Solvent production by Clostridium beijerinckii NRRL B592 growing on different potato media

Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media. The increase in whole potato concentration contributed to the increased final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects. To obtain good solvent productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch was necessary, indicating high activity of the clostridial amylases produced by the strain applied. Received: 17 April 1998 / Received revision: 22 June 1998 / Accepted: 27 June 1998     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Dialysis cultures

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a “nutrient-split” feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective. Received: 24 March 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Repeated application of carvone-induced bacteria to enhance biodegradation of polychlorinated biphenyls in soil

Carvone, the principal component of spearmint oil, induces biodegradation of polychlorinated biphenyls (PCB) by Arthrobacter sp. strain B1B. This study investigated the effectiveness of the repeated application of carvone-induced bacteria for bioremediation of Aroclor-1242-contaminated soil. Control treatments compared a single inoculation of carvone-induced cells, repeated applications of noninduced cells, and repeated applications of cell-free carvone/fructose medium. The results showed that repeated application of carvone-induced bacteria was the most effective treatment for mineralizing PCB, resulting in 27 ± 6% degradation of Aroclor 1242 after 9 weeks; whereas a single application of cells resulted in no significant degradation. Addition of cell-free, carvone/fructose medium resulted in 10% degradation of PCB, which suggests that this treatment stimulated biodegradation of PCB by the indigenous microflora. The di- and trichlorobiphenyls were the most readily degraded congeners. More highly chlorinated congeners, which had been previously shown to be degraded in liquid culture, were not substantially degraded in soil, indicating that low bioavailability may have limited their degradation. With the development of new technology, which permits automated in situ fermentation and delivery of degrader microorganisms, the repeated application of carvone-induced bacteria may facilitate bioremediation of PCB-contaminated soils. Received: 7 January 1998 / Received revision: 18 June 1998 / Accepted: 27 June 1998     

Pullulan content of the ethanol precipitate from fermented agro-industrial wastes

Ethanol-precipitated substances after fermentation of various agro-industrial wastes by Aureobasidium pullulans were examined for their pullulan content. Grape skin pulp extract, starch waste, olive oil waste effluents and molasses served as substrates for the fermentation. A glucose-based defined medium was used for comparison purposes. Samples were analysed by an enzyme-coupled assay method and by high-performance anion-exchange chromatography with pulsed amperometric detection after enzymic hydrolysis with pullulanase. Fermentation of grape skin pulp extract gave 22.3 g l⁻¹ ethanol precipitate, which was relatively pure pullulan (97.4% w/w) as assessed by the coupled-enzyme assay. Hydrolysed starch gave only 12.9 g l⁻¹ ethanol precipitate, which increased to 30.8 g l⁻¹ when the medium was supplemented with NH₄NO₃ and K₂HPO₄; this again was relatively pure pullulan (88.6% w/w). Molasses and olive oil wastes produced heterogeneous ethanol-precipitated substances containing small amounts of pullulan, even when supplemented with nitrogen and phosphate. Overall, grape skin pulp should be considered as the best substrate for pullulan production. Starch waste requires several hydrolyis steps to provide a usable carbon source, which reduces its economic attraction as an industrial process. Received: 24 October 1997 / Received revision: 10 February 1998 / Accepted: 15 February 1998     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Biosynthesis of tannin acyl hydrolase from tannin-rich forest residue under different fermentation conditions

Caesalpinia digyna, a tannin-rich forest residue, was used as substrate for production of tannase and gallic acid. Media engineering was carried out under solid-state fermentation, submerged fermentation and modified solid state fermentation conditions for optimum synthesis of tannase and gallic acid (based on 58% tannin content in the raw material). Tannase vis-à-vis gallic acid recovery under modified solid-state fermentation condition was maximum. Conversions of tannin to gallic acid under solid-state fermentation, submerged fermentation and modified solid-state fermentation conditions were 30.5%, 27.5% and 90.9%, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 29–38. Received 02 November 1999/ Accepted in revised form 12 February 2000     

