Fermentation optimization for the production of lovastatin by Aspergillus terreus: use of response surface methodology

A Box–Behnken experimental design was used to investigate the effects of five factors—ie oxygen content in the gas phase; concentrations of C, N and P; and fermentation time—on the concentrations of biomass and lovastatin produced in batch cultures of Aspergillus terreus. The values of the various factors in the experiment ranged widely, as follows: 20–80% (v/v) oxygen in the aeration gas; 8–48 g dm^?3 C‐concentration; 0.2–0.6 g dm^?3 N‐concentration; 0.5–2.5 g dm^?3 phosphate‐concentration; and 7–11 days fermentation time. No previous work has used statistical analysis in documenting the interactions between oxygen supply and nutrient concentrations in lovastatin production. The Box–Behnken design identified the oxygen content in the gas phase as the principal factor influencing the production of lovastatin. Both a limitation and excess of oxygen reduced lovastatin titers. A medium containing 48 g dm^?3 C supplied as lactose, 0.46 g dm^?3 N supplied as soybean meal, and 0.79 g dm^?3 phosphate supplied as KH₂PO₄, was shown to support high titers (～230 mg dm^?3) of lovastatin in a 7‐day fermentation in oxygen‐rich conditions (80% v/v oxygen in the aeration gas). Under these conditions, the culture medium had excess carbon but limiting amounts of nitrogen. The optimized fermentation conditions raised the lovastatin titer by four‐fold compared with the worst‐case scenario within the range of factors investigated. Copyright © 2004 Society of Chemical Industry     

Maximum volumetric production of β‐galactosidase by Kluyveromyces fragilis in fed‐batch culture with automatic control

The production of β‐galactosidase by Kluyveromyces fragilis was studied in different culture systems, with dissolved oxygen concentration control and using defined media. An operating strategy of fed‐batch culture with automatic control of substrate addition regulated by dissolved oxygen concentration, consisting of the replacement of variable volumes of broth by fresh medium (once the fed‐batch culture has finished), was designed. The volumetric enzyme productivity (Q_p, 13 600 UI dm^?3 h^?1) obtained was 38% higher than that reached in continuous culture of K fragilis with dissolved oxygen concentration control and far higher than that obtained by batch culture of K fragilis under the same aeration conditions. © 2002 Society of Chemical Industry     

Production of β‐galactosidase from recombinant Saccharomyces cerevisiae grown on lactose

Improved productivity and costs reduction in fermentation processes may be attained by using flocculating cell cultures. The production of extracellular heterologous β‐galactosidase by recombinant flocculating Saccharomyces cerevisiae cells, expressing the lacA gene (coding for β‐galactosidase) of Aspergillus niger under the ADHI promotor and terminator in a bioreactor was studied. The effects of lactose concentration and yeast extract concentration on β‐galactosidase production in a semi‐synthetic medium were analysed. The extracellular β‐galactosidase activity increased linearly with increasing initial lactose concentrations (5–150 g dm^?3). β‐Galactosidase production also increased with increased yeast extract concentration. During the entire fermentation, no accumulation of the hydrolysed sugars, glucose and galactose, was observed. The catabolic repression of the recombinant strain when cultured in a medium containing equal amounts of glucose and galactose was confirmed. In complete anaerobiosis, the fermentation of lactose resulted in a very slow fermentation pattern with lower levels of β‐galactosidase activity. The bioreactor operation together with optimisation of culture conditions (lactose and yeast extract concentration) led to a 21‐fold increase in the extracellular β‐galactosidase activity produced when compared with preliminary Erlenmeyer fermentations. Copyright © 2004 Society of Chemical Industry     

Kinetic modelling of lactic acid production from whey by Lactobacillus casei (NRRL B‐441)

The biomass growth, lactic acid production and lactose utilisation kinetics of lactic acid production from whey by Lactobacillus casei was studied. Batch fermentation experiments were performed at controlled pH and temperature with six different initial whey lactose concentrations (9‐77 g dm^?3) in a 3 dm³ working volume bioreactor. Biomass growth was well described by the logistic equation with a product inhibition term. In addition, biomass and product inhibition effects were defined with corresponding power terms, which enabled adjustment of the model for low‐ and high‐substrate conditions. The Luedeking‐Piret equation defined the product formation kinetics. Substrate consumption was explained by production rate and maintenance requirements. A maximum productivity of 2.5 g dm^?3 h^?1 was attained with an initial lactose concentration of 35.5 g dm^?3. Copyright © 2006 Society of Chemical Industry     

