Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae

The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead.     

Comparison between immobilized Kluyveromyces fragilis and Saccharomyces cerevisiae coimmobilized with beta-galactosidase, with respect to continuous ethanol production from concentrated whey permeate

Kluyveromyces fragilis immobilized in calcium alginate gel was compared to Saccharomyces cerevisiae coimmobilized with beta-galactosidase, for continuous ethanol production from whey permeate in packed-bed-type columns. Four different whey concentrations were studied, equivalent to 4.5, 10, 15, and 20% lactose, respectively. In all cases the coimmobilized preparation produced more ethanol than K. fragilis. The study went on for more than 5 weeks. K. fragilis showed a decline in activity after 20 days, while the coimmobilized preparation was stableduring the entrire investigation. Under experimental conditions theoretical yields of ethanol were obtained from 4.5 and 10% lactose substrates with the coimmobilized system. Using 15% lactose substrate, theoretical yields were only obtained when a galactose-adapted immobilized S. cerevisiae column was run in series with the coimmobilized column. Then a maximum of 71 g/L ethanol was produced with a productivity of 2.5 g/L h. The coimmobilized column alone gave a maximum ethanol concentration of 52 g/L with a productivity of 4.5 g/L h, whereas immobolized K. fragilis only produced 13 g/L ethanol with a productivity of 1.1 g/L h. It was not possible to obtain theoretical yields of ethanol from the highest substrate concentration.     

Kappa-carrageenan/gelatin gel beads for the co-immobilization of aerobic and anaerobic microbial communities degrading 2,4,6-trichlorophenol under air-limited conditions

Alginate and kappa-carrageenan gels were tested as bead materials for the co-immobilization of anaerobic and aerobic microorganisms for the mineralization of 2,4,6-trichlorophenol under air-limited conditions. Chemical, mechanical and thermal culture constraints were pre-defined and the gel resistances were established. Alginate was quickly eliminated because of its chemical instability in the culture media. In anaerobic conditions, the microorganisms transformed the substrates into CH4 and CO2. The kappa-carrageenan gel did not enable these gases to diffuse. They remained as bubbles in the core of the beads and made the beads float. Gelatin was added to kappa-carrageenan in order to change the carrier properties. No biogas bubbles appeared in the bead core during the cultures in anaerobic conditions and the beads reacted well to the culture conditions in the reactor. The co-immobilization of the anaerobic and aerobic microbial communities was successfully performed with the kappa-carrageenan/gelatin gel (2% (w/w) of each polymer). The biological activities, measured by the impedancemetry technique, were preserved in the beads.     

Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity

Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. (c) 1997 John Wiley & Sons, Inc.     

Coimmobilization of Nitrosomonas europaea and Paracoccus denitrificans cells using polyelectrolyte complex-stabilized calcium alginate gel

Calcium alginate gel (CAG) that withstands phosphate ions in the medium was prepared by reinforcing a network structure of the gel with a polyelectrolyte complex (PEC) consisting of potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide. The PEC-stabilized CAG beads were used as a supporting matrix for the coimmobilization of Nitrosomonas europaea ATCC 25978 cells and Paracoccus denitrificans IFO 12442 cells. The coimmobilized cells were aerobically cultured on a medium containing 3 mM of phosphate ions, using (NH₄)₂SO₄ as a substrate and ethanol as a carbon source. Ammonia was consumed without forming nitrite, indicating the concurrence of nitrification and denitrification in the same system. No breakage of the gel beads was observed during the cultivation. Repeated aerobic cultivation using a column packed with beads of coimmobilized cells had stable initial activity for at least one month.     

Production of ethanol directly from potato starch by mixed culture of Saccharomyces cerevisiae and Aspergillus niger using electrochemical bioreactor

When cultivated aerobically, Aspergillus niger hyphae produced extracellular glucoamylase, which catalyzes the saccharification of unliquified potato starch into glucose, but not when grown under anaerobic conditions. The Km and Vmax of the extracellular glucoamylase were 652.3 mg starch l-1 and 253.3 mg glucose l-1 min-1, respectively. In mixed culture of A. niger and Saccharomyces cerevisiae, oxygen had a negative influence on the alcohol fermentation of yeast, but activated fungal growth. Therefore, oxygen is a critical factor for ethanol production in the mixed culture, and its generation through electrolysis of water in an electrochemical bioreactor needs to be optimized for ethanol production from starch by coculture of fungal hyphae and yeast cells. By applying pulsed electric fields (PEF) into the electrochemical bioreactor, ethanol production from starch improved significantly: Ethanol produced from 50 g potato starch l-1 by a mixed culture of A. niger and S. cerevisiae was about 5 g l-1 in a conventional bioreactor, but was 9 g l-1 in 5 volts of PEF and about 19 g l-1 in 4 volts of PEF for 5 days.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Continuous ethanol fermentation using immobilized yeast cells

Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/L gel h and prolonged life stability for more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4 kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10%(v/v)ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.     

