Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

Production of xylitol by Candida peltata

Candida peltata NRRL Y-6888 to ferment xylose to xylitol was evaluated under different fermentation conditions such as pH, temperature, aeration, substrate concentration and in the presence of glucose, arabinose, ethanol, methanol and organic acids. Maximum xylitol yield of 0.56 g g⁻¹ xylose was obtained when the yeast was cultivated at pH 6.0, 28°C and 200 rpm on 50 g L⁻¹ xylose. The yeast produced ethanol (0.41 g g⁻¹ in 40 h) from glucose (50 g L⁻¹) and arabitol (0.55 g g⁻¹ in 87 h) from arabinose (50 g L⁻¹). It preferentially utilized glucose > xylose > arabinose from mixed substrates. Glucose (10 g L⁻¹), ethanol (7.5 g L⁻¹) and acetate (5 g L⁻¹) inhibited xylitol production by 61, 84 and 68%, respectively. Arabinose (10 g L⁻¹) had no inhibitory effect on xylitol production. Received 24 December 1998/ Accepted in revised form 18 March 1999     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens were systematically studied for the production of succinic acid from whey. Addition of 2.5 g l⁻¹ yeast extract and 2.5 g l⁻¹ polypeptone per 10 g l⁻¹ whey was most effective for succinic acid production from both treated and nontreated whey. When 20 g l⁻¹ nontreated whey and 7 g l⁻¹ glucose were used as cosubstrates, the yield and productivity of succinic acid reached at the end of fermentation were 95% and 0.46 g (l h)⁻¹, respectively. These values were higher than those obtained using nontreated whey alone [93% and 0.24 g (l h)⁻¹ for 20 g l⁻¹ whey]. Continuous fermentation of A. succiniciproducens at an optimal dilution rate resulted in the production of succinic acid with high productivity [1.35 g (l h)⁻¹], high conversion yield (93%), and higher ratio of succinic acid to acetic acid (5.1:1) from nontreated whey. Received: 23 July 1999 / Received revision: 17 November 1999 / Accepted: 24 December 1999     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation

1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μ _max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l⁻¹). For glycerol at 20 g l⁻¹, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l⁻¹. However, for an initial 1,3-propanediol concentration of 50 g l⁻¹ and glycerol at 70 g l⁻¹, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l⁻¹(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l⁻¹). The addition of 1,2-propanediol or 2,3-butanediol (50 g l⁻¹) in the presence of glycerol (50–100 g l⁻¹), showed that 2-diols reduced the μ _max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered. Received: 15 November 1999 / Revision received: 1 February 2000 / Accepted: 4 February 2000     

Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis

Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l⁻¹ h⁻¹ at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h. Received: 20 April 1998 / Received revision: 29 May 1998 / Accepted: 19 June 1998     

Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources

Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l⁻¹ d-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose consumption rate increased a further threefold when 20 g l⁻¹ d-glucose or raffinose was used as co-substrate together with 50 g l⁻¹ d-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after 82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and much less acetic acid formation. Received: 12 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999     

Lactic acid production from xylose by the fungus Rhizopus oryzae

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure l(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g⁻¹. By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l⁻¹ showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l⁻¹) and glucose (19.2 g l⁻¹) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l⁻¹ h⁻¹ and 0.5 g xylose l⁻¹ h⁻¹. This resulted mainly into the product lactic acid (6.8 g l⁻¹) and ethanol (5.7 g l⁻¹).     

Metabolic evolution of non-transgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> SZ420 for enhanced homoethanol fermentation from xylose

Efficient utilization of pentose sugars (xylose and arabinose) is an essential requirement for economically viable ethanol production from cellulosic biomass. The desirable pentose-fermenting ethanologenic biocatalysts are the native microorganisms or the engineered derivatives without recruited exogenous gene(s). We have used a metabolic evolution (adaptive selection) approach to improve a non-transgenic homoethanol Escherichia coli SZ420 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) for xylose fermentation. An improved mutant, E. coli KC01, was evolved through a 3 month metabolic evolution process. This evolved mutant increased pyruvate dehydrogenase activity by 100%, cell growth rate (h⁻¹) by 23%, volumetric ethanol productivity by 65% and ethanol tolerance by 200%. These improvements enabled KC01 to complete 50 g xylose l⁻¹ fermentations with an ethanol titer of 23 g l⁻¹ and a yield of 90%. The improved cell growth and ethanol production of KC01 are likely attributed to its three fold increased ethanol tolerance.     

