Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanol by Pichia stipitis

Fermentation of acid hydrolysate of water-hyacinth (Eichhornia crassipes), a free floating aquatic plant has been investigated for ethanol production. The dilute acid treatment has been applied to utilize the maximum hemicellulosic content of the water-hyacinth. The goal of this work was to investigate, both experimentally and theoretically using mathematical tools, a fermentative system utilizing water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate as a substrate for ethanol production using Pichia stipitis. It was found that 72.83% of xylose was converted to ethanol with a yield of 0.425 g_p/g_s and productivity of 0.176 g_p/L/h. An appropriate mathematical model was developed to explain theoretically the bioconversion of this hemicellulose acid hydrolysate to ethanol and the model was tested statistically to check the validity of the model.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Crabtree-negative characteristics of recombinant xylose-utilizing Saccharomyces cerevisiae

For recombinant xylose-utilizing Saccharomyces cerevisiae, ethanol yield and productivity is substantially lower on xylose than on glucose. In contrast to glucose, xylose is a novel substrate for S. cerevisiae and it is not known how this substrate is recognized on a molecular level. Failure to activate appropriate genes during xylose-utilization has the potential to result in sub-optimal metabolism and decreased substrate uptake. Certain differences in fermentative performance between the two substrates have thus been ascribed to variations in regulatory response. In this study differences in substrate utilization of glucose and xylose was analyzed in the recombinant S. cerevisiae strain TMB3400. Continuous cultures were performed with glucose and xylose under carbon- and nitrogen-limited conditions. Whereas biomass yield and substrate uptake rate were similar during carbon-limited conditions, the metabolic profile was highly substrate dependent under nitrogen-limited conditions. While glycerol production occurred in both cases, ethanol production was only observed for glucose cultures. Addition of acetate and 2-deoxyglucose pulses to a xylose-limited culture was able to stimulate transient overflow metabolism and ethanol production. Application of glucose pulses enhanced xylose uptake rate under restricted co-substrate concentrations. Results are discussed in relation to regulation of sugar metabolism in Crabtree-positive and -negative yeast.     

Growth characteristics and oxidative capacity of<Emphasis Type="Italic"> Acetobacter aceti</Emphasis> IFO 3281: implications for <Emphasis Type="SmallCaps">l</Emphasis>-ribulose production

We studied the growth characteristics and oxidative capacities of Acetobacter aceti IFO 3281 in batch and chemostat cultures. In batch culture, glycerol was the best growth substrate and growth on ethanol occurred only after 6 days delay, although ethanol was rapidly oxidized to acetic acid. In continuous culture, both glycerol and ethanol were good growth substrates with similar characteristics. Resting cells in a bioreactor oxidized ribitol to l-ribulose with a maximal specific rate of 1.2 g g^–1 h^–1). The oxidation of ribitol was inhibited by ethanol but not by glycerol. Biomass yield (Y_SX; C-mmol/C-mmol) on ethanol and glycerol was low (0.21 and 0.17, respectively). In the presence of ribitol the yield was somewhat higher (0.25) with ethanol but lower (0.13) with glycerol, with respectively lower and higher CO₂ production. In chemostat cultures the oxidation rate of ribitol was unaffected by ethanol or glycerol. Cell-free extract oxidized ethanol very slowly but not ribitol; the oxidative activity was located in the cell membrane fraction. Enzymatic activities of some key metabolic enzymes were determined from steady-state chemostat with ethanol, glycerol, or ethanol/glycerol mixture as a growth limiting substrate. Based on the measured enzyme activities, metabolic pathways are proposed for ethanol and glycerol metabolism.     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.     

Effects of the carbon source and the interaction between carbon sources on the physiology of the industrial Saccharomyces cerevisiae CAT-1

Abstract

During industrial fermentation, wild isolates are able to persist and even predominate in the bioreactors. Saccharomyces cerevisiae CAT-1 was one of these isolates and now is one of the yeasts mostly used in industrial ethanol processes in Brazil due to its efficient fermentation capacity. Despite it, the strain’s physiology has been marginally studied so far. Since strains of the same species may have different responses to a specific cultivation condition, this work aimed to evaluate the physiology of S. cerevisiae CAT-1 in batch cultures using different carbon sources (glucose, fructose, sucrose, maltose, and galactose) as a sole carbon source and in binary mixtures, at 30 and 37?°C. The results showed that the fructose, sucrose, and maltose were the sugars that presented the highest ethanol yields on the substrate (0.40?g_ethanol g_substrate^?1) at both temperatures. Galactose was the sugar that the yeast had the lowest affinity given the lowest maximum specific growth rate (0.28?h^?1). Despite the influence of a variety of mechanisms for sugar transport, the cells consume first substrates with fewer metabolic steps to catabolism and are susceptible to adaptive evolution depending on the availability of substrate.     