The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminaria japonica utilization

Brown seaweed contains various carbohydrates, such as alginate, laminaran, and mannitol, therefore ethanol fermentation was attempted with Nuruk and a mixed culture that included Laminaria japonica. Nuruk is used to make Korean traditional alcohol. In the research, four microorganisms that produced ethanol and had the ability to achieve alginate degradation were obtained on the L. japonica medium. Nuruk 4 was found to produce a better result than the other tested microorganisms, and the optimal substrate for ethanol production was found to be mannitol (2.59 g/L at 96 h). Nuruk 4 was more than three times better compared with Candida tropicalis in regards to ethanol production. When alginate lyase activity occurred, it appeared as a clear zone around Nuruk 3. The maximal ethanol production yield conditions were comprised of Nuruk 3 and 4 on the anaerobic culture. In this case, 2.0 g/L of ethanol were efficiently produced under the same conditions.     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures.

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica)

l-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp. lactis immobilized in loofa sponge. A. awamori was immobilized directly in cylindrical loofa sponge while the L. lactis was immobilized in a loofa sponge alginate gel cube. In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells. The alginate gel formed a hard outer layer covering the soft porous gel inside. By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition. Repeated fed-batch l-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g^–1 starch and 1.6 g lactic acid l^–1 h^–1, respectively.     

Degradation of 2,4,6-trichlorophenol (2,4,6-TCP) by co-immobilization of anaerobic and aerobic microbial communities in an upflow reactor under air-limited conditions

The co-immobilization and the culture of anaerobic and aerobic communities was tested for the mineralization of 2,4,6-trichlorophenol (2,4,6-TCP). At first, the anaerobic microorganisms (aggregated into granules) were cultivated in an upflow anaerobic sludge blanket (UASB) reactor, in a continuous mode, with glucose, propionate, acetate (COD loading rate = 0.5-2.0 g COD/l per day, ratio 1:1:1) and 2,4,6-TCP (2,4,6-TCP loading rate = 25-278 micromol/l per day) as substrates. 2,4,6-TCP was degraded into 2,4-DCP and 4-CP, but it was not mineralized because of the low degradation rates of 4-CP. Furthermore, the highest loading rates of 2,4,6-TCP (>126 micromol/l per day) caused the inhibition of the strains degrading the propionate. The granules were therefore tested in association with the aerobic community. They were immobilized in kappa-carrageenan/gelatin [2% (w/w) of each polymer] gel beads and cultivated in a reactor, on their own (to test the influence of the gel), and then with the aerobic community, under anaerobic and air-limited conditions, respectively. The results showed that (1) the gel did not influence the activity of the granules, (2) the anaerobic and aerobic communities could be easily co-immobilized in gel beads and cultivated in a reactor, (3) the mineralization of 2,4,6-TCP (2,4,6-TCP loading rate = 10-506 micromol/l per day), its intermediates of degradation and the other substrates [glucose + acetate + propionate (ratio 1:1:1) = COD loading rate = 500 mg COD/l per day] could be obtained under air-limited conditions if the culture parameters were strictly controlled [airflow = 36-48 vvd (volume of air/volume of liquid in the reactor per day), pH value at around 7.5]. Finally, the gel did not retain its structure during the whole culture (263 days) in the air-limited reactor, but the anaerobic and aerobic communities retained their activities and worked together for the mineralization.     

Ethanol from whey: Continuous fermentation with cell recycle

The production of ethanol from cheese whey lactose has been demonstrated using a single-stage continuous culture fermentation with 100% cell recycle. In a two-step process, an aerobic fed batch operation was used initially to allow biomass buildup in the absence of inhibitory ethanol concentrations. In the anaerobic ethanol-producing second step, a strain of Kluyveromyces fragilis selected on the basis of batch fermentation data had a maximum productivity of 7.1 g ethanol/L/h at a dilution rate of 0.15 h(-1), while achieving the goal of zero residual sugar concentration. The fermentation productivity diminished when the feed sugar concentation exceeded 120 g/L despite the inclusion of a lipid mixture previous shown to enhance batch fermentation productivities.     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

Microvinification--how small can we go?