A bioprocessing mode for simultaneous fungal biomass protein production and wastewater treatment using an external air‐lift bioreactor

A pilot plant investigation for bioprocessing has been undertaken to develop a simple, non‐aseptic, low‐cost single process for production of fungal biomass protein (FBP) and wastewater treatment using starch processing wastewater. It has been confirmed that the newly developed external air‐lift bioreactor was very suitable for bioconversion of starch materials and FBP production by the microfungi Aspergillus oryzae and Rhizopus arrhizus. Bioproduct yields of 8.5 g dm^?3 of FBP that contained 46–50% protein were obtained within a comparatively short retention time. A fungal biomass productivity in a range of 0.85–0.92 g dm^?3 h^?1 and removals of total suspended solids and 95% COD were achieved in batch, semi‐continuous and continuous processes. The operation modes of the semi‐continuous and continuous processes demonstrated a high biological dynamics in fungal biomass productivity and COD reduction. The semi‐continuous process appeared to be the most practical mode. © 2001 Society of Chemical Industry     

D‐arabitol production from lactose by Kluyveromyces lactis at different aerobic conditions

BACKGROUND: The pentitol D‐arabitol has been produced from D‐glucose utilizing osmophilic yeast strains, however, there are remarkably few reports available on the production of D‐arabitol from lactose. Previous studies in the laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 can convert lactose to extracellular D‐arabitol without extracellular accumulation of D‐glucose or D‐galactose. The present study was undertaken to determine the participation of aeration on the D‐arabitol synthesis in K. lactis NBRC 1903. RESULTS: The highest D‐arabitol concentration of 91.7 mmol L^?1 was achieved after 120 h cultivation in medium containing 555 mmol L^?1 of lactose with initial volumetric liquid‐phase mass transfer coefficient of oxygen (k_La)₀ of 85.5 h^?1. The fractional yield of D‐arabitol was affected by not only aeration but also growth phase. The highest fractional yield of D‐arabitol in terms of lactose consumption was 0.255 that was obtained at stationary phase with (k_La)₀ of 85.5 h^?1. CONCLUSION: It was found that oxygen supply is a key factor in the production of D‐arabitol. Patterns of metabolism were classified according to the level of oxygen supply and the growth phase. Copyright © 2010 Society of Chemical Industry     

Research note: Effect of dissolved oxygen level on lactase production by Kluyveromyces fragilis

The influence of dissolved oxygen (% DO) on lactase production by Kluyveromyces fragilis (NRRL-Y-1109) in chemostat culture using a defined medium was studied. The aim was to determine conditions for both high specific enzyme activity and high volumetric enzyme productivity. Significant differences in the specific enzyme activity and specific and volumetric enzyme productivity were found at the corresponding steady states when the DO was varied between 0 and 90%. Maximum lactase production was attained at 10% DO. Under this condition the best results were an enzyme activity of 5910 IU g⁻¹, specific production rate of 1810 IU g⁻¹ h⁻¹ and volumetric production rate of 1530 IU dm⁻³ h⁻¹. This seem to be due to the fact that at low aeration conditions the yeast metabolism is more reductive and as a consequence it verifies both higher specific lactose consumption rate and higher enzyme expression than in full aeration conditions. The results of this investigation are also compared with those of other studies of lactase production by Kluyveromyces sp. © 1998 Society of Chemical Industry     

Synthesis and characterization of cassava starch graft poly(acrylic acid) and poly[(acrylic acid)‐co‐acrylamide] and polymer flocculants for wastewater treatment

