SCP production by mixed culture of Rhodocyclus gelatinosus and Rhodobacter sphaeroides from Cassava Waste

The amylolytic enzymes produced by Rhodocyclus gelatinosus hydrolyzed cassava starch mainly to maltose and a small amount of glucose. The organism utilized maltose at the specific growth rate of 0.15 l/h, but in the presence of glucose, maltose consumption rate was retarded. Therefore, a series of mixed cultures was conducted with Rhodobacter sphaeroides P47, which showed a high growth rate of 0.2 l/h on glucose and contained 29.5 μg/g cell of vitamin B12 and 0.49 mg/g cell of carotenoid compared with the 18.4 μg/g cell and 0.23 mg/g cell respectively of Rc. gelatinosus. Mixed cultures with three different inoculum ratios of the two organisms based on cell number all gave higher growth yields and contents of vitamin B12 and carotenoid in the total cell mass than single cultures. When the inoculum ration of Rc. gelatinosus to Rb. sphaeroides P47 was over 1.0, the culture time was shortened due to the synergistic effect of sugar consumption. Therefore, it was suggested that the mixed culture of these two organisms would be practically profitable for more nutritive SCP production from cassava waste.     

Production of salmosin, a snake venom-derived disintegrin, in recombinant Pichia pastoris using high cell density fed-batch fermentation

Salmosin, a snake venom-derived disintegrin, was successfully expressed in the methylotrophic yeast Pichia pastoris and secreted into the culture supernatant, as a 6 kDa protein. High-cell density fermentation of recombinant P. pastoris was optimized for the mass production of salmosin. In a 5 L jar fermentor, recombinant P. pastoris was fermented in growth medium containing 5% (w/v) glycerol at the controlled pH of 5.0. After culturing for 21 h, glycerol feeding medium was fed at one time into the culture broth. After 7 h (a total of 28 h), induction medium that contained methanol was increasingly added until the culture time totaled 75 h. Finally, these optimized culture conditions produced a high cell density of recombinant P. pastoris (dry cell weight of 113.38 g/L) and led to the mass production of salmosin (a total protein concentration of 369.2 mg/L). The culture supernatant containing salmosin inhibited platelet aggregation, resulting in a platelet aggregation of 9% compared to that of 94% in the control experiment, without culture supernatant. These results demonstrate that recombinant salmosin in culture supernatant from high cell density fed-batch fermentation can serve as a platelet aggregation inhibitor.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

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     

Cultivation of thermophilic methanogen KN-15 on H2-CO2 under pressurized conditions

Cultivation of the thermophilic methanogen KN-15 was carried out under pressurized batch and continuous conditions. In pressurized batch culture, both the turning point at which cell growth changed from exponential to linear and the growth rate during the linear growth phase increased with the rise of total pressure of the gas phase in the fermentor. The cell concentration reached 18.5 g dry cell/l after 10 h of batch cultivation under 3.0 × 10⁵ Pa pressurized conditions. Under pressurized continuous conditions, it was also observed that the cell concentration and cell productivities increased with the rise of total pressure of the gas phase. Cell and methane productivities of 3.0 g dry cell/l/h and 1.28 mol/l/h, respectively, were achieved in 3.0 × 10⁵ Pa pressurized continuous culture. According to the results from the Monod model application, the achievable cell productivities (V_max) and the Monod type saturation constant for cell productivities (K_s^′) were 12.8 g dry cell/l/h and 9.7 × 10⁵ Pa, respectively.     

A factorial approach for a sugarcane juice-based low cost culture medium: increasing the astaxanthin production by the red yeast Phaffia rhodozyma

A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300?μg astaxanthin/g of dry yeast and 6500?μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26?°C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418?μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987?μg/l).     

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45° to 47°C, maximum protein yield at 45°C, and no growth at 25°C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45°C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10⁶ to 10⁸ per animal) injected by various routes into normal and γ-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation

The potential application of dry biomass of a cyanobacterium Anacystis nidulans as a supplement in SSF for the production of laccase from Pleurotus ostreatus was evaluated. Experiments were carried out in solid culture using groundnut shell as a basic substrate supplemented with four independent nitrogen sources (ammonium sulphate, urea, yeast extract and dry powder of cyanobacteria). All the four supplements enhanced the enzyme yield, and yeast extract showed precedence over inorganic nitrogenous sources. However, when dry biomass of A. nidulans was used as an additive to groundnut shell (agricultural residues), it supported maximum cell growth (56.83 ± 5.56 mg/g dry substrate) and laccase production (49.21 ± 4.89 U/g dry substrate). Addition of 1 mM copper salt in the medium containing groundnut shell supplemented with yeast extract gave laccase activity of 32.64 ± 3.4 U/g dry substrate. When dry powder of cyanobacterial biomass was used as N-supplement, laccase production enhanced to 65.42 ± 6.48 U/g dry substrate. In addition to the enhancement to enzyme production inhibitory effects of high concentrations of copper was also diminished in the medium having dry cyanobacterial biomass. This study, forms the first report on the potential application of cyanobacterial biomass as an additive for production of laccase by Pleurotus ostraetus MTCC 1804 in solid state fermentation and has relevance in scale-up production of this fungal enzyme of commercial significance.     

