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.     

Correlation of Biological Activity and Reactor Performance in Biofiltration of Toluene with the Fungus Paecilomyces variotii CBS115145

A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m³ of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O₂/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O₂/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms.     

Electro-stimulation of tofu wastewater for the production of single cell protein from various microorganisms

Tofu wastewater can be utilized as a substrate for microorganisms that produce single-cell proteins (SCPs). Because different microorganisms have different cellular components, the composition of SCPs varies. Electro-stimulation has the potential to speed up fermentation and increase product yield. The goal of this study was to find the best way to produce SCPs from Aspergillus awamori, Rhizopus oryzae, and Saccharomyces cerevisiae in the tofu wastewater substrate using electro-stimulation. The experimental method was used in the study, the data were analyzed using independent t-test statistical analysis, and the best treatment was identified using the effective index method. This treatment consisted of producing SCP with electro-stimulation of −1.5 V and without electro-stimulation for 72 h for the yeast and 96 h for the mold at 25 °C in tofu wastewater that had already been conditioned to a pH of 5. The parameters measured included measurement of population of microorganism, change in pH, dry biomass weight, carbohydrate content, and protein content. Electro-stimulation reduced the optimum fermentation time of A. awamori SCP from 56 to 32 h, resulting in 0.0406 g/50 mL of dry biomass, 30.09% carbohydrate content, and 6.86% protein content. Meanwhile, the optimal fermentation time on R. oryzae and S. cerevisiae were not accelerated by electro-stimulation. The best treatment was A. awamori without electro-stimulation, which produced 0.0931 g/50 mL of dry biomass, 20.29% carbohydrate, and 7.55% protein.     

Kinetic behaviour of waste tyre rubber as microorganism support in an anaerobic digester treating cane molasses distillery slops

The kinetics of anaerobic digestion of cane molasses distillery slops was investigated using a continuous-flow bioreactor which contained waste tyre rubber as support, to which the microorganisms became immobilized. Hydraulic retention times (HRT) ranging from 1 to 10 days were investigated at an average influent chemical oxygen demand (COD) concentration of 47.7?g/l. The maximum substrate utilization rate, k, and half saturation coefficient, K _L, were determined to be 1.82?kg COD_removed/kg VSS day and 0.33?kg COD/kg VSS day. The yield coefficient, Y, and sludge decay rate coefficient, K _d, were also determined to be 0.06?kg VSS/kg COD_removed and 0.05?day^-1, respectively. Methane production was maximum (6.75?l/l day) at a 2 day HRT corresponding to a biomass loading rate of 2.578?kg COD/kg VSS day. Biogas yield ranged between 0.51?l/g COD (HRT=2 days) and 0.25?l/g COD (HRT=1?day). In addition, the methane percentage in the biogas varied between 70.5% (HRT=10?days) and 47.5% (HRT=1?day). The close relationship between biomass loading rate and specific substrate utilization rate supported the use of Monod equations. Finally, the experimental values of effluent substrate concentration were reproduced with deviations equal to or less than 10% in every case.     

Conversion of cheese whey to single-cell protein

Thirteen yeast species belonging to nine genera were screened for the production of single-cell protein (SCP) using cheese whey as the substrate. Cheese whey supplemented with minerals and yeast extract proved to the best medium for yield, lactose utilization, biomass production, and conversion efficiency. Production of beta-galactosidase was studied in Brettanomyces anomalus, Kluyveromyces fragilis, Trichosporon cutaneum, and Wingea robertsii; the last proved to be the best strain combining high yield with shorter incubation period.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

SCP production and removal of organic load from cassava starch industry waste by yeasts

Wastewater obtained from the cassava processing industry was used to produce single cell protein (SCP). A few selected strains of yeasts were analysed for the SCP production based on the criteria of best biomass yield and high reduction in Chemical Oxygen Demand (COD) under nitrogen limitation. The monoculturing of Candida tropicalis gave the highest μmax with a protein enhancement of 4 fold and 50% decrease in COD. The performance of cocultures have shown an improvement in the COD removal of the wastewater and in the percent increase of protein. The effects of coculturing were better in a lab fermentor under controlled conditions.     