Simultaneous fermentation and isomerization of xylose

Summary Ethanol was produced from xylose by converting the sugar to xylulose, using commercial xylose isomerases, and simultaneously converting the xylulose to ethanol by anaerobic fermentation using different yeast strains. The process was optimized with the yeast strain Schizosaccharomyces pombe (Y-164). The data show that the simultaneous fermentation and isomerization of 6% xylose can produce final ethanol concentrations of 2.1% w/v within 2 days at temperatures as high as 39°C.Nomenclature SFIX simultaneous fermentation and isomerization of xylose - V _p volumetric production (g ethanol·l^-1 per hour) - Q _p specific rate (g ethanol·g^-1 cells per hour) - Y _s yield from substrate consumed (g ethanol, g^-1 xylose) - ET ethanol concentration (% wt/vol) - XT xylitol concentration (% wt/vol) - Glu glucose - Xyl xylose - --m maximum - --f final     

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.     

Apparent loss of sugar transport activity inSaccharomyces cerevisiae may mainly account for maximum ethanol production during alcoholic fermentation

Summary Maximum ethanol productions of two enological yeast strains (Saccharomyces cerevisiae K1 and 738-2) were compared during alcoholic fermentation under conditions where substrate was not a limiting factor. Although strain 738-2 seemed to exhibit the lowest sensitivity to ethanol, the strain K1 showed a higher production of ethanol, and a higher CO₂ production rate in presence of ethanol than the strain 738-2.The main differences between these two strains were their kinetics of apparent loss of the hexose transport activity: this phenomenon is sufficient to explain the observed differences in maximum ethanol production. Moreover, these kinetics seemed to be biphasic for the strain K1. This result may be an indication of the existence of two different low-affinity components of hexose transport system in this strain.     

Filipin and its interaction with cholesterol in aqueous media studied using static and dynamic light scattering

Aggregation of filipin in aqueous medium and filipin-induced changes in cholesterol micelles have been studied using intensity and dynamic light scattering. The dependencies of filipin aggregate dimensions on concentration, solvent, and temperature were studied, and revealed that the aggregates do not have a well-defined geometry, i.e., a critical micelle concentration cannot be detected and stable structures are not formed. The aggregates are of size R_g ≈ 110 nm and R_h ≈ 63 nm, referring to the radius of gyration and hydrodynamic radius, respectively. In the concentration range studied (1 μM < C < 30 μM), a low molecular weight species (monomer/dimer) is always present together with the aggregates. In ethanol/ water mixtures, large (R_g ≈ 500 nm), narrow distribution aggregates are formed in the water volume fraction range 0.45 < Φ_H2_O < 0.65. Aggregation also occurs on changing the temperature; In the range 7–37°C, smaller aggregates (10–30 nm) form and the process is only partially reversible. No pronounced effect of filipin on the structure of the cholesterol micelles was observed (a small increase in R_g and R_h is noted). These results rule out any “specificity” for the filipin interactions with cholesterol, which has been considered a key event in the filipin biochemical mode of action. A reevaluatiori Of this question is suggested and some alternatives are advanced. © 1994 John Wiley & Sons, Inc.     

Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production

Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification. Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase, 0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g_p/g_s by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO₂ released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail was good, and enzymatic hydrolyzate could be fermented to produce ethanol.     