Benzene/toluene/p-xylene degradation. Part II. Effect of substrate interactions and feeding strategies in toluene/benzene and toluene/p-xylene fermentations in a partitioning bioreactor

A two-phase aqueous/organic partitioning bioreactor scheme was used to degrade mixtures of toluene and benzene, and toluene and p-xylene, using simultaneous and sequential feeding strategies. The aqueous phase of the partitioning bioreactor contained Pseudomonas sp. ATCC 55595, an organism able to degrade benzene, toluene and p-xylene simultaneously. An industrial grade of oleyl alcohol served as the organic phase. In each experiment, the organic phase of the bioreactor was loaded with 10.15 g toluene, and either 2.0 g benzene or 2.1 g p-xylene. The resulting aqueous phase concentrations were 50 mg/l, 25 mg/l and 8 mg/l toluene, benzene and p-xylene respectively. The simultaneous fermentation of benzene and toluene consumed these compounds at volumetric rates of 0.024 g l⁻¹ h⁻¹ and 0.067 g l⁻¹ h⁻¹, respectively. The simultaneous fermentation of toluene and p-xylene consumed these xenobiotics at volumetric rates of 0.066 g l⁻¹ h⁻¹ and 0.018 g l⁻¹ h⁻¹, respectively. A sequential feeding strategy was employed in which toluene was added initially, but the benzene or p-xylene aliquot was added only after the cells had consumed half of the initial toluene concentration. This strategy was shown to improve overall degradation rates, and to reduce the stress on the microorganisms. In the sequential fermentation of benzene and toluene, the volumetric degradation rates were 0.056 g l⁻¹ h⁻¹ and 0.079 g l⁻¹ h⁻¹, respectively. In the toluene/p-xylene sequential fermentation, the initial toluene load was consumed before the p-xylene aliquot was consumed. After 12 h in which no p-xylene degradation was observed, a 4.0-g toluene aliquot was added, and p-xylene degradation resumed. Excluding that 12-h period, the microbes consumed toluene and p-xylene at volumetric rates of 0.074 g l⁻¹ h⁻¹ and 0.025 g l⁻¹ h⁻¹, respectively. Oxygen limitation occurred in all fermentations during the rapid growth phase. Received: 16 November 1998 / Received revision: 29 March 1999 / Accepted: 9 April 1999     

Adaptive evolution of nontransgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l⁻¹ ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l⁻¹) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l⁻¹ and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.     

Analysis and optimization of a two-substrate fermentation for xylitol production using Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose using Candida tropicalisATCC 13803. A two-substrate fermentation was designed in order to increase xylitol yield and volumetric productivity. Glucose was used initially for cell growth followed by conversion of xylose to xylitol without cell growth and by-product formation after complete depletion of glucose. High glucose concentrations increased volumetric productivity by reducing conversion time due to high cell mass, but also led to production of ethanol, which, in turn, inhibited cell growth and xylitol production. Computer simulation was undertaken to optimize an initial glucose concentration using kinetic equations describing rates of cell growth and xylose bioconversion as a function of ethanol concentration. Kinetic constants involved in the equations were estimated from the experimental results. Glucose at 32 g L⁻¹ was estimated to be an optimum initial glucose concentration with a final xylose concentration of 86 g L⁻¹ and a volumetric productivity of 5.15 g-xylitol L⁻¹ h⁻¹. The two-substrate fermentation was performed under optimum conditions to verify the computer simulation results. The experimental results were in good agreement with the predicted values of simulation with a xylitol yield of 0.81 g-xylitol g-xylose⁻¹ and a volumetric productivity of 5.06 g-xylitol L⁻¹ h⁻¹. Received 16 June 1998/ Accepted in revised form 28 February 1999     

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Thermophilic acidification of dairy wastewater

