassava starch hydrolysate on cell growth and lipid accumulation of the heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis>

Heterotrophic fermentation of microalgae has been shown to accumulate high amounts of microalgal lipids, which are regarded as one of the most promising feedstocks for sustainable biodiesel production. To increase the biomass and reduce the cost of microalgal culture, the purpose of this study was to evaluate the possibility of using cassava starch hydrolysate (CSH) instead of glucose as carbon source for heterotrophic culture of Chlorella protothecoides in flasks. First, the two-step enzymatic process of hydrolysis of cassava starch by α-amylase and glucoamylase was optimized; the conversion efficiency for cassava starch was up to 97.7%, and over 80% of CSH was glucose. Subsequently, we compared heterotrophic cultures of C. protothecoiedes using glucose or CSH as carbon source. The results demonstrated that when using CSH as the organic carbon source, the highest biomass and the maximum total lipid yield obtained were 15.8 and 4.19 g/L, representing increases of 42.3 and 27.7%, respectively, compared to using glucose as the organic carbon source. This suggests that CSH is a better carbon source than glucose for heterotrophic Chlorella protothecoides.     

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α‐amylase from Aspergillus oryzae as well as α‐amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:338–347, 2014     

Direct fermentative production of lactic acid on cassava and other starch substrates

Lactobacillus amylovorus utilized raw corn, rice and wheat starch medium to produce lactic acid with a productivity of 10.1, 7.9 and 7.8 g lactic acid/L, but had lower productivities of 4.8 g/L and 4.2 g/L on cassava and potato starch in basal medium respectively. When peptone (1%) is added to basal medium with cassava starch as substrate, conversion rate increased from 43% conversion to 70% conversion (7.7 g lactic acid/L). The availability of some components of protein in corn starch is assumed to be the reason for high lactic acid production as compared to that of cassava starch.     

Dark hydrogen fermentation from hydrolyzed starch treated with recombinant amylase originating from Caldimonas taiwanensis On1

Starch is one of the most abundant resources on earth and is suited to serve as a cost-effective feedstock for biological hydrogen production. However, producing hydrogen from direct fermentation of starch is usually inefficient, as the starch hydrolysis is often the rate-limiting step. Therefore, in the present work, enzymatic starch hydrolysis was conducted to enhance the feasibility of using starch feedstock for H2 production. The amylase (with a molecular weight of ca. 112 kDa) used for starch hydrolysis was produced from a recombinant E. coli harboring an amylase gene originating from Caldimonas taiwanensis On1. Using statistical experimental design, the optimal pH and temperature for starch hydrolysis with the recombinant amylase was pH 6.86 and 52.4 degrees C, respectively, at an initial starch concentration of 7 g/L. The hydrolyzed products contained mainly glucose, maltotriose, and maltotetrose, while a tiny amount of maltose was also detected. The enzymatically hydrolyzed products of soluble starch and cassava starch were used as the substrate for dark hydrogen fermentation using Clostridium butyricum CGS2 and Clostridium pasteurianum CH4. The highest H2 production rate (vH2) and yield (YH2) of C. butyricum CGS2 was 124.0 mL/h/L and 6.32 mmol H2/g COD, respectively, both obtained with the hydrolysate of cassava starch. The best H2 production rate (63.0 mL/h/L) of C. pasteurianum CH4 occurred when using hydrolyzed cassava starch as the substrate, whereas the highest yield (9.95 mmol H2/g COD) was obtained with the hydrolyzed soluble starch.     

DO-stat fed-batch production of 2-keto-d-gluconic acid from cassava using immobilized Pseudomonas aeruginosa

Bioconversion of cassava-derived glucose to 2-keto-d-gluconic acid (2-KDG) using resting cells of immobilized Pseudomonas aeruginosa IFO 3448 was investigated. The tuberous roots of cassava were selected as the feedstock as they are inexpensive and widely available, and possess high amounts of starch (approximately 70% (w/w) of dry mass). Immobilized bacteria was used in a fed-batch fermenter and recycled over a period of 2 weeks. Given that the formation of 2-KDG from glucose requires oxygen as a reagent, and that high glucose concentrations are detrimental to the production yield of 2-KDG by resting cells, a DO-stat control strategy was used, whereby the feed rate of cassava hydrolysate was regulated by coupling it with the control variable, dissolved oxygen. For 319 h of operation including three cycles of repeated fed batch, 72 g of 2-KDG was produced from hydrolysate derived from 110 g of dried cassava at a maximum production rate of 0.55 g/L/h and an average concentration of 35 g/L.     

Production of ethanol from cassava whey

Investigations were conducted into the potential use of enzyme hydrolysed cassava whey for ethanol production by Saccharomyces cerevisiae Aspergillus niger grown on whct bran was used as crude enzyme source to saccharify the whey starch. The whey with an initial HCN concentration of 54.0μg/ml was fermented at pH 4.5 and 30°C in a one-step process to produce ethanol. A maximum ethanol concentration of 4.5% (v/v) was obtained in 120 h with a decrease in HCN level to 4.0 μg/ml. In a two-stage fermentation, in which the raw whey was pre-hydrolysed and under the same fermentation conditions, the unsterilized hydrolysate yielded alcohol content of 5.5% (v/v), while the sterilized hydrolysate gave higher alcohol yield, 7.5% (v/v), in 48 h. No HCN was detected in the fermented liquour at the end of the two-stage process.     

Optimization of single-cell-protein production from cassava starch using Schwanniomyces castellii

Schwanniomyces castellii B5285 grew faster and produced greater biomass and higher protein yield than either S. alluvius ATCC 26074 or S. alluvius 81Y when these amylolytic yeasts were grown with 2% (w/v) cassava starch as sole C source. With 0.5% (w/v) glutamate as N source, S. castellii reached 7.12 g cell dry mass/l, with a protein yield of 6.4 g/100 g starch. The optimal agitation speed, aeration rate and pH for growth of this yeast in a fermenter were 400 rev/min, 1.67 vol./vol.min. and 5.0, respectively. Tween 80 at 0.1% increased cell dry mass to 8.90 g/l, cell yield to 44 g/100 g starch and protein yield to 7.4 g/100 g starch.The authors are with the Department of industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90110, Thailand