Starch‐g‐poly(acrylic acid) and poly[(acrylic acid)‐co‐acrylamide] synthesized via chemically crosslinking polymerization were then each mixed with inorganic coagulants of aluminum sulfate hydrate [Al₂(SO₄)₃·18H₂O], calcium hydroxide [Ca(OH)₂], and ferric sulfate [Fe₂(SO₄)₃] in a proper ratio to form complex polymeric flocculants (CPFs). All CPFs exhibited low water absorbency than those of the uncomplexed superabsorbent copolymers. The color reduction by the CPFs was tested with both synthetic wastewater and selected wastewater samples from textile industries. The synthetic wastewater was prepared from a direct dye in a concentration of 50 mg dm^?3 at pH 7. The CPFs of poly[(acrylic acid)‐co‐acrylamide] with calcium hydroxide at a ratio of 1:2 is the most effective CPF for the wastewater color reduction. The CPF concentration of 500 mg dm^?3 could reduce the color of the synthetic wastewater containing the direct dye solution by 95.4% and that of the industrial wastewater by 76%. Starch‐g‐poly(acrylic acid)/Ca(OH)₂ CPF can reduce the synthetic direct dye and the industrial wastewater by 74% and 18%, respectively. Chemical oxygen demand, residual metal ion concentrations, pHs, turbidity of the wastewater were also investigated and the potential use of the complex polymer flocculants for textile wastewater treatment was indicated. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 2915–2928, 2006     

Optimization of lactic acid production from whey by L casei NRRL B‐441 immobilized in chitosan stabilized Ca‐alginate beads

The production of lactic acid from whey by Lactobacillus casei NRRL B‐441 immobilized in chitosan‐stabilized Ca‐alginate beads was investigated. Higher lactic acid production and lower cell leakage were observed with alginate–chitosan beads compared with Ca‐alginate beads. The highest lactic acid concentration (131.2 g dm^?3) was obtained with cells entrapped in 1.3–1.7 mm alginate–chitosan beads prepared from 2% (w/v) Na‐alginate. The gel beads produced lactic acid for five consecutive batch fermentations without marked activity loss and deformation. Response surface methodology was used to investigate the effects of three fermentation parameters (initial sugar, yeast extract and calcium carbonate concentrations) on the concentration of lactic acid. Results of the statistical analysis showed that the fit of the model was good in all cases. Initial sugar, yeast extract and calcium carbonate concentrations had a strong linear effect on lactic acid production. The maximum lactic acid concentration of 136.3 g dm^?3 was obtained at the optimum concentrations of process variables (initial sugar 147.35 g dm^?3, yeast extract 28.81 g dm^?3, CaCO₃ 97.55 g dm^?3). These values were obtained by fitting of the experimental data to the model equation. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactic acid production using alginate–chitosan‐immobilized cells. Copyright © 2005 Society of Chemical Industry     

Zinc uptake by Streptomyces rimosus biomass using a packed‐bed column

The ability of Streptomyces rimosus biomass to bind zinc ions in batch mode was shown recently. The aim of this study was to determine the zinc uptake capacity by Streptomyces rimosus biomass in continuous mode. Bacterial biomass was able to bind more Zn(II) after pretreatment with sodium hydroxide (1 mol dm⁻³) than without treatment. The maximum adsorption capacity and the adsorption capacity at the saturation point calculated by means of both the exchange zone model and the Thomas model were practically identical of about 2.9 mg_Zn(II) g⁻¹_biomass. This result was lower than the batch adsorption capacity of Streptomyces rimosus, indicating that the packed‐bed is not the most appropriate process to exploit the bacterial biomass adsorption capacity. The effect of zinc concentration in the range of 10 to 200 mg_Zn(II) dm⁻³ on the biosorption capacity of the packed‐bed was not significant. Biomass regeneration with 0.1 mol dm⁻³ HCl gave a 90% recovery of the adsorbed Zn(II). © 1999 Society of Chemical Industry     

Batch production of L(+) lactic acid from whey by Lactobacillus casei (NRRL B‐441)

The effects of temperature, pH, and medium composition on lactic acid production by Lactobacillus casei were investigated. The highest lactic acid productivity values were obtained at 37 °C and pH 5.5. The productivity was 1.87 g dm^?3 h^?1 at 37 °C in shake flasks. In the fermenter, a productivity of 3.97 g dm^?3 h^?1 was obtained at pH 5.5. The most appropriate yeast extract concentration was 5.0 g dm^?3. Whey yielded a higher productivity value than the analytical lactose and glucose. Initial whey lactose concentration did not affect lactic acid productivity. MnSO₄ ·H₂O was necessary for lactic acid production by L casei from whey. Product yields were approximately 0.93 g lactic acid g lactose^?1. Copyright © 2004 Society of Chemical Industry     