New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation

Clostridial acetone/butanol fermentation used to rank second only to ethanol fermentation by yeast in its scale of production and thus is one of the largest biotechnological processes known. Its decline since about 1950 has been caused by increasing substrate costs and the availability of much cheaper feedstocks for chemical solvent synthesis by the petrochemical industry. The so-called oil crisis in 1973 led to renewed interest in novel fermentation and product recovery technologies as well as in the metabolism and genetics of the bacterial species involved. As a consequence, almost all of the enzymes leading to solvent formation are known, their genes have been sequenced (in fact, Clostridium acetobutylicum has been recently included in the microbial genome sequencing project), the regulatory mechanisms controlling solventogenesis have begun to emerge and recombinant DNA techniques have been developed for these clostridia to construct specific production strains. In parallel, cheap agricultural-waste-based feedstocks have been exploited for their potential as novel substrates, continuous culture methods have been successfully established and new on-line product recovery technologies are now available, such as gas stripping, liquid/liquid extraction, and membrane-based methods. In combination with these achievements, a reintroduction of acetone/butanol fermentation on an industrial scale seems to be economically feasible, a view that is supported by a new pilot plant in Austria recently coming into operation. Received: 18 December 1997 / Received revision: 27 January 1998 / Accepted: 27 January 1998     

Kinetics of Thermoascus aurantiacus solid-state fermentation on sugar-beet pulp – polysaccharide alteration and production of related enzymatic activities

 The fungal solubilization of cell wall components of sugar-beet pulp, during solid-state fermentation of Thermoascus aurantiacus, is reported here. The extracellular fungal enzyme activities related to the substrate degradation were also studied. In 120 h, more than 60% of the main sugar-beet pulp polysaccharides, i.e. pectins, arabinose- and glucose-containing polysaccharides, were rapidly brought into solution by the fungus. The slow accumulation of monosaccharides compared to the fast degradation of the polysaccharides suggested that most of the released sugars were consumed by the fungus. The analysis of the enzymes present in the water extracts of the solid-state cultures proved that the fungus was able to synthesize a complete enzymatic system required for the hydrolysis of the main sugar-beet pulp polysaccharides. The highest enzyme activities measured were β-glucosidase and α-L-arabinofuranosidase. Received: 22 September 1995/Received revision: 15 January 1996/Accepted: 22 January 1996     

The formation of 1-hydroxymethylnaphthalene and 6-hydroxymethylquinoline by both oxidative and reductive routes in Cunninghamella elegans

Extraction of medium after incubation of the fungus, Cunninghamella elegans, with 0.03% (w/v) 1-methylnaphthalene produced mainly 1-hydroxymethylnaphthalene together with some 1-naphthoic acid and hydroxynaphthoic acid. Higher concentrations of substrate were inhibitory to biotransformation. Similar incubations with 1-naphtoic acid as substrate resulted in reduction of the carboxyl group to give 1-hydroxymethylnaphthalene. When 6-methylquinoline was used, the main product was 6-hydroxymethylquinoline but also some quinoline-6-carboxylic acid and some 6-methylquinoline-N-oxide were identified. In a 2-l fermenter 2.5 g substrate was transformed in 324 h. The 6-hydroxymethylquinoline was also produced by reduction of quinoline-6-carboxylic acid by the organism. Received: 9 March 1998 / Received revision: 15 June 1998 / Accepted: 19 June 1998     

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agar-plate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 °C respectively. Received: 2 February 1998 / Received revision: 20 April 1998 / Accepted: 27 April 1998     

Degradation of cyanide in agroindustrial or industrial wastewater in an acidification reactor or in a single-step methane reactor by bacteria enriched from soil and peels of cassava

During cassava starch production, large amounts of cyanoglycosides were released and hydrolysed by plant-borne enzymes, leading to cyanide concentrations in the wastewater as high as 200 mg/l. For anaerobic degradation of the cyanide during pre-acidification or single-step methane fermentation, anaerobic cultures were enriched from soil residues of cassava roots and sewage sludge. In a pre-acidification reactor this culture was able to remove up to 4 g potassium cyanide/l of wastewater at a hydraulic retention time (t _HR) of 4 days, equivalent to a maximal cyanide space loading of 400 mg CN⁻ l⁻¹ day⁻¹. The residual cyanide concentration was 0.2–0.5 mg/l. Concentrated cell suspensions of the mixed culture formed ammonia and formate in almost equimolar amounts from cyanide. Little formamide was generated by chemical decay. A concentration of up to 100 mmol ammonia/l had no inhibitory effect on cyanide degradation. The optimal pH for cyanide degradation was 6–7.5, the optimal temperature 25–37 °C. At a pH of 5 or lower, cyanide accumulated in the reactor and pre-acidification failed. The minimal t _HR for continuous cyanide removal was 1.5 days. The enriched mixed culture was also able to degrade cyanide in purely mineralic wastewater from metal deburring, either in a pre-acidification reactor with a two-step process or in a one-step methanogenic reactor. It was necessary to supplement the wastewater with a carbon source (e.g. starch) to keep the population active enough to cope with any possible inhibiting effect of cyanide. Received: 29 April 1998 / Received revision: 8 June 1998 / Accepted: 14 June 1998     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Towards rice genome scanning by map-based AFLP fingerprinting