Temperature profile ofCandida cacaoi SS-2566

Summary Candida cacaoi SS-2566 was found to have a dissociative temperature profile and thermotolerant growth yield behaviour. With temperature-independent growth rates up to 42°C, its utilization at relatively high temperature for the production of cells (SCP, SCL) and/or metabolites may well be of interest, particularly in view of its hydrocarbon utilization.     

Hydrogen utilization by Methylocystis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs

Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H₂ to the culture headspace had the most significant positive effect on the growth yield under CH₄ (6%) and O₂ (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (±0.52) to 13.82 (±0.69) mg cell dry weight per mmol CH₄, while CH₄ consumption was significantly reduced. Regardless of H₂ addition, CH₄ utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O₂/CH₄ mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H₂ addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H₂ (37 d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H₂ may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH_4, such as the production of high-quality feed protein.     

Bioenergetic consequences of glucoamylase production in carbon-limited chemostat cultures ofAspergillus niger

Aspergillus niger has been grown in glucose- and maltose-limited continuous cultures to determine the bioenergetic consequences of the production of the extracellular enzyme glucoamylase. Growth yields (g biomass per mol substrate) were high, indicating that growth was very efficient and protein production for biomass was not exceedingly energy consuming. It has been found that the energy costs for the production of this extracellular enzyme is very high. Depending on the efficiency of energy conservation the glucoamylase protein yield on ATP is between 1.3 and 2.6 g protein per mol ATP, which is equal or less than 10% of the theoretical maximum of 25.5. These high energy costs most probably have to be invested in the process of excretion. A comparison between an industrial over-producing strain and the wild typeAspergillus niger showed that this over-producing strain most probably is a regulatory mutant. Two regions of specific growth rates could be determined (one at specific growth rates lower and one at specific growth rates higher than 0.1 h^-1), which are characterized by differences in mycelium morphology and a significant deviation from linearity in the linear equation for substrate utilization. Analysis of the region of specific growth rates higher than 0.1 h^-1 yielded maintenance requirements of virtual zero. It has been concluded that for a good analysis of the growth behaviour of filamentour fungi the linear equation for substrate utilization is not suitable, since it contains no term for the process of differentiation.     

Production of single cell protein from agro-waste using <Emphasis Type="Italic">Rhodococcus opacus</Emphasis>

Livestock and fish farming are rapidly growing industries facing the simultaneous pressure of increasing production demands and limited protein required to produce feed. Bacteria that can convert low-value non-food waste streams into singe cell protein (SCP) present an intriguing route for rapid protein production. The oleaginous bacterium Rhodococcus opacus serves as a model organism for understanding microbial lipid production. SCP production has not been explored using an organism from this genus. In the present research, R. opacus strains DSM 1069 and PD630 were fed three agro-waste streams: (1) orange pulp, juice, and peel; (2) lemon pulp, juice, and peel; and (3) corn stover effluent, to determine if these low-cost substrates would be suitable for producing a value-added product, SCP for aquafarming or livestock feed. Both strains used agro-waste carbon sources as a growth substrate to produce protein-rich cell biomass suggesting that that R. opacus can be used to produce SCP using agro-wastes as low-cost substrates.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

Evaluation of hardboard manufacturing process wastewater as a feedstream for ethanol production

