Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

Nitrate removal from drinking water using a membrane-fixed biofilm reactor

Biological treatment of drinking water is a cost-effective alternative to conventional physico/chemical processes. A new concept was tested to overcome the main disadvantage of biological denitrification, the intensive post-treatment process to remove microorganisms and remnant carbon source. The biological reaction zone and carbon supply were separated from the raw water stream by a nitrate-permeable membrane. Denitrification takes place in a biofilm, which is immobilized at the membrane. In a series of bench-scale runs, different types of membranes and reactor configurations were investigated. The best denitrification rates achieved were 1230 mg NO₃ ⁻-N m⁻² day⁻¹. In one run, raw water containing 100 mg NO₃ ⁻ l⁻¹ was completely freed from nitrate. The membrane and the attached biofilm also represent a barrier against the passage of the C source and nutrients into the raw water. At concentrations of 20 mg l⁻¹ ethanol and 15 mg l⁻¹ phosphate in the bioreactor no diffusion through the membrane into the treated water was observed. Without any post-treatment, the effluent met nearly all the relevant criteria for drinking water; only the colony count was slightly increased. Received: 18 December 1996 / Received last revision: 14 April 1997 / Accepted: 19 April 1997     

Treatment of cyanide-containing wastewater from the food industry in a laboratory-scale fixed-bed methanogenic reactor

During the process of producing cassava starch from Manihot esculenta roots, large amounts of cyanoglycosides were released, which rapidly decayed to CN⁻ following enzymatic hydrolysis. Depending on the varying cyanoglycoside content of the cassava varieties, the cyanide concentration in the wastewater was as high as 200 mg/l. To simulate anaerobic stabilization, a wastewater with a chemical oxygen demand (COD) of about 20 g/l was prepared from cassava roots and was fermented in a fixed-bed methanogenic reactor. The start-up phase for a 99% degradation of low concentrations of cyanide (10 mg/l) required about 6 months. After establishment of the biofilm, a cyanide concentration of up to 150 mg CN⁻/l in the fresh wastewater was degraded during anaerobic treatment at a hydraulic retention time of 3 days. All nitrogen from the degraded cyanide was converted to organic nitrogen by the biomass of the effluent. The cyanide-degrading biocoenosis of the anaerobic reactor could tolerate shock concentrations of cyanide up to 240 mg CN⁻/l for a short time. Up to 5 mmol/l NH₄Cl (i.e. 70 mg N/l = 265 mg NH₄Cl/l) in the fresh wastewater did not affect cyanide degradation. The bleaching agent sulphite, however, had a negative effect on COD and cyanide removal. For anaerobic treatment, the maximum COD space loading was 12 g l⁻¹ day⁻¹, equivalent to a hydraulic retention time of 1.8 days. The COD removal efficiency was around 90%. The maximum permanent cyanide space loading was 50 mg CN⁻ l⁻¹ day⁻¹, with tolerable shock loadings up to 75 mg CN⁻ l⁻¹day⁻¹. Under steady-state conditions, the cyanide concentration of the effluent was lower than 0.5 mg/l. Received: 15 August 1997 / Received revision: 10 October 1997 / Accepted: 14 October 1997     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

Biodegradation of the pesticide 4,6-dinitro-ortho-cresol by microorganisms in batch cultures and in fixed-bed column reactors

A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and 84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly ¹⁴C-labelled DNOC was recovered as ¹⁴CO₂. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol, 2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads in fixed-bed column reactors were 30 mmol day⁻¹ (l reactor volume)⁻¹, leaving an effluent concentration of less than 5 μg l⁻¹ DNOC in the outflowing medium. The apparent K _s value of the immobilized mixed culture for DNOC was 17 μM. Degradation was inhibited at DNOC concentrations above 30 μM and it ceased at 340 μM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing mechanism. Received: 27 March 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997     