Map-based DNA fingerprinting with AFLP markers provides a fast method for scanning the rice genome. Three hundred AFLP markers identified with ten primer combinations were mapped in two rice populations. The genetic maps were aligned and almost full coverage of the rice genome was obtained. The transferability of AFLP markers between indica × japonica and indica × indica crosses was tested. The chromosomes were divided into DNA Fingerprint Linkage Blocks (DFLBs) defined by specific AFLP markers. Using these blocks, the degree of similarity or divergence within specific chromosome regions was calculated for nine varieties. Applications of map-based fingerprinting for biodiversity studies and maker-assisted selection are discussed. Received: 6 June 1998 / Accepted: 11 November 1998     

Critical factors in chitin production by fermentation of shrimp biowaste

Factors affecting Lactobacillus fermentation of shrimp waste for chitin and protein liquor production were determined. The objective of the fermentation is medium conditioning by Lactobacillus through production of proteases and lowering of the pH. The efficiency was tested by conducting fermentation of biowaste in 1-l beakers with or without pH adjustment using different acids. Addition of 5% glucose to the biowaste supported the growth of lactic acid bacteria and led to better fermentation. Among four acids tested to control pH at the start and during fermentation, acetic acid and citric acid proved to be the most effective. In biowaste fermented with 6.7% L. plantarum inoculum, 5% glucose, and pH 6.0 adjusted with acetic acid, 75% deproteination and 86% demineralization was achieved. Replacement of acetic acid by citric acid gave 88% deproteination and 90% demineralization. The fermentation carried out in the presence of acetic acid resulted in a protein fraction that smelled good and a clean chitin fraction. Received: 4 April 2000 / Received revision: 9 June 2000 / Accepted: 9 June 2000     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Development of markers for product formation and cell cycle in batch cultivation of Clostridium acetobutylicum ATCC 824

Experiments were performed to identify relationships between morphological and physiological events during batch fermentation of Clostridium acetobutylicum ATCC 824. Differing fermentation conditions were obtained by manipulation of the culture pH value during the process. The bacterium showed marked changes in morphology during its cultivation, similar to those previously observed for other strains. However, although the acidogenic phase was characterized by the presence of rod-shaped cells, and the solventogenic phase by clostridial forms, there was no simple relationship between the proportion of clostridial forms present and the ratio of solvents to acids. Nevertheless, the shift from acidogenesis to solventogenesis was always coincident with the presence of some clostridial forms and with the accumulation of granulose within the cells. In addition, the “solvent shift” was associated with major changes in the cellular protein pattern, as analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Hence, the potential solventogenic ability of any particular culture may be recognised by its morphological appearance and/or its cellular protein pattern. Received: 19 September 1997 / Received revision: 9 February 1998 / Accepted: 9 February 1998     

A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation

We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisherilux operon. This recA–lux fusion (pMOE15) was introduced into wild-type P. aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production. The RM4440 strain responded to increasing doses of ultraviolet radiation by an increase in its bioluminescence. RM4440 has the potential to be useful as a biosensor for the presence of DNA-damaging agents in the environment. Received: 18 February 1998 / Received revision: 18 June 1998 / Accepted: 27 June 1998     

Biotechnology of succinic acid production and markets for derived industrial products

Succinic acid, derived from fermentation of agricultural carbohydrates, has a specialty chemical market in industries producing food and pharmaceutical products, surfactants and detergents, green solvents and biodegradable plastics, and ingredients to stimulate animal and plant growth. As a carbon-intermediate chemical, fermentation-derived succinate has the potential to supply over 2.7 × 10⁸ kg industrial products/year including: 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, adipic acid, n-methylpyrrolidone and linear aliphatic esters. Succinate yields as high as 110 g/l have been achieved from glucose by the newly discovered rumen organism Actinobacillus succinogenes. Succinate fermentation is a novel process because the greenhouse gas CO₂ is fixed into succinate during glucose fermentation. New developments in end-product recovery technology, including water-splitting electrodialysis and liquid/liquid extraction have lowered the cost of succinic acid production to U.S. $ 0.55/kg at the 75 000 tonne/year level and to $ 2.20/kg at the 5000 tonne/year level. Research directions aimed at further improving the succinate fermentation economics are discussed. Received: 27 October 1998 / Received revision: 22 January 1999 / Accepted: 22 January 1999