Waste streams from the wood processing industry can serve as feedstream for ethanol production from biomass residues. Hardboard manufacturing process wastewater (HPW) was evaluated on the basis of monomeric sugar recovery and fermentability as a novel feedstream for ethanol production. Dilute acid hydrolysis, coupled with concentration of the wastewater resulted in a hydrolysate with 66 g/l total fermentable sugars. As xylose accounted for 53 % of the total sugars, native xylose-fermenting yeasts were evaluated for their ability to produce ethanol from the hydrolysate. The strains selected were, in decreasing order by ethanol yields from xylose (Y _p/s, based on consumed sugars), Scheffersomyces stipitis ATCC 58785 (CBS 6054), Pachysolen tannophilus ATCC 60393, and Kluyveromyces marxianus ATCC 46537. The yeasts were compared on the basis of substrate utilization and ethanol yield during fermentations of the hydrolysate, measured using an HPLC. S. stipitis, P. tannophilus, and K. marxianus produced 0.34, 0.31, and 0.36 g/g, respectively. The yeasts were able to utilize between 58 and 75 % of the available substrate. S. stipitis outperformed the other yeast during the fermentation of the hydrolysate; consuming the highest concentration of available substrate and producing the highest ethanol concentration in 72 h. Due to its high sugar content and low inhibitor levels after hydrolysis, it was concluded that HPW is a suitable feedstream for ethanol production by S. stipitis.     

Bioconversion of acid- and gamma-ray-treated sweet potato residue to microbial protein by mixed cultures

Sweet potato residue, a starchy agricultural waste, was used as a substrate to produce microbial protein by Fusarium moniliforme and Saccharomyces cerevisiae in submerged fermentation. Acid- and gamma-irradiation-pretreated sweet potato residue enhanced the biomass yield and protein production when the residue was fermented with F. moniliforme and S. cerevisiae. A mixed culture of F. moniliforme and S. cerevisiae efficiently and rapidly utilized free sugars; the maximal biomass yield (13.96 g/l) and protein production (65.8%) were obtained after 3 days fermentation. Lower carbon utilization by the two microbial strains occurred in the waste-containing media as compared to control, increasing the economic value of the waste usage. Received 25 October 2001/ Accepted in revised form 22 June 2002     

Production of SCP and cellulase by Aspergillus terreus from bagasse substrate

The fermentation of 1.0% untreated bagasse under optimum cultural and nutritional conditions with Aspergillus terreus GN1 indicated that the maximum rate of protein and cellulase production could be obtained during three days of submerged fermentation. Even though 16.4% protein recovery, 0.55 units CMCase/mL, and 0.027 FPase units/mL were obtained on the seventh day, the rates of increase in protein recovery and cellulase production were slower than those obtained up to these days, which were 14.3% protein recovery, 0.45 units CMCase/mL, and 0.019 units FPase/mL. There was an initial lag in the utilization of cellulose up to two days due to the utilization of the water-soluble carbohydrate present in untreated bagasse. Cellulose utilization and water-soluble carbohydrate content during fermentation were correlated with protein recovery and enzyme production. The protein and cellulase production during three days fermentation with 1.0% untreated and treated bagasse were compared and the protein content of the total biomass was calculated and treated bagasse were compared and the protein content of the biomass was calculated into constituent protein contributed by the fungal mycelium and the under graded bagasse. The total biomass recovered with untreated and treated bagasse was 1020 and 820 mg/g bagasse substrate, respectively, and contained 14.3 and 20.6% crude protein, respectively. The contribution of fungal biomass and under graded bagasse was 309 and 711, and 373 and 447 mg/g untreated and treated bagasse substrates, respectively. In an 8-L-flask trial during three days of fermentation, the recovery of SCP and cellulase were 66 g and 32,400 units (Sigma) for treated bagasse and 82 g and 8200 units (Sigma) for untreated bagasse, respectively.     

Utilization of oil extracted from spent coffee grounds for sustainable production of polyhydroxyalkanoates

Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R ²?=?0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y _P/S yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the process.     

