Ayaaki Ishizaki

Biodiesel production from crude palm oil and evaluation of butanol extraction and fuel properties

Three principal variables, molar ratio of methanol to oil, amount of catalyst, and reaction temperature, affecting the yield of acid-catalyzed production of methyl ester (biodiesel) from crude palm oil were investigated. The biodiesel was then used as an extractant in batch and continuous acetone–butanol–ethanol fermentation, and its fuel properties and that of the biodiesel–ABE product mix extracted from the batch culture analyzed. The optimized variables, 40:1 methanol/oil (mol/mol) with 5% H2S04 (vol/wt) reacted at 95°C for 9 h, gave a maximum ester yield of 97%. Biodiesel preferentially extracted butanol, and enhanced its production in the batch culture from 10 to 12 g l−1. The fuel properties of biodiesel and the biodiesel–ABE mix were comparable to that of No.2 diesel, but their cetane numbers and the boiling points of the 90% fractions were higher. Therefore, they could serve as efficient No. 2 diesel substitutes. The biodiesel–ABE mixture had the highest cetane number.

High butanol production by Clostridium saccharoperbutylacetonicum N1-4 in fed-batch culture with pH-Stat continuous butyric acid and glucose feeding method.

A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. The specific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyric acid to 0.42 g-butanol/g-cells/h with 5.0 g/l butyric acid. The pH value in broth decreases with butyric acid production during acidogenesis, and then butyric acid reutilization and butanol production result in a pH increase during solventogensis. The pH-stat fed-batch culture was performed to maintain a constant pH and butyric acid concentration in the culture broth, but feeding only butyric acid could not support butyric acid utilization and butanol production. Subsequently, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol was produced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), several B/G ratio solutions were fed. The maximum butanol production was 16 g/l and the residual glucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol in relation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch culture with butyric acid than that of conventional batch culture, respectively.

Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth.

The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30°C and in the pH range 5.0–5.5. The addition of Ca2+ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl2. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production.

Class IIa bacteriocins from lactic acid bacteria: Antibacterial activity and food preservation

In the last decade, a variety of ribosomally synthesized antimicrobial peptides, or bacteriocins, produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into various fundamental aspects of biology and biochemistry such as bacteriocin processing and secretion, mechanisms of cell immunity, and structure-function relationships. In parallel, there has been a growing awareness that bacteriocins may be developed into useful antimicrobial food additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their significant biological activities and potential applications. The present review provides an overview of the knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances. The activity and potential food applications of class IIa bacteriocins are a major focus of this review.

Extractive acetone-butanol-ethanol fermentation using methylated crude palm oil as extractant in batch culture of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

The possibility of employing methylated crude palm oil (CPOE) as an extraction solvent to reduce end-product inhibition and to enhance solvent productivity in acetone-butanol-ethanol (ABE) fermentation was evaluated using oleyl alcohol as the standard butanol extractant. Fermentation was carried out at an initial glucose concentration of 90 g/l. CPOE did not inhibit the growth of the fermentative organism. Without solvent extraction, butanol production ceased after 30 h at a concentration of 15.4 g/l limiting cell growth to 3.98 g/l and glucose consumption to 62%. Applying CPOE as the extraction solvent, about 47% of the total butanol produced was extracted, glucose consumption was increased to 83% and relatively high glucose consumption rates and solvent productivities were obtained. Butanol production increased to 20.9 g/l; total ABE solvents and yield also increased from 21.2 g/l and 38% (in conventional fermentation) to 29.8 g/l and 40.4%, respectively.

Microbial production of poly-D-3-hydroxybutyrate from CO2

Abstract. This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H2, O2 and CO2 by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha. Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R. eutropha for the mass production of P(3HB) from CO2 should be designed on the basis of a recycled-gas, closed-circuit culture system. Also, maintaining the O2 concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding. Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O2 concentration was maintained below 6.9%. However, a study on the continuous production of P(3HB) from CO2 by chemostat culture of R. eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture. It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO2.

Production of poly-β-hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC 17697T

The characteristics of PHB production from carbon dioxide by autotrophic culture of Alcaligenes eutrophus ATCC 17697T using a recycled gas closed circuit culture system under the condition of oxygen limitation were investigated. Cell concentration increased to more than 60 g/l after 60 h of cultivation, while the PHB concentration reached 36 g/l. PHB accumulation in the oxygen-limited culture was superior than that in an ammonium-deficient culture. The PHB produced was identified as a homopolymer of d-3-hydroxybutyrate by 1H and 13C NMR analysis. The stoichiometry for PHB production from CO2 under the oxygen limitation condition was indicated to be as follows: 33H2 + 12O2 + 4CO2 → C4H6O2 + 30H2O. This stoichiometry shows that the hydrogen consumption per one mole of CO2 for PHB production is larger than that for cell formation.

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean.

Aims:  Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin.

A Novel Lantibiotic, Nukacin ISK-1, of Staphylococcus warneri ISK-1: Cloning of the Structural Gene and Identification of the Structure

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.

Investigation of the utility of pineapple juice and pineapple waste material as low-cost substrate for ethanol fermentation by Zymomonas mobilis

The utility of the juice of rotten or discarded pineapples and the waste material of the production of pineapple juice as low-cost substrates for ethanol production by Zymomonas mobilis was investigated. Z. mobilis ATCC 10988 produced 59.0 g.l(-1) ethanol in undiluted pineapple juice without nutritional supplementation and without the regulation of the pH while 42.5 g.l(-1) ethanol was obtained using a 125 g.l(-1) sucrose medium supplemented with 10 g.l(-1) yeast extract and mineral salts. Ethanol fermentation using unhydrolyzed and enzymatically hydrolyzed pineapple waste material was also investigated under various culture conditions. When a 15% (v/v) dilution of unhydrolyzed waste material without nutritional supplementation was used, more than 3.5 g.l(-1) ethanol was produced. When the media containing 15, 30, and 40% (v/v) of the hydrolyzate consisting of a 60% (v/v) suspension of pineapple waste material were used, final concentrations of ethanol were 5.0 g.l(-1), 7.6 g.l(-1), and 9.3 g.l(-1), respectively. These results suggest that pineapple juice and the waste material can be useful low-cost substrates for ethanol production by Z. mobilis without supplementation with expensive organic nitrogen complexes such as yeast extract and without the regulation of the pH during cultivation, leading to the reduction in the production costs.