Nitrate and phosphate ion removal from water by Phormidium laminosum immobilized on hollow fibres in a photobioreactor

Removal of nitrate and phosphate ions from water, by using the thermophilic cyanobacterium Phormidium laminosum, immobilized on cellulose hollow fibres in the tubular photobioreactor at 43 °C, was studied by continuously supplying dilute growth medium for 7 days and then secondarily treated sewage (STS) for 12 days. The concentrations of NO⁻ ₃ and PO³⁻ ₄ in the effluent from the dilute growth medium decreased from 5.0 mg N/l to 3.1 mg N/l, and from 0.75 mg P/l to 0.05 mg P/l respectively, after a residence time of 12 h. The concentrations of NO⁻ ₃ and PO³⁻ ₄ in the effluent from STS decreased from 11.7 mg N/l to 2.0 mg N/l, and from 6.62 mg P/l to 0.02 mg P/l respectively, after a residence time of 48 h. The removal rates of nitrogenous␣and phosphate ions from STS were 0.24 and 0.11 mmol day⁻¹ l reactor⁻¹ respectively, under the same conditions. Although, among nitrogenous ions, nitrate and ammonium ions were efficiently removed by P.␣laminosum, the nitrite ion was released into the effluent when STS was used as influent. Treatment of water with thermophilic P. laminosum immobilized on hollow fibres thus appears to be an appropriate means for the removal of inorganic nitrogen and phosphorus from treated wastewater. Received: 15 August 1997 / Received last revision: 18 November 1997 / Accepted: 29 November 1997     

Bioconversion of limonene to α-terpineol by immobilized Penicillium digitatum

Bioconversion of (4R)-(+)-limonene to (4R)-(+)-α-terpineol by immobilized fungal mycelia of Penicillium digitatum was investigated in batch, repeated-batch and continuously fed systems. The fungi were immobilized in calcium alginate beads. These beads remained active for at least 14 days when they were stored at 4 °C. Three different aeration rates were tested. The highest yield was obtained at a dissolved oxygen level of 50.0 μmol/l. α-Terpineol production by this fungus was 12.83 mg (g beads)⁻¹ day⁻¹, producing a 45.81% bioconversion of substrate. Repeated-batch bioconversion showed yield decreases in the second and the third cycles. Regeneration with nutrient media after the third cycle improved the bioconversion yields. With continuous bioconversion, the half-life was dependent on the aeration. The optimum conditions with a continuous reactor were at an aeration rate of 0.3 standard l/min and a dilution rate of 0.0144 h⁻¹. Received: 10 June 1997 / Received revision: 18 August 1997 / Accepted: 11 September 1997     

Toluene degradation in the recycle liquid of biotrickling filters for air pollution control

Pollutant degradation in biotrickling filters for waste air treatment is generally thought to occur only in the biofilm. In two experiments with toluene degrading biotrickling filters, we show that suspended microorganisms in the recycle liquid may substantially contribute to the overall pollutant removal. Two days after reactor start up, the overall toluene elimination capacity reached a maximum of 125 g m⁻³ h⁻¹, which was twice that found during prolonged operation. High biodegradation activity in the recycle liquid fully accounted for this short-term peak of pollutant elimination. During steady-state operation, the toluene degradation in the recycle liquid was 21% of the overall elimination capacity, although the amount of suspended biomass was only 1% of the amount of immobilized biomass. The results suggest that biotrickling filter performance may be improved by selecting operating conditions allowing for the development of an actively growing suspended culture. Received: 16 June 1999 / Received revision: 17 November 1999 / Accepted: 15 December 1999     

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     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

Degradation of naphthalene by cells of Pseudomonas sp. strain NGK 1 immobilized in alginate, agar and polyacrylamide

A Pseudomonas sp. strain NGK 1 (NCIM 5120) was immobilized in various matrices, namely, alginate, agar (1.8 × 10¹¹ cfu g⁻¹ beads) and polyacrylamide (1.6 × 10¹¹ cfu g⁻¹ beads). The degradation of naphthalene was studied, by freely suspended cells (4 × 10¹⁰ cfu ml⁻¹) and immobilized cells in batches, with shaken culture and continuous degradation in a packed-bed reactor. Free cells brought about the complete degradation of 25 mmol naphthalene after 3 days of incubation, whereas, a maximum of 30 mmol naphthalene was degraded by the bacteria after 3–4 days of incubation with 50 mmol and 75 mmol naphthalene, and no further degradation was observed even after 15 days of incubation. Alginate-entrapped cells had degraded 25 mmol naphthalene after 3.5 days of incubation, whereas agar- and polyacrylamide-entrapped cells took 2.5 days; 50 mmol naphthalene was completely degraded by the immobilized cells after 6–7 days of incubation. Maximum amounts of 55 mmol, 70 mmol and 67 mmol naphthalene were degraded, from an initial 75 mmol naphthalene, by the alginate-, agar- and polyacrylamide-entrapped cells after 15 days of incubation. When the cell concentrations were doubled, 25 mmol and 50 mmol naphthalene were degraded after 2 and 5.5 days of incubation by the immobilized cells. Complete degradation of 75 mmol naphthalene occurred after 10 days incubation with agar- and polyacrylamide-entrapped␣cells, whereas only 60 mmol naphthalene was degraded by alginate-entrapped cells after 15 days of␣incubation. Further, with 25 mmol naphthalene, alginate-, agar- and polyacrylamide-entrapped cells (1.8 × 10¹¹ cfu g⁻¹ beads) could be reused 18, 12 and 23 times respectively. During continuous degradation in a packed-bed reactor, 80 mmol naphthalene 100 ml⁻¹ h⁻¹ was degraded by alginate- and polyacrylamide-entrapped cells whereas 80 mmol naphthalene 125 ml⁻¹␣h⁻¹ was degraded by agar-entrapped cells. Received: 21 October 1997 / Received revision: 15 January 1998 / Accepted: 18 January 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     

Fate and transformation of thiocyanate and cyanate under methanogenic conditions

The fate of thiocyanate (SCN⁻) and cyanate (OCN⁻) under methanogenic conditions was investigated at 35 °C. Thiocyanate and cyanate were added to mixed methanogenic cultures along with an organic mixture. Thiocyanate was stable under these conditions, and had no adverse effect on methanogenesis at a concentration as high as 2.5 mM. In contrast, cyanate at a concentration as low as 0.3 mM initially inhibited methanogenesis but, after the complete removal of cyanate, methanogenesis gradually recovered. The inhibitory effect of cyanate on methanogenesis became more profound with repeated additions of cyanate. The transformation of cyanate followed the hydrolytic route to ammonia and bicarbonate under anaerobic conditions and its hydrolysis rate was enhanced by microbial activity. Cyanide was not detected as a cyanate transformation product under the methanogenic conditions of this study. Received: 13 June 1997 / Received revision: 29 August 1997 / Accepted: 15 September 1997     

The use of silica gel prepared by sol-gel method and polyurethane foam as microbial carriers in the continuous degradation of phenol

A mixed microbial culture was immobilized by entrapment into silica gel (SG) and entrapment/ adsorption on polyurethane foam (PU) and ceramic foam. The phenol degradation performance of the SG biocatalyst was studied in a packed-bed reactor (PBR), packed-bed reactor with ceramic foam (PBRC) and fluidized-bed reactor (FBR). In continuous experiments the maximum degradation rate of phenol (q _s ^max) decreased in the order: PBRC (598 mg l⁻¹h⁻¹) > PBR (PU, 471 mg l⁻¹h⁻¹) > PBR (SG, 394 mg l⁻¹h⁻¹) > FBR (PU, 161 mg l⁻¹h⁻¹) > FBR (SG, 91 mg l⁻¹ h⁻¹). The long-term use of the SG biocatalyst in continuous phenol degradation resulted in the formation of a 100–200 μm thick layer with a high cell density on the surface of the gel particles. The abrasion of the surface layer in the FBR contributed to the poor degradation performance of this reactor configuration. Coating the ceramic foam with a layer of cells immobilized in colloidal SiO₂ enhanced the phenol degradation efficiency during the first 3 days of the PBRC operation, in comparison with untreated ceramic packing. Received: 2 December 1999 / Revision received: 2 February 2000 / Accepted: 4 February 2000     

Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h⁻¹ and 1.0 h⁻¹ and glycerol input concentrations of 32 g l⁻¹ and 56 g l⁻¹. The near-to-optimum propanediol concentration of 26.5 g l⁻¹ (for 56 g l⁻¹ glycerol) was maintained up to a dilution rate of 0.5 h⁻¹ and then decreased while the propanediol productivity was highest at 0.7 h⁻¹. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved. Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population

The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l⁻¹ to 20 g l⁻¹ the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l⁻¹ day⁻¹ compared to 320 mg l⁻¹ day⁻¹ at 10 g l⁻¹ peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH₃/g peptone in the mesophilic cultures. If 0.5–6.5 g l⁻¹ ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l⁻¹ was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a K _i (50%) for NH₃ of 92, 95 and 88 mg l⁻¹ at 37 °C and 251, 274 and 297 mg l⁻¹ at 55 °C respectively. This indicated that the thermophilic flora tolerated significantly more NH₃ than the mesophilic flora. In the mesophilic reactor effluent 4.6 × 10⁸ peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 × 10⁷ cfu/ml was observed. Received: 24 April 1998 / Received revision: 26 June 1998 / Accepted: 27 June 1998     

Nitrate reduction by Citrobacter diversus under aerobic environment

A new aerobic denitrifier, Citrobacter diversus, was isolated from both nitrification and denitrification sludge. To monitor the variation in the concentration of nitrogen oxides, aerobic denitrification by C. diversus was carried out in a batch reactor. When the nitrate concentration was greater than 180 mg N l⁻¹, the nitrate reduction rate became stable. The effect of the C/N ratio on the denitrification activity was also investigated. The results showed that the optimum denitrification activity was obtained when the C/N ratio was 4–5. The range of the C/N ratio was higher than that for traditional anoxic denitrification. The effect of the dissolved oxygen concentration was further studied; and it was found that the range of dissolved oxygen concentrations, both for specific growth rates and for specific denitrification rates, was 2–6 mg⁻¹. From these results, it can be concluded that both the concentration of dissolved oxygen and the C/N ratio are key factors in the aerobic denitrification by C. diversus. Received: 23 November 1999 / Received revision: 4 February 2000 / Accepted: 13 February 2000     

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     

Use of immobilized bacteria to treat industrial wastewater containing a chlorinated pyridinol

 Pseudomonas sp. strain M285 immobilized on diatomaceous earth beads was used to remove 3,5,6-trichloro-2-pyridinol (TCP) from industrial wastewater. Batch studies showed that immobilized Pseudomonas sp. strain M285 mineralized [2,6-¹⁴C]TCP rapidly; about 75% of the initial radioactivity was recovered as ¹⁴CO₂. Transformation of TCP was inhibited by high concentrations of salt, and addition of osmoprotectants (proline and betaine at 1 mM) did not reduce the adverse effect of salt. TCP-containing wastewater (60–140 mg/l) was passed through columns containing immobilized Pseudomonas sp. strain M285 at increasing flow rates and increasing TCP concentrations; TCP removal of 80%–100% was achieved. Addition of nutrients, such as glucose and yeast extract, retarded TCP degradation. Growing cell cultures were found to be better inocula for immobilization than resting cells. Received: 5 February 1996 / Received last revision: 12 August 1996 / Accepted: 24 August 1996