Effect of dilution rates on the amount of wasted substrate during alcohol fermentation

Continuous ethanol production in a one-stage continuous stirred tank fermentor without recycle was carried out using a yeast strain Saccharomyces cerevisiae. Different dilution rates were used. Cell and ethanol concentrations in the culture medium decreased with increasing dilution rates, and the maximum value of 3.0 g l⁻¹h⁻¹was found at a dilution rate of 0.340 h⁻¹. Specific ethanol productivities increased as dilution rates were increased, and the highest value appeared at about the same dilution rate as that for the maximum fermentor productivity. A material balance equation, which relates total amount of spent medium to cell synsthesis, ethanol production, and overall maintenance, was introduced. The cellular yield and overall maintenance coefficients increased with increasing dilution rates. The fraction of limiting substrate utilized for overall maintenance, which includes the limiting substrate spent for purposes other than cell synthesis and ethanol production, decreased with increasing dilution rates. The non-product associated substrate utilization can be minimized if correct dilution rate is chosen.     

Optimization of solid substrate fermentation of wheat straw

Optimal conditions for solid substrate fermentation of wheat straw with Chaetomium cellulolyticum in laboratory-scale stationary layer fermenters were developed. The best pretreatment for wheat straw was ammonia freeze explosion, followed by steam treatment, alkali treatment, and simple autoclaving. The optimal fermentation conditions were 80% (w/w) moisture content; incubation temperature of 37 degrees C; 2% (w/w) unwashed mycelial inoculum; aeration at 0.12 L/h/g; substrate thickness of 1 to 2 cm; and duration of three days. Technical parameters for this optimized fermentation were: degree of substance utilization, 27.2%; protein yield/substrate, 0.09 g; biomass yield/bioconverted substrate, 0.40 g; degree of bioconversion of total available sugars in the substrate, 60.5%; specific efficiency of bioconversion, 70.8%; and overall efficiency of biomass production from substrate, 42.7%. Mixed culturing of Candida utilis further increased biomass production by 20%. The best mode of fermentation was a semicontinuous fed-batch fermentation where one-half of the fermented material was removed at three-day intervals and replaced by fresh substrate. In this mode, protein production was 20% higher than in batch mode, protein productivity was maintained over 12 days, and sporulation was prevented.     

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on apple processing wastes

Sequential cultures of the yeasts Saccharomycopsis fibuliger and Candida utilis were grown on selected wastes from the processing of apples. Effluent from cider manufacture supported the growth of 45.4 g cells/100 g substrate and C. utilis formed 96% of the viable cells in the harvested biomass. Whole, unripe apples yielded 44 g cells/100 g substrate with a reduction in the substrate viscosity of 84%. C. utilis formed 56% of the viable cells in the harvested biomass. Effluent from pectin manufacture contained a substantial proportion of reducing compounds and supported the growth of C. utilis without prehydrolysis by S. fibuliger, to yield 33 g cells/100 g substrate.     

Enhanced cellulase production through random mutagenesis of Talaromyces pinophilus OPC4-1 and fermentation optimization

Reducing cellulase cost remains a major challenge for lignocellulose to fuel and chemical industries. In this study, mutants of a novel wild-type cellulolytic fungal strain Talaromyces pinophilus OPC4-1 were developed by consecutive UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG) and ethylmethane sulfonate (EMS) treatment. A potential mutant EMM was obtained and displayed enhanced cellulase production. Using Solka Floc cellulose as the substrate, through fed-batch fermentation, mutant strain T. pinophilus EMM generated crude enzymes with an FPase activity of 27.0 IU/mL and yield of 900 IU/g substrate. When corncob powder was used, strain EMM produced crude enzymes with an FPase activity of 7.3 IU/mL and yield of 243.3 IU/g substrate. In addition, EMM crude enzymes contained 29.2 and 16.3 IU/mL β-glucosidase on Solka Floc cellulose and corncob power, respectively. The crude enzymes consequently displayed strong biomass hydrolysis performance. For corncob hydrolysis, without supplement of any commercial enzymes, glucose yields of 591.7 and 548.6 mg/g biomass were obtained using enzymes produced from Solka Floc cellulose and corncob powder, respectively. It was 553.9 mg/g biomass using the commercial enzyme mixture of Celluclast 1.5 L and Novozyme 188. Strain T. pinophilus EMM was therefore a potential fungus for on-site enzyme production in biorefinery processes.