Yorikazu Sonoda

Repeated-batch fermentation process using a thermotolerant flocculating yeast constructed by protoplast fusion

Abstract A thermotolerant flocculating yeast, Saccharomyces cerevisiae KF-7 (KF-7), was constructed by protoplast fusion of the flocculating yeast S. cerevisiae IR-2 (IR-2) and the thermotolerant yeast S. cerevisiae EP-1 (EP-1). In repeated-batch fermentation with KF-7 at 30°C in a molasses medium containing 20% (w/v) total sugar, stable fermentations could be performed continuously and an ethanol productivity of 5 g/l·h was obtained. Even at 35°C a productivity of 3.6 g/l·h was achieved, which was 1.7 times higher than that achieved with IR-2. In a flask-cultivation test, the maximum growth rate and the decay constant of KF-7 were not influenced by an increase in temperature, unlike the results for IR-2. Thus, the fusant KF-7 had acquired thermotolerance. However, KF-7 may be inferior to IR-2 in terms of osmotic tolerance.

Support media for microbial adhesion in an anaerobic fluidized-bed reactor

Abstract The most significant variable in anaerobic digestion in an anaerobic fluidized-bed reactor (AFBR) is the selection of the support medium for microbial adhesion. Using eight kinds of media, cristobalite, zeolite, vermiculite, granular active carbon, granulated clay, pottery stone, volcanic ash, and slag, we examined the physical properties of each medium, microbial adhesion, loading rates of organic matter and removal efficiencies in an AFBR. It appeared that good performance as a support medium was associated with rougher surfaces rather than with larger surface areas, because, although cristobalite had a much smaller surface area (50 m 2 /g) than that of the granular active carbon (1,125 m 2 /g), it had a very rough surface with many tubercular processes, by which a maximum loading rate of TOC of 8 g/ l ·d could be achieved in a synthetic wastewater. Moreover, it appeared to be important that the surface of the medium has a positive charge judging from the difference in performance between cristobalite and zeolite. That is, the two media were charged positively and negatively at pH 7, respectively. As a result, microorganisms, charged negatively in general, could adhere more easily to cristobalite than to zeolite, which was confirmed under a scanning electron microscope (SEM) and by amounts of microbial cells adhering (85 mg cells/g). The upflow linear velocity to allow twice the expanded volume (defined as the ratio of the expanded height to the static height) was decreased to half (0.13 cm/s) by microbial adhesion. In conclusion, a suitable medium for adherence of microorganisms in AFBR should have a rough and positively charged surface rather than a large surface area.

ACCUMULATION OF PROPIONIC ACID DURING ANAEROBIC TREATMENT OF DISTILLERY WASTEWATER FROM BARLEY-SHOCHU MAKING

Abstract Synthetic wastewater consisting aliphatic acids contained in distillery wastewater from barley- shochu making was treated anaerobically. It was suggested that propionic acid was produced from lactic acid and citric acid via succinic acid. Since it appears to be difficult to treat anaerobically wastewater in which propionic acid is accumulated, we attempted to repress the production of propionic acid during acidification. The amount of propionic acid produced increased with an increase in the hydraulic retention time (HRT) at pH 7. Although the treatment was examined using different pHs at a shorter HRT of 10 h, it was difficult to repress the production of propionic acid.

The effect of aeration on stability of continuous ethanol fermentation by a flocculating yeast.

Abstract Continuous fermentation by a flocculating fusant, Saccharomyces cerevisiae HA2, was carried out and a high ethanol productivity of 25 g/l·h, with an ethanol concentration of 63 g/l being achieved at a dilution rate of 0.4 h−1 at 30°C in molasses medium. However, during 4 d operation at a dilution rate of 0.5 h−1 the continuous fermentation became unstable because the flocculating activity of the yeast decreased. The deflocculation is believed to be caused by the action of proteases secreted from lysed cells as judged from the results of treatment tests of flocculating cells with reagents, protease and boiling. Aeration stabilized the flocculating activity of yeasts, allowing a long-term operation of the fermentor at a high dilution rate of 0.5 h−1. In this condition, ethanol productivity of 30 g/l·h, with an ethanol concentration of 60 g/l, could be achieved. The conversion ratio of total sugar to ethanol was 0.4, a value which corresponds to 78% of the theoretical value of 0.51.

Continuous high-ethanol fermentation from cane molasses by a flocculating yeast.

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μmax) and the maximum specific production rate of ethanol (qmax) were 0.12 h−1 and 0.1 g ethanol/109 cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 109 cells/ml for production of 85 g/l ethanol at a dilution rate (D1) of 0.2 h−1. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D1) of 0.2 h−1 by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 109 cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (Dt) of 0.25 h−1, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (Dt) of 0.25 h−1 was achieved, just as it was in the laboratory-scale fermentation test.

Influence of Mineral Nutrients on High Performance during Anaerobic Treatment of Wastewater from a Beer Brewery

Abstract The BOD of wastewater from beer breweries is around 2,000–3,000 mg/l, which is considered to be low-strength wastewater; however, high-strength wastewater, known sometimes as trube wastewater (BOD, about 80,000 mg/l), is also produced as a result of the filtration of wort. This high-strength wastewater was found not to respond well to treatment by a mono-phase thermophilic methane fermentation process using an anaerobic fluidized-bed reactor. Considering the composition of the wastewater and the pathway for methane fermentation, NH4+ at 500 mg/l, Ni2+ at 7 mg/l, Co2+ at 2 mg/l and Fe3+ at 30 mg/l were added to the wastewater. After five-fold dilution by volume, the diluted wastewater treated anaerobically in the same manner. As a result, the TOC (total organic carbon) concentration in the effluent was constant at about 400 mg/l, and no further problems were encountered. The volumetric loading rate of TOC was studied by treating variously diluted samples of high-strength wastewater (TOC 15,000, 23,500, 47,000 mg/l). The results obtained with the raw wastewater (TOC 47,000 mg/l) were remarkably good, with a maximum volumetric loading rate of TOC of 14 g/l·d, a gas yield of 1.2 l/g TOC consumed and a methane content of 59%. From tests involving omission of the mineral nutrients, it appears that Ni2+ and Co2+ play important roles in the anaerobic treatment of the wastewater.

