Ethanol production from biomass by repetitive solid-state fed-batch fermentation with continuous recovery of ethanol.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

Lactic acid bacteria display on the cell surface cytosolic proteins that recognize yeast mannan.

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast.

Effect of CO2 concentration on the growth and exopolysaccharide production of Bifidobacterium longum cultivated under anaerobic conditions.

For substantial cell growth and exopolysaccharide (EPS) production of Bifidobacterium longum JBL05, anaerobic conditions (dissolved oxygen concentrations below 0.05 ppm) and the supplement of CO2 (> or = 20%) were required. Under these conditions, B. longum JBL05 produced EPS amounts of up to 2.9 g/l.

Beer volatile compounds and their application to low-malt beer fermentation.

Low-malt beers, in which the amount of wort is adjusted to less than two-thirds of that in regular beer, are popular in the Japanese market because the flavor of low-malt beer is similar to that of regular beer but the price lesser than that of regular beer. There are few published articles about low-malt beer. However, in the production process, there are many similarities between low-malt and regular beer, e.g., the yeast used in low-malt beer fermentation is the same as that used for regular beer. Furthermore, many investigations into regular beer are applicable to low-malt beer production. In this review, we focus on production of volatile compounds, and various studies that are applicable to regular and low-malt beer. In particular, information about metabolism of volatile compounds in yeast cells during fermentation, volatile compound measurement and estimation methods, and control of volatile compound production are discussed in this review, which concentrates on studies published in the last 5-6 years.

Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production.

Hydrolysates of lignocelluloses hydrolyzed by diluted sulfuric acid contain toxic compounds that inhibit ethanol production by Saccharomyces cerevisiae and the ethanologenic recombinant Escherichia coli KO11. We investigated the biological detoxification of a hydrolysate of waste house wood (WHW) by a thermophilic bacterium, Ureibacillus thermosphaericus. When the hydrolysate was treated with this bacterium at 50 degrees C for 24 h, the ethanol production rate by S. cerevisiae increased markedly and was comparable to that for the hydrolysate treated with an excess amount of calcium hydroxide (overliming). Chromatographic analysis of synthetic hydrolysates containing furfural or 5-hydroxymethyl furfural that are considered to be major toxic compounds in hydrolysates revealed that U. thermosphaericus degrades these compounds. In the WHW hydrolysates, however, the concentrations of these compounds were not decreased markedly by the bacterium. These results suggest that the bacterium degrades minor but more toxic compounds or phenolic compounds in the WHW hydrolysates. The combination of bacterial and overliming treatments of hydrolysates minimized significantly the decrease in ethanol production rate by E. coli KO11 as fermentation proceeded. Because the bacterium grows rapidly and does not consume sugars, our biological detoxification should be useful for bioethanol production from acid hydrolysates of lignocelluloses.

Characterization of new isolated Ralstonia eutropha strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production.

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and gamma-hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of gamma-hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments.

New strategy for enhancement of microbial viability in simulated gastric conditions based on display of starch-binding domain on cell surface.

The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 fused to the linker region and the starch-binding domain (SBD) of the *-amylase of Streptococcus bovis 148 was produced intracellularly in Escherichia coli. The fusion protein (CPH-SBD) was able to bind to the cell surface of Lactobacillus casei NRRL B-441 and to corn starch. Therefore, adhesion of cells to corn starch was mediated by the fusion protein. At a cell density of 10(9) cfu/ml and a starch concentration of 5 mg/ml, CPH-SBD-displaying L. casei cells aggregated with corn starch, whereas the free cells of L. casei did not form any aggregates with corn starch. After incubation in simulated gastric juice (pH 3.0, 1 h), the survival percentages of free cells, amylose-coated free cells, and free cells mixed with corn starch were 0.074%, 7.2%, and 3.1% respectively. When CPH-SBD-displaying bacteria aggregated with corn starch, their survival percentage was 8% higher than that of free cells mixed with corn starch. The survival of the amylose-coated CPH-SBD-displaying L. casei cells was comparable to that of amylose-coated free cells, whereas the survival percentage of amylose-coated aggregates of CPH-SBD-displaying bacteria with corn starch was 28% higher than that of amylose-coated mixture of free cells with corn starch. These results demonstrate the potential usefulness of the cell-surface display technique for enhancement of the delivery of viable microorganisms to the intestinal tract.