Utilization of cellulose from waste paper by Myrothecium verrucaria

Extensive screening studies on cellulolytic bacteria and fungi led to the selection of Myrothecium verrucaria as the organism producing the maximum rate of protein biosynthesis from ball-milled newspaper. Studies in aerated stirred-jar fermentors were carried out to determine the conditions for maximum protein synthesis rate and maximum final protein concentration. The optimum aeration rate was 250 to 374 mM of oxygen at 300 to 400 rpm stirring rate. The pH optimum was broad, from 3.9 to 6.5. Urea at 0.03% and yeast autolysate at 0.1% stimulated growth rate and protein production. The maximum rate of protein biosynthesis and the maximum protein yield were 0.3 g/liter/day and 1.42 g/liter, respectively, from medium G3 with 4% ball-milled newspaper. The final product, obtained by evaporation of the total culture, was 33.7 g from one liter of medium which originally contained 40 g of ball-milled newspaper and 11.3 g of other dissolved materials. The protein content of this final product was 3.3 g, calculated from total organic N × 6.25 or 1.42 g calculated from the biuret method. Both the synthesis rate and the final cell yield are below those obtainable by growing Fungi Imperfecti, yeasts or bacteria on soluble materials such as glucose.     

Continuous single cell protein production fromCellulomonas sp. andCandida utilis grown in mixture on barley straw

Summary Continuous fermentations with mixed cultures of the cellulolytic bacteriaCellulomonas sp. and the yeastCandida utilis were examined. Fermentations were carried out in an aerated 5-l fermenter with different preparations of wet disintegrated barley straw as the cellulose source (3.6–4.2%). The straw was pretreated with NaOH (3.2–8.5 kg NaOH/100 kg dry straw) under high pressure and temperature in a feedstuff pellet press. The quantity of dry cell mass produced and the breakdown of the straw were measured. Crude protein and ash content in cell dry matter and residual fiber were determined. The experiments showed thatCellulomonas sp. andCandida utilis may be grown together in a continuous culture (dilution rate D=0.12–0.14 h^–1) for at least 3 days without washing out one of the organisms. Highest productivity was 1.39 g cell dry matter/l/h when using straw pretreated with 5.7% NaOH. The dry cell product contained 58–66% crude protein and up to 51% of the organic fiber dry matter was solubilized. The yield constants were 0.32–0.61 g cell dry matter per g solubilized organic fibers.     

Extended culture: The growth of Candida utilis at controlled acetate concentrations

Extended culture, a special type of semicontinuous culture, permits prolonged maintenance of a constant or programmed environment in a growing culture by a controlled addition of one or more substrates. Differences between extended culture and continuous culture data are a measure of differences in the properties of cell populations with different cell age distributions but identical steady-state environments. Both extended culture and continuous culture were used to study the growth kinetics of Candida utilis (ATCC 9226) under conditions of substrate inhibition at controlled concentrations of sodium acetate in a carbon-limited mineral salts medium supplemented with 0.01 g/1 yeast extract. Acetate concentrations ranged from 1.2 g/l to 10.8 g/l (expressed as acetic acid), while yeast concentrations varied from 0.3 to 7.8 (g dry cells)/1. Rate parameters such as growth yields (Y), specific growth rates (μ), and linear growth rates (K), were calculated by computer from the data and theory presented herein. Specific growth rates as high as 0.54/hr were observed, although extended culture growth was more nearly linear than exponential in these experiments. Growth yields usually varied between 0.2 and 0.4 (g dry cells)/(g acetate), although values were as high as 0.8 for a brief period during one experiment. Growth yields at a given acetate concentration were correlated by an equation of the form 1/Y = 1/Y_G + m/μ. A maintenance coefficient (m) of 0.17 (g acetate)/(g dry cell-hr) was observed at acetate concentrations of 4.5 and 10. g/1. A typical maximum growth yield (Y_G) of 0.51 (g dry cell)/(g acetate) was obtained at 4.5 g/1 acetate, but an unusually high Y_G of 1.33 was found at 10. g/1 acetate. Oxygen uptake measurements are compared with these cell yield measurements. Linear growth rates in expended culture were correlated by the equation K = 0.89–0.70 (S/S₀) where K has units of (g dry cell)/(l-hr), S is the instantaneous acetate concentration, and S₀ is the initial acetate concentration. The extended culture kinetic data are shown to be substantially different from continuous culture kinetic data. Reason for these differences are discussed in light of diffrences in the cell age distributions, as well as possible differences in experimental conditions.     