High-throughput methodologies to screen large numbers of microorganisms necessitate the use of small-scale culture vessels. In this context, an increasing number of researchers are turning to microtiter plate (MTP) formats to conduct experiments. MTPs are now widely used as a culturing vessel for phenotypic screening of aerobic laboratory cultures, and their suitability has been assessed for a range of applications. The work presented here extends these previous studies by assessing the metabolic footprint of MTP fermentation. A comparison of Chardonnay grape juice fermentation in MTPs with fermentations performed in air-locked (self-induced anaerobic) and cotton-plugged (aerobic) flasks was made. Maximum growth rates and biomass accumulation of yeast cultures grown in MTPs were indistinguishable from self-induced anaerobic flask cultures. Metabolic profiles measured differed depending on the metabolite. While glycerol and acetate accumulation mirrored that of self-induced anaerobic cultures, ethanol accumulation in MTP ferments was limited by the increased propensity of this volatile metabolite for evaporation in microlitre-scale culture format. The data illustrates that microplate cultures can be used as a replacement for self-induced anaerobic flasks in some instances and provide a useful and economical platform for the screening of industrial strains and culture media.     

Cultivation characteristics of immobilized Aspergillus oryzae for kojic acid production

Aspergillus oryzae in situ grown from spores entrapped in calcium alginate gel beads was used for the production of kojic acid. The immobilized cells in flask cultures produced kojic acid in a linear proportion while maintaining the stable metabolic activity for a prolonged production period. Kojic acid was accumulated up to a high concentration of 83 g/L, at which the kojic acid began to crystallize, and, thus, the culture had to be replaced with fresh media for the next batch culture. The overall productivities of two consecutive cultivations were higher than that of free mycelial fermentation. However, the production rate of kojic acid by the immobilized cells was suddenly decreased with the appearance of central cavernae inside the immobilized gel beads after 12 days of the third batch cultivation.     

Screening of bacterial strains capable of converting biodiesel‐derived raw glycerol into 1,3‐propanediol, 2,3‐butanediol and ethanol

The ability of bacterial strains to assimilate glycerol derived from biodiesel facilities to produce metabolic compounds of importance for the food, textile and chemical industry, such as 1,3‐propanediol (PD), 2,3‐butanediol (BD) and ethanol (EtOH), was assessed. The screening of 84 bacterial strains was performed using glycerol as carbon source. After initial trials, 12 strains were identified capable of consuming raw glycerol under anaerobic conditions, whereas 5 strains consumed glycerol under aerobiosis. A plethora of metabolic compounds was synthesized; in anaerobic batch‐bioreactor cultures PD in quantities up to 11.3 g/L was produced by Clostridium butyricum NRRL B‐23495, while the respective value was 10.1 g/L for a newly isolated Citrobacter freundii. Adaptation of Cl. butyricum at higher initial glycerol concentration resulted in a PD_max concentration of ～32 g/L. BD was produced by a new Enterobacter aerogenes isolate in shake‐flask experiments, under fully aerobic conditions, with a maximum concentration of ～22 g/L which was achieved at an initial glycerol quantity of 55 g/L. A new Klebsiella oxytoca isolate converted waste glycerol into mixtures of PD, BD and EtOH at various ratios. Finally, another new C. freundii isolate converted waste glycerol into EtOH in anaerobic batch‐bioreactor cultures with constant pH, achieving a final EtOH concentration of 14.5 g/L, a conversion yield of 0.45 g/g and a volumetric productivity of ～0.7 g/L/h. As a conclusion, the current study confirmed the utilization of biodiesel‐derived raw glycerol as an appropriate substrate for the production of PD, BD and EtOH by several newly isolated bacterial strains under different experimental conditions.     

Physiology and enzymology of thermophilic anaerobic bacteria degrading starch

Abstract The capability of secreting thermoactive enzymes exhibiting α-amylase and pullulanase with debraching activity, seems to be widely distributed amongst anaerobic thermophilic bacteria. Interestingly, pullulanase formed by these bacteria displays dual specificity by attacking α-1,6- as well as α-1,4-glycosidic linkages in branched glucose polymers. Unlike the enzyme system of aerobic microorganisms the majority of starch hydrolysing enzymes of anaerobic bacteria is metal indepedent and is extremely thermostable. This enzyme system is controlled by substrate induction and catabolite repression; enzyme expression is accomplished when maltose or maltose-containing carbohydrates are used as substrates. By developing a process in continuous culture we were able to greatly enhance enzyme synthesis and release by anaerobic thermophilic bacteria. An elevation in the specific activities of cell-free amylases and pullulanases could also be achieved by entrapping of bacteria in calcium alginate beads. The unique properties of extracellular enzymes of thermophilic anaerobic bacteria makes this group of organisms suitable candidates for inductrial application.     

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

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