Limitations in substrate utilization efficiency by Zymomonas mobilis

Summary Optimal growth conditions for Zymomonas mobilis have been established using continuous cultivation methods. Optimal substrate utilization efficiency occurs with 2.5 g l^–1 yeast extract, 2.0 g l^–1 ammonium sulfate and 6.0 g l^–1 magnesium sulfate in the media. Catabolic activity is at its maximum with glucose uptake rates of 16–18 g l^–1 h^–1 and ethanol production rates of 8–9 g l^–1 h^–1, Q_g values of 22–26 and Q_p values between 11 and 13, which results in 40 g l^–1 h^–1 ethanol yields using a 100 g l^–1 substrate feed. Any increase in these parameters goes on cost of substrate utilization efficiency. Calcium pantothenate can not substitute yeast extract.Abbreviations G Glucose (%) - Pant Calcium pantothenate (mg l^–1) - D Dilution rate (h^–1) - NH₄ Ammonium sulfate (%) - M_g Magnesium sulfate (%) - S₁ Residual glucose in the fermenter (g l^–1) - S₀ Glucose feed (g l^–1) - Eth Ethanol concentration (g l^–1) - GUR Glucose uptake rate (g l^–1 h^–1) - Q_g Specific glucose uptake rate (g g^–1 h^–1) - Q_p Specific ethanol production rate (g g^–1 h^–1) - EPR Ethanol production rate (g l^–1 h^–1) - Y_g Yield coefficient for glucose (g g^–1) - Y_p Conversion efficiency (%) - C Biomass concentration (g l^–1) Present address: (Until June 1982) Institut für Mikrobiologie, TH Darmstadt, 6100 Darmstdt, Federal Republic of Germany     

abetd-Xylose metabolism in Aureobasidium pullulans: effects of aeration and vitamins

Summary The yeast-like organism Aureobasidium pullulans efficiently converted abetd-xylose to cell mass (Y _X/S=0.45 g·g^–1) with negligible production of polyols (Y _P/S=0.003 g·g^–1) under aerobic conditions. A. pullulans grown semiaerobically exhibited different fermentation capacities in seven basal (vitaminless) medium and medium containing a mixture of seven vitamins. It was found that under semiaerobic conditions a mixture of vitamins significantly enhanced production of ethanol from abetd-xylose, resulting in a 15-fold higher yield coefficient of ethanol (Y _E/S=0.22 g·g^–1) as compared to that achieved in vitaminless medium. This increase in ethanol production was accomplished at the expense of cell mass. A. pullulans produced extremely low amounts of polyols throughout all aerobic and semiaerobic experiments. A. pullulans displayed strictly NADPH-linked xylose reductase and NAD⁺-linked xylitol dehydrogenase activities.     

Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405

We have investigated hydrogen (H₂) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H₂-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H₂, carbon dioxide (CO₂), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H₂/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H₂, CO₂, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H₂-production continued even in stationary phase and H₂/CO₂ ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H₂ production rate of 14.6 mmol g dry cell⁻¹ h⁻¹ was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.     

Fermentation of hexoses and pentoses from sugarcane bagasse hydrolysates into ethanol by Spathaspora hagerdaliae

The present study evaluated 13 strains of yeast for ethanol and xylitol production from xylose. Among them, Spathaspora hagerdaliae UFMG-CM-Y303 produced ethanol yields (Y_P/S) of 0.25 g g^− 1 and 0.39 g g^− 1 under aerobic and microaerophilic conditions, respectively, from a mixture of glucose and xylose in flasks. A pH of 5.0 and an inoculum of 3.0 × 10⁸ cells mL^− 1r resulted in the highest ethanol yields. These conditions were tested in a bioreactor for fermenting a medium containing an enzymatic hydrolysate of sugarcane bagasse with 15.5 g L^− 1 of glucose and 3 g L^− 1 of xylose, and achieved a Y_P/S of 0.47 g g^− 1, in relation to total available sugar. These results suggest that S. hagerdaliae UFMG-CM-Y303 has potential for use in second-generation ethanol studies.

     

The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis

Summary The fermentation by Candida shehatae and Pichia stipitis of xylitol and the various sugars which are liberated upon hydrolysis of lignocellulosic biomass was investigated. Both yeasts produced ethanol from d-glucose, d-mannose, d-galactose and d-xylose. Only P. stipitis fermented d-cellobiose, producing 6.5 g·l^-1 ethanol from 20 g·l^-1 cellobiose within 48 h. No ethanol was produced from l-arabinose, l-rhamnose or xylitol. Diauxie was evident during the fermentation of a sugar mixture. Following the depletion of glucose, P. stipitis fermented galactose, mannose, xylose and cellobiose simultaneously with no noticeable preceding lag period. A similar fermentation pattern was observed with C. shehatae, except that it failed to utilize cellobiose even though it grew on cellobiose when supplied as the sole sugar. P. stipitis produced considerably more ethanol from the sugar mixture than C. shehatae, primarily due to its ability to ferment cellobiose. In general P. stipitis exhibited a higher volumetric rate and yield of ethanol production. This yeast fermented glucose 30–50% more rapidly than xylose, whereas the rates of ethanol production from these two sugars by C. shehatae were similar. P. stipitis had no absolute vitamin requirement for xylose fermentation, but biotin and thiamine enhanced the rate and yield of ethanol production significantly.Nomenclature _max Maximum specific growth rate, h^-1 - Q _p Maximum volumetric rate of ethanol production, calculated from the slope of the ethanol vs. time curve, g·(l·h)^-1 - q _p Maximum specific rate of ethanol production, g·(g cells·h) - Y _p/s Ethanol yield coefficient, g ethanol·(g substrate utilized)^-1 - Y _x/s Cell yield coefficient, g biomass·(g substrate utilized)^-1 - E Efficiency of substrate utilization, g substrate consumed·(g initial substrate)^-1·100     