Obtaining and Study of Callus and Suspension Plant Cell Cultures of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> L., a Producer of Steroidal Glycosides

Callus and suspension plant cell cultures of Tribulus terrestris L., a valuable medicinal plant producing steroidal glycosides, were obtained. The seeds from an American population of T. terrestris were used as explants. Regulation of the production and growth of cell cultures, as well as the biosynthetic characteristics of the cell lines, were studied. The combination of phytohormones of 2,4-D (2.0 mg/L) and BAP (1.0 mg/L) was found to be optimal for callus induction and cultivation. Suspension cell culture obtained in liquid medium of the same composition showed such high growth characteristics during prolonged cultivation (more than 2 years) as a maximum accumulation of dry biomass of 13 g/L, specific growth rate at exponential phase of 0.24 day^–1, and economical coefficient of 0.39. A semicontinuous mode of cultivation was used to grow the plant cell suspension in a lab-scale bioreactor. Screening of the steroidal glycosides in the obtained cell cultures was carried out. Steroidal glycosides were not found in the callus cultures. However, as was demonstrated by TLC and UPLC ESI MS methods, the suspension culture contained furostanol glycosides, and their amount increased during the cultivation process. These results support the hypothesis of the autoselection of cultivated cells containing compounds promoting their proliferation in vitro.     

Selenium-enriched Candida utilis: Efficient preparation with l-methionine and antioxidant capacity in rats

Selenium-enriched Candida utilis has attracted much attention due to its expanding application in food and feed additives. The objective of this study was to efficiently prepare selenium-enriched C. utilis and to investigate the effects of the prepared yeast on antioxidant capacity in rats. A batch culture of selenium-enriched C. utilis was first carried out, and the addition of sodium selenite (Na₂SeO₃) after all glucose had been consumed was found to favor higher intracellular glutathione and organic selenium content. Moreover, l-methionine boosted yeast cell growth and glutathione biosynthesis, and prevented glutathione from leaking to the extracellular space that can be caused by Na₂SeO₃. We therefore developed a two-stage culture strategy involving supplementation with l-methionine and Na₂SeO₃ at separate culture phases to improve the performance of selenized C. utilis. Using this two-stage culture strategy, intracellular glutathione content reached 18.6 mg/g and 15.5 mg/g, respectively, in batch and fed-batch systems, and organic selenium content reached 905.2 μg/g and 984.7 μg/g, respectively. The effects of selenium-enriched C. utilis on the activities of antioxidant related enzymes in rats were investigated, and the prepared selenium-enriched C. utilis was shown to be an optimal dietary supplement for enhancing antioxidant capacity in rats.     

Mathematical model for aerobic culture of a recombinant yeast

A mathematical model was formulated to simulate cell growth, plasmid loss and recombinant protein production during the aerobic culture of a recombinant yeast S. cerevisiae. Model development was based on three simplified metabolic events in the yeast: glucose fermentation, glucose oxidation and ethanol oxidation. Cell growth was expressed as a composite of these metabolic events. Their contributions to the total specific growth rate depended on the activities of the pacemaker enzyme pools of the individual pathways. The pacemaker enzyme pools were regulated by the specific glucose uptake rate. The effect of substrate concentrations on the specific growth rate was described by a modified Monod equation. It was assumed that recombinant protein formation is only associated with oxidative pathways. Plasmid loss kinetics was formulated based on segregational instability during cell division by assuming constant probability of plasmid loss. Experiments on batch fermentation of recombinant S. cerevisiae C468/pGAC9 (ATCC 20690), which expresses Aspergillus awamori glucoamylase gene and secretes glucoamylase into the extracellular medium, were carried out in an airlift bioreactor in order to evaluate the proposed model. The model successfully predicted the dynamics of cell growth, glucose consumption, ethanol metabolism, glucoamylase production and plasmid instability. Excellent agreement between model simulations and our experimental data was achieved. Using published experimental data, model agreement was also found for other recombinant yeast strains. In general, the proposed model appears to be useful for the design, scale-up, control and optimization of recombinant yeast bioprocesses.     

Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His₆) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.     

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.     

Effect of culture conditions of Kluyveromyces marxianus on its autolysis, and process optimization

Cultivation of the lactose-metabolizing yeast Kluyveromyces marxianus var. marxianus (formerly K.?fragilis) on supplemented whey permeate resulted in cellular yield little affected by culture conditions in the ranges pH?=?2.3–5 and T?=?30–40?°C. When autolysis was induced only by energy source deficiency and thermal shock, cellular material solubilization depended slightly on autolysis temperature in the range T?=?45–60?°C. On the contrary, the process was under tight control of culture conditions; when autolysis was carried out at 50?°C with an initial dry cellular concentration of 50?g l^?1, a clear optimum was observed for cells cultivated at pH?=?4.5 and T?=?35?°C. So the critical step of the autolytic process consisted in biosynthesis of lytic enzymes (during cell growth) rather than enzymatic progress (during autolysis). These results were compatible with a model previously proposed for Saccharomyces cerevisiae [1].     

Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on alpha-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His(6)) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-alpha-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley beta-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and beta-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing beta-glucan as the sole carbon source and could directly ferment 45 g of beta-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.