Rational screening of antibodies and design of sandwich enzyme linked immunosorbant assay on the basis of a kinetic model.

A rational strategy for the rapid establishment of a sensitive sandwich enzyme linked immunosorbant assay was developed. The kinetic properties required for the solid-phase and enzyme-conjugated antibodies of sandwich ELISA were determined rationally on the basis of a kinetic model describing antibody-antigen interaction. Some antibodies possessing the required kinetic properties against a model antigen, C-reactive protein (CRP), were successfully isolated from a phage antibody library under the screening conditions that were designed on the basis of simulation results. The best combination of solid-phase and enzyme-conjugated antibodies that gives the most sensitive sandwich ELISA was determined by simulation on the basis of the apparent association and dissociation rate constants of the isolated antibodies. It was confirmed by experiment that the sandwich ELISA using the best combination of antibodies was actually the most sensitive one. Our strategy would be useful for the rapid establishment of sensitive sandwich ELISAs compared with the traditional hybridoma method in which the best condition is determined by trial and error.

Strategies for reducing supplemental medium cost in bioethanol production from waste house wood hydrolysate by ethanologenic Escherichia coli: inoculum size increase and coculture with Saccharomyces cerevisiae.

In this paper, we report a simultaneous realization of both efficient ethanol production and saving medium nutrient (corn steep liquor [CSL]) during bioethanol fermentation of overliming-treated hydrolysate of waste house wood (WHW) using ethanologenic Escherichia coli KO11. In cultivation using WHW hydrolysate supplemented with 4% (v/v) CSL and 0.2 g-dry cell weight (DCW)/l E. coli KO11 cells, the overall ethanol yield reached 84% of the theoretical value at 61 h. When we conducted the cultivation with 1% CSL to reduce the supplemental medium cost, the overall ethanol yield remained in the range of 66-72% even at 90 h. We proposed two alternative methods for increasing the overall yield even with 1% CSL. The first method involved increasing the inoculum size of E. coli KO11 up to 0.8 g-DCW/l, where 83% of the overall yield was attained at 60 h of cultivation. The second method involved the coculture of 0.2 g-DCW/l E. coli KO11 together with 0.02 g-DCW/l of Saccharomyces cerevisiae TJ1, and the overall yield reached 81% at 47 h of cultivation.

Bidirectional cell-surface anchoring function of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403.

With the aim of constructing an efficient protein display system for lactic acid bacteria (LABs), the effect of fusion direction on the cell-surface binding activity of the C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 was studied. CPH fused to the alpha-amylase (AMY) of Streptococcus bovis 148 either at its C-terminus (CPH-AMY) or at its N-terminus (AMY-CPH) was expressed intracellularly in Escherichia coli. This domain was able to direct binding of AMY to the surface of L. lactis ATCC 19435 in both constructs. However, the number of bound molecules per cell and the specific activity for starch digestion in the case of CPH-AMY were 3 and 14 times greater than those in the case of AMY-CPH, respectively. Of the LABs tested, L. lactis ATCC 19435 showed the highest binding capability for CPH-AMY, up to 6 x 10(4) molecules per cell, with a dissociation rate constant of 5.00 x 10(-5) s(-1). The binding of CPH-AMY to the surface of Lactobacillus delbrueckii ATCC 9649 cells was very stable with a dissociation rate constant of 6.96 x 10(-6) s(-1). The production of CPH-AMY in the soluble form increased 3-fold as a result of coexpression with a molecular chaperone, trigger factor. The results of this study suggest the usefulness of CPH as a bidirectional anchor protein for the production of cell-surface adhesive enzymes in E. coli. Furthermore, the importance of the fusion direction of CPH in determining cell-surface binding and enzymatic activities was shown.