Studies on the kinetic parameters for alcoholic fermentation by flocculent Saccharomyces cerevisiae strains and non-hydrogen sulfide-producing strains

Summary The growing demand for high quality products and the immense export potential that cacha?a represents, demonstrated especially during the past few years, have clearly indicated the necessity of establishing well-defined standards of quality, as well as effective means of controlling the process of production of this beverage. The objective of this study was the selection of S. cerevisiae yeast strains and the investigation of their influence on the kinetic parameters of fermentation. Ninety strains of S. cerevisiae isolated from distilleries of the state of Minas Gerais were evaluated with respect to the following parameters: flocculation capacity, production of H₂S and kinetic parameters of fermentation. The UFMGA 905 strain was used as a reference because it presented desirable characteristics for the production of cacha?a. Five strains presented high specific sedimentation velocities (SSV), indicating a high flocculation capacity, and two did not produce H₂S. The strains presented significant statistical differences for fermentation parameters: yield of ethanol; efficiency of substrate conversion to ethanol; ratio of substrate conversion to ethanol (Y _p/s), to cells (Y _x/s), to organic acids (Y _ac/s), and to glycerol (Y _g/s); and productivity. In general, the strains presented a good fermentative potential, with ethanol yields varying from 74.7 to 82.1% and an efficiency of 76.1–84.4%. All strains presented high productivities (4.6–6.6 g l⁻¹ h⁻¹), indicating that this parameter can be used in the selection of strains for the production of cacha?a.     

Modelling the kinetics of growth of Acetobacter aceti in discontinuous culture: influence of the temperature of operation

Acetic acid fermentation is the biochemical process by which, under strict conditions of aerobiosis, Acetobacter aceti oxidises the ethanol contained in alcoholic substrates into acetic acid. This paper studies the effect of temperature on the specific growth rate of the microorganisms (μ _C), in particular, the mathematical modelling of this process, with the aim of developing previous studies of the mathematical relationships between μ _C of A. aceti and the concentrations of substrate (ethanol), product (acetic acid) and dissolved oxygen. Until now this relationship has not been widely studied, and only a few studies have looked at the influence of temperature on growth kinetics of this bacteria. We have developed an extensive experimental system, to determine precisely the influence of temperature on the maximum specific growth rate. Received: 15 July 1997 / Received revision: 7 October 1997 / Accepted: 19 October 1997     

STUDIES ON THE PROPERTIES OF RETINAL ALCOHOL DEHYDROGENASE FROM THE RAT

An NAD-dependent alcohol dehydrogenase (alcohol:NAD oxidoreductase; EC 1.1.1.1) has been isolated and partially purified from the retinal cytosol of the rat. Its substrate specificity and sensitivity to inhibitors of hepatic alcohol dehydrogenase have been investigated. Ethanol, 1-propanol and 1-butanol served as substrates for this enzyme but the K_m values were more than 100-fold higher than those reported for hepatic alcohol dehydrogenase. Methanol and retinol were unreactive with this alcohol dehydrogenase. Inhibition by pyrazole was observed but the K_t was about 100-fold higher than the value observed for hepatic alcohol dehydrogenase. n-Butyraldoxime inhibited retinal alcohol dehydrogenase with a K_t of 2 μM, a value which approximates its K_t for hepatic alcohol dehydrogenase. 1, 10-Phenanthroline was ineffective as an inhibitor. Oxidation of retinol was observed in retinal homogenates in the presence of NADP but no inhibition was observed with ethanol, methanol or pyrazole. We conclude that oxidation of retinol is not catalysed by soluble retinal alcohol dehydrogenase.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.