Acidification of simulated dairy wastewater was conducted in an upflow reactor at 55 °C. Results showed that the degree of acidification decreased with the increase in chemical oxygen demand (COD) loading rate, from 60.8% at 4 g l⁻¹ day⁻¹ to 27.1% at 24 g l⁻¹ day⁻¹. Carbohydrate was readily degraded at all loading rates, but degradation of protein and lipid decreased with the increase in loading rate. Most carbohydrate degradation occurred at the reactor bottom, whereas protein was degraded mainly after the carbohydrate became depleted. The predominant acidification products were acetate, propionate, butyrate and ethanol, whereas formate, i-butyrate, valerate, i-valerate, caproate, lactate, methanol, propanol and butanol were present in lesser quantities. The increase in loading rate resulted in the increase of propionate and the decrease of acetate, but had little effect on ethanol and butyrate productions. Only 2.5–8.8% of influent COD was converted to hydrogen and methane. The biomass yield was 0.30–0.43 mg VSS mg⁻¹ COD. Received: 8 December 1999 / Received revision: 14 February 2000 / Accepted: 25 February 2000     

<Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> mutant with high inhibitor tolerance obtained by low-energy ion implantation

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l⁻¹ of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g⁻¹. When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l⁻¹ of ABE with a yield of 0.34 g g⁻¹, including 2.2 g l⁻¹ acetone, 6.8 g l⁻¹ butanol, and 0.5 g l⁻¹ ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.     

Towards a high-yield bioconversion of ferulic acid to vanillin

Natural vanillin is of high interest in the flavor market. Microbial routes to vanillin have so far not been economical as the medium concentrations achieved have been well below 1 g l⁻¹. We have now screened microbial isolates from nature and known strains for their ability to convert eugenol or ferulic acid into vanillin. Ferulic acid, in contrast to the rather toxic eugenol, was found to be an excellent precursor for the conversion to vanillin, as doses of several g l⁻¹ could be fed. One of the isolated microbes, later identified as Pseudomonas putida, very efficiently converted ferulic acid to vanillic acid. As vanillin was oxidized faster than ferulic acid, accumulation of vanillin as an intermediate was not observed. A completely different metabolic flux was observed with Streptomyces setonii. During the metabolism of ferulic acid, this strain accumulated vanillic acid only to a level of around 200 mg l⁻¹ and then started to accumulate vanillin as the principal metabolic overflow product. In shake-flask experiments, vanillin concentrations of up to 6.4 g l⁻¹ were achieved with a molar yield of 68%. This high level now forms the basis for an economical microbial production of vanillin that can be used for flavoring purposes. Received: 15 October 1998 / Received revision: 13 January 1999 / Accepted: 18 January 1999     

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Butanol, a four-carbon primary alcohol (C₄H₁₀O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l⁻¹ butanol from 80 g l⁻¹ glycerol as compared to 7.6 g l⁻¹ butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l⁻¹, 0.30 g g⁻¹, and 0.43 g l⁻¹ h⁻¹ could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone–butanol–ethanol productivity and the butanol productivity of 8.3 and 7.8 g l⁻¹ h⁻¹, respectively, could be achieved at the dilution rate of 0.9 h⁻¹. This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.     

Reclamation of an activated-sludge microbial consortium by selective biostimulation

Our previous study showed that an activated-sludge process broke down at the phenol-loading rate of 1.5 g l⁻¹ day⁻¹, when non-flocculating bacteria (called R6T and R10) overgrew the sludge, resulting in a sludge washout. In this study, we attempted to circumvent this breakdown problem by reclaiming the consortium structure. Activated sludge was fed phenol, and the phenol-loading rate was increased stepwise from 0.5 g l⁻¹ day⁻¹ to 1.0 g l⁻¹ day⁻¹ and then to 1.5 g l⁻¹ day⁻¹. Either galactose or glucose (at 0.5 g l⁻¹ day⁻¹) was also supplied to the activated sludge from the phenol-loading rate of 1.0 g l⁻¹ day⁻¹. Pure culture experiments have suggested galactose to be a preferential substrate for a floc-forming bacterium (R6F) that predominantly degrades phenol under low phenol-loading conditions. Supplying galactose allowed sustainment of the R6F population and suppression of the overgrowth of R6T and R10 at the phenol-loading rate of 1.5 g l⁻¹ day⁻¹. This measure allowed the activated-sludge process to treat phenol at a phenol-loading rate up to 1.5 g l⁻¹ day⁻¹, although it broke down at 2.0 g l⁻¹ day⁻¹. In contrast, supplying glucose reduced the R6F population and allowed the activated-sludge process to break down at the phenol-loading rate of 1.0 g l⁻¹ day⁻¹. This study demonstrated that reclamation of the activated-sludge consortium by selective biostimulation of the floc-forming population improved the phenol-treating ability of the process. Received: 13 January 2000 / Received revision: 10 March 2000 / Accepted: 7 April 2000