Treatment of coffee waste by slurry-state anaerobic digestion

Slurries containing 20% (w/v) coffee waste solids were treated anaerobically in one- and two-phase thermophilic methane fermentation systems (53°C) with or without pH control. In one-phase methane fermentation using a roller bottle reactor, the maximum gas evolution rate of 0.87 l/l·d was achieved during treatment for 91 d. However, this one-phase methane fermentation did not yield reproducible data. In a two-phase methane fermentation system consisting of a completely stirred tank reactor type (CSTR-type) liquefaction reactor without pH control and an anaerobic fluidized bed type gasification reactor, three-repetitions of treatment were conducted. Each treatment was very stable and the average gas evolution rate per volume of the gasification reactor was about 2.4 l/l·d. Two-repetitions of treatment were then done while controlling pH in the liquefaction at more than 6. The average gas evolution rate per volume of gasification reactor was found to have increased to 10.2 l/l·d, a value which corresponded to 0.84 l/l·d per total volume, including the liquefaction reactor. It was observed that treatment in a two-phase methane fermentation could be repeated in a stable fashion even in the closed system without discharging anything but the coffee waste residues.

Production of protease using wastewater from the manufacture of Shochu

The production of protease using wastewater from a shochu distillery was investigated in order to devise a process for the treatment of shochu distillery wastewater. Aspergillus usami mut. shirousami IFO 6082 was selected from among eight strains for production of protease. Production of 240 U/ml of proteolytic activity was achieved after 72 h in a jar-fermentor culture under the following conditions: initial pH, 5; 30°C; aeration, 1 vvm; and agitation, 600 rpm. The protease was purified by column chromatography on Sephadex G-150 and isoelectrofocusing. The molecular weight of the purified enzyme determined by SDS-PAGE was 56 kDa, and the isoelectric point was pH 4.0. The optimum pH for the reaction was about 4.0, so the enzyme is therefore an acid protease. The optimum temperature for proteolysis ranged between 55 and 60°C, while the enzyme was unstable at temperatures above 60°C.

Repeated-Batch Ethanol Fermentation by a Flocculating Yeast, Saccharomyces cerevisiae IR-2

Abstract Repeated-batch fermentation by a flocculating yeast, Saccharomyces cerevisiae IR-2, was set up in a molasses medium using an automatically controlled system, and the effect of temperature and mash density were studied. At 30°C, a concentration of ethanol of 106g/l with a productivity of 2.5 g/l·h, which was 2.5 times higher than that of the fed-batch fermentation process used commercially in Japan, and a fermentation efficiency of 83% were obtained in a molasses medium that initially contained 25% (w/v) sugar. However, raising the temperature from 30°C to 33°C and then to 35°C had a very detrimental effect of S. cerevisiae IR-2, even in a molasses medium with 20% (w/v) sugar. The specific growth rate (μ) and the specific production rate of ethanol (q) could be represented in terms of ethanol concentration as follows: μ=μ max (1 − P P g ) , and q= q max (1 − P P e ) , where P represents the concentration of ethanol. Increasing the initial concentration of sugar had little effect on the values of Pg (the concentration of ethanol above which yeast cells do not grow) and Pe (the concentration of ethanol above which yeast cells do not produce ethanol), which were about 78 and 104 g/l, respectively. Raising the temperature caused a decrease in Pg and Pe, which fell to 32 and 65 g/l at 35°C, respectively. The repeated-batch fermentation process using S. cerevisiae IR-2 gave good results at 30°C, but it did not proceed well at higher temperatures.

Production of saccharifying enzyme using the wastewater of a Shochu distillery

A saccharifying enzyme was produced by Aspergillus awamori var. kawachi using wastewater generated by a Shochu distillery. The production of the saccharifying enzyme was of the non-growth associated type, and 80 U of activity per ml of broth was obtained in about 6 d of flask cultivation. Since the Shochu distillery wastewater contained high concentrations of volatile fatty acids that were converted to their free forms and severely inhibited cell growth at low pH, the optimum initial pH ranged from 4.5 to 6.0. It is suggested that cell autolysis facilitated the release of the saccharifying enzyme, but a released protease digested the saccharifying enzyme with a subsequent decrease in activity. The saccharifying enzyme was easy to purify, and the purified enzyme was homogeneous when analyzed by disc electrophoresis. The molecular weight was estimated to be 54,000 Da by SDS-PAGE, and the isoelectric point was found to be pH 3.6 by isoelectric focusing. The optimum temperature and pH for the reaction ranged from 50 to 55°C and 4.5 to 5.5, respectively. The saccharifying enzyme could not digest raw starch. The hydrolyzate of soluble starch hydrolyzed by the saccharifying enzyme was composed of two to four oligosaccharides. From these results and the amino acid sequence in the N-terminal, the enzyme produced was concluded to be α-amylase.