Intermediate accumulation and efficiency of anaerobic digestion treatment of surfactant (alcohol sulfate)‐rich wastewater at increasing surfactant/biomass ratios

Textile (eg cotton) finishing industry wastewater is characterised by high concentrations of surfactants (up to 2 g dm^?3) and of readily biodegradable biopolymers (COD 5–15 g dm^?3). The anionic surfactant decyl sulfate (DS) was chosen as model surfactant and soluble starch (size) as model compound for the readily biodegradable fraction of the wastewater. Twenty‐two batch experiments with increasing DS/biomass ratio (and starch/biomass ratio) were started simultaneously. Biomass concentrations ranged from 50 to 15 000 mg dm^?3. Minor inhibition effects were found for the surfactant degradation itself at all DS/biomass ratios (maximum biodegradation rate 7.7 mg_DS g_biomass^?1 h^?1). The starch hydrolysis started without a lag‐phase at DS/biomass ratios of up to 0.15 g_DS g_biomass^?1. The lag‐phase was prolonged to about 100 h at a very high DS/biomass ratio (3 g_DS g_biomass^?1). The relative importance of the accumulated intermediates was dependent on the DS/biomass ratio. Above 0.3 g_DS g_biomass^?1 10% of the substrate organic carbon accumulated as ethanol, but no ethanol accumulation was observed at low DS/biomass ratios. Moderate DS/biomass ratios caused a considerable delay of the methanogenesis; high DS/biomass ratios prevented the methanogenesis almost completely. © 2002 Society of Chemical Industry     

Removal of aqueous phenolic compounds from a model system by oxidative polymerization with turnip (Brassica napus L var purple top white globe) peroxidase

Turnip roots, which are readily available in Mexico, are a good source of peroxidase, and because of their kinetic and biochemical properties have a high potential as an economic alternative to horseradish peroxidase (HRP). The efficiency of using turnip peroxidase (TP) to remove several different phenolic compounds as water‐insoluble polymers from synthetic wastewater was investigated. The phenol derivatives studied included phenol, 2‐chlorophenol, 3‐chlorophenol, o‐cresol, m‐cresol, 2,4‐dichlorophenol and bisphenol‐A. The effect of pH, substrate concentration, amount of enzyme activity, reaction time and added polyethylene glycol (PEG) was investigated in order to optimize reaction conditions. A removal efficiency ≥85% was achieved for 0.5 mmol dm^?3 phenol derivatives at pH values between 4 and 8, after a contact time of 3 h at 25 °C with 1.28 U dm^?3 of TP and 0.8 mmol dm^?3 H₂O₂. Addition of PEG (100–200 mg dm^?3) significantly reduced the reaction time required (to 10 min) to obtain >95% removal efficiency and up to 230% increase in remaining TP activity. A relatively low enzyme activity (0.228 U dm^?3) was required to remove >95% of three phenolic solutions in the presence of 100–200 mg dm^?3 PEG. TP showed efficient and fast removal of aromatic compounds from synthetic wastewaters in the presence of hydrogen peroxide and PEG. These results demonstrate that TP has good potential for the treatment of phenolic‐contaminated solutions. © 2002 Society of Chemical Industry     

Semi‐specific biosensors for measuring BOD in dairy wastewater

BACKGROUND: Semi‐specific microbial biochemical oxygen demand (BOD) biosensors were constructed using living cells of Escherichia coli R17.1.3 (E. coli) and Raoultella terrigena P74.3 (R. terrigena) immobilized in agarose gel matrix. The research involved comparison with the Pseudomonas fluorescens P75 (P. fluorescens) biosensor that had no specificity in decomposing lactose and milk derivates. The constructed BOD biosensors were calibrated with OECD synthetic wastewater and tested with different wastewater samples. RESULTS: The linear range of the calibration curve was 5–200 mg L^?1 BOD₇ for R. terrigena and P. fluorescens based biosensors and 150 mg L^?1 BOD₇ for the E. coli based biosensor. Service life was 60 days for E. coli and P. fluorescens based biosensors and 40 days for R. terrigena based biosensors. BOD values for dairy industry wastewater obtained with current semi‐specific biosensors considerably overestimate BOD₇, while universal biosensors underestimate BOD₇ obtained by the conventional 7‐day BOD test. CONCLUSION: In spite of extensive overestimation of BOD₇ the semi‐specific biosensors enabled better estimation of BOD in dairy industry wastewater than a reference P. fluorescens biosensor. The best result, in terms of service life, stability, sensitivity and reproducibility was accomplished with semi‐specific E.coli biosensor. Copyright © 2010 Society of Chemical Industry     