Expression of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403 in methylotrophic yeast Pichia pastoris.

The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 produced intracellularly in Escherichia coli was able to attach to the surface of cells of Lactobacillus casei NRRL B-441, Bacillus subtilis 168, E. coli XL1-blue and Saccharomyces cerevisiae IFO0216. Therefore, this domain is a suitable fusion partner for the adhesion of proteins to cell surfaces. The production of cell-surface adhesive proteins using this domain in Pichia pastoris is particularly attractive, because this organism has better capability to allow the correct folding of the recombinant proteins than prokaryotic hosts. However, when this domain is produced in this yeast, its cell-surface binding activity may be limited by glycosylation. In this study, therefore, we constructed a CPH mutant (CPHM) devoid of the potential N-glycosylation sites by site-directed mutagenesis. CPHM was successfully expressed extracellularly in P. pastoris (GS115) using the methanol inducible AOX1 promoter with an alpha-mating factor signal sequence, whereas the native CPH was not produced in this host. Western blot analysis revealed that the apparent molecular size of CPHM was 18 kDa greater than that of CPH produced in E. coli (32 kDa), which is attributed to O-glycosylation. However, CPHM produced in P. pastoris was capable of binding to the cell surfaces despite its modification by the yeast, and its dissociation rate constant from the surface of L. casei NRRL B-441 cells was 3.5-fold lower than that of CPH produced in E. coli. These results demonstrate the applicability of the constructed domain (CPHM) for the production of cell-surface adhesive proteins in P. pastoris.

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis.

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.

Analysis of hemin effect on lactate reduction in Lactococcus lactis.

Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.

Fed-batch coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for effective production of kefiran.

In a batch coculture of kefiran-producing lactic acid bacteria Lactobacillus kefiranofaciens and lactate-assimilating yeast Saccharomyces cerevisiae, lactate accumulation in the medium was observed, which inhibited kefiran production. To enhance kefiran productivity by preventing lactate accumulation, we conducted lactose-feeding batch operation with feedforward/feedback control during the coculture, so that the lactate production rate of L. kefiranofaciens was balanced with the lactate consumption rate of S. cerevisiae. The lactate concentration was maintained at less than 6 g l(-1) throughout the fed-batch coculture using a 5 l jar fermentor, although the concentration reached 33 g l(-1) in the batch coculture. Kefiran production was increased to 6.3 g in 102 h in the fed-batch coculture, whereas 4.5 g kefiran was produced in 97 h in the batch coculture. The kefiran yield on lactose basis was increased up to 0.033 g g(-1) in the fed-batch coculture, whereas that in the batch coculture was 0.027 g g(-1).

Simulation model for predicting limit of detection and range of quantitation of competitive enzyme-linked immunosorbent assay.

The limit of detection (LOD) and range of quantitation (ROQ) of competitive enzyme-linked immunosorbent assay (ELISA) were determined from a model describing the calibration curve and precision profile of the assay. The calibration curve is given by solving the differential equations describing the change in the concentrations of an antigen-antibody complex and an enzyme-conjugated antigen-antibody complex by a Runge-Kutta method. The precision profile is described in terms of possible error sources such as the pipetting volumes of the analyte, enzyme-conjugated antigen, antibody and substrate solutions, calibration curve and inherent absorbances between the wells in an ELISA plate. An appropriate concentration of the enzyme-conjugated antigen that balances a low detection limit and sufficient color development was found to be in a narrow range, which is consistent with the empirical rule. The optimum conditions for competitive ELISA using an antibody with a kinetic property can be designed from our model.

Physiological analysis of yeast cells by flow cytometry during serial-repitching of low-malt beer fermentation.

At the end of beer brewing fermentation, yeast cells are collected and repitched for economical reasons. Although it is generally accepted that the physiological state of inoculated yeast cells affects their subsequent fermentation performance, the effect of serial-repitching on the physiological state of such yeast cells has not been well clarified. In this study, the fermentation performance of yeast cells during serial-repitching was investigated. After multiple repitchings, the specific growth rate and maximum optical density (OD(660)) decreased, and increases in isoamyl alcohol, which causes an undesirable flavor, and residual free amino acid nitrogen (FAN) concentrations were observed. The physiological state of individual cells before inoculation was characterized by flow cytometry using the fluorescent dyes dehydrorhodamine 123 (DHR) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). The fluorescence intensities of DHR, an indicator of reactive oxygen species (ROSs), and OXN, which indicates membrane potential, gradually increased as the number of serial-repitching cycles increased. Fluorescence intensity correlated strongly with cell growth. The subsequent fermentation performance can be predicted from this correlation.

Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis.

BACKGROUND: Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition. RESULTS: We performed a metabolic flux analysis (MFA) with [1-13C]- and [U-13C]-labeled glucose in the glutamate production phase of C. glutamicum, based on the analysis of the time courses of 13C incorporation into proteinogenic amino acids by gas chromatography-mass spectrometry (GC-MS). The flux from phosphoenolpyruvate (PEP) to oxaloacetate (Oxa) catalyzed by phosphoenolpyruvate carboxylase (PEPc) was active in the growth phase not producing glutamate, whereas that from pyruvate to Oxa catalyzed by pyruvate carboxylase (Pc) was inactive. In the glutamate overproduction phase induced by the addition of the detergent material Tween 40, the reaction catalyzed by Pc also became active in addition to the reaction catalyzed by PEPc. CONCLUSION: It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40. This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains. The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.

Microaeration enhances productivity of bioethanol from hydrolysate of waste house wood using ethanologenic Escherichia coli KO11.

This is the first study showing the successful application of waste house wood (WHW) to the pilot-scale production of bioethanol by hydrolysis using diluted acid and fermentation using the ethanologenic recombinant Escherichia coli KO11. The major sugars in the WHW hydrolysate were glucose, mannose and xylose; the percentages were approximately 35%, 35% and 20% (w/w), respectively. In anaerobic fermentation using a 5-l reactor in which the oxygen transfer rate (OTR) was 0 mmol/(l x h), KO11 consumed only 25% of the xylose in the WHW hydrolysate over the examined fermentation time of 100 h; however, hexoses such as glucose and mannose were consumed completely. Microaeration at an OTR of 4 mmol/(l x h) enhanced the xylose utilization ratio of KO11 to 100%, at which the ethanol concentration was 35.4 g/l and the ethanol yield was 0.42, although the maximum ethanol concentrations were 28.8 and 26.6 g/l at OTRs of 0 mmol/(l x h) and 15 mmol/(l x h), respectively. Moreover, this microaerobic fermentation at OTR of 4 mmol/(l x h) was applied to 1000-l scale bioethanol production using the WHW hydrolysate. The xylose utilization ratio reached 100% and the ethanol yield was determined to be 0.45 for a 63-h fermentation, which were comparable to those obtained from the laboratory-scale fermentation.

Enhancement of 1,4-dihydroxy-2-naphthoic acid production by Propionibacterium freudenreichii ET-3 fed-batch culture.

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.

Enhancement of ethanol production by promoting surface contact between starch granules and arming yeast in direct ethanol fermentation.

The specific ethanol production rate of raw starch by arming yeast cells displaying alpha-amylase and glucoamylase increased significantly when the cells and starch granules settled together. The specific ethanol production rate also increased when the size distribution of starch granules was almost same as that of the yeast cells. These results indicate that the surface contact between starch granules and yeast cells is important for increasing the apparent specific activity of alpha-amylase, which was the rate-limiting factor of the direct fermentation.