Characteristics of gold biosorption from cyanide solution

Gold adsorption from cyanide solution by bacterial (Bacillus subtilis), fungal (Penicillium chrysogenum) and seaweed (Sargassum fluitans) biomass was examined. At pH 2.0, these biomass types were capable of sequestering up to 8.0 µmol g⁻¹, 7.2 µmol g⁻¹ and 3.2 µmol g⁻¹, respectively. An adverse effect of increasing solution ionic strength (NaNO₃) on gold biosorption was observed. Gold‐loaded biomass could be eluted with 0.1 mol dm⁻³ NaOH with efficiencies higher than 90% at pH 5.0 at the Solid‐to‐Liquid ratio, S/L, = 4 (g dm⁻³). Cyanide mass balances for the adsorption, desorption as well as for the AVR process indicated the stability of the gold‐cyanide which did not dissociate either upon acidification or upon binding by biomass functional groups. Gold biosorption mainly involved anionic AuCN₂⁻ species bound by ionizable biomass functional groups carrying a positive charge when protonated. FTIR analyses indicated that the main biomass functional groups involved in gold biosorption are most probably nitrogen‐containing weak base groups. The present results confirmed that waste microbial biomaterials have some potential for removing and concentrating gold from solutions where it occurs as a gold‐cyanide complex. © 1999 Society of Chemical Industry     

Design of different bioreactor configurations: application to ligninolytic enzyme production in semi‐solid‐state cultivation

The production of ligninolytic enzymes by Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) in laboratory‐scale bioreactors was studied. The cultivations were carried out in semi‐solid‐state conditions, employing corncob as carrier, which functioned both as a place of attachment and as a source of nutrients. Several bioreactor configurations were investigated in order to determine the most suitable one for ligninolytic enzyme production: a 1‐dm³‐static‐bed bioreactor, a 1‐dm³‐static‐bed bioreactor with air diffusers into the bed, a 0.5‐dm³‐static‐bed bioreactor with air diffusers into the bed and a tray bioreactor. Although the static‐bed configurations produced maximum individual lignin peroxidase (LiP) activities about 400 U dm⁻³ (1.0‐dm³ bioreactor) and about 700 U dm⁻³ (0.5‐dm³ bioreactor), manganese‐dependent peroxidase (MnP) was not detected throughout the cultures. Nevertheless, the tray configuration led to maximum individual MnP and LiP activities of about 200 U dm⁻³ and 300 U dm⁻³, respectively. Therefore, this configuration is the most adequate of the different bioreactor configurations tested in the present work, since the ligninolytic complex formed by MnP and LiP is more efficient for its application to bio‐processing systems. In addition, the results indicated the influence of the oxygen in ligninolytic enzyme production. © 2001 Society of Chemical Industry     

A three‐phase fluidized bed reactor in the combined anaerobic/aerobic treatment of wastewater

Aerobic degradation or polishing is an essential step in the combined anaerobic/aerobic treatment of wastewater. In this study, a type of porous glass beads was used for immobilization of microbial cells in a three‐phase aerobic fluidized bed reactor (AFBR) with an external liquid circulation. The effects of superficial gas and liquid velocities on bed expansion, solid and gas hold‐ups and specific oxygen mass transfer rate, k_La, were investigated. A tracer study showed that the mixing and flow pattern in the 8 dm³ reactor could be simulated by a non‐ideal model of two continuous stirred tank reactors (CSTRs) in series. By treating an effluent from an upflow anaerobic sludge blanket (UASB) digester, the distribution of suspended and immobilized biomass in the reactor as well as the kinetics of COD removal were determined. The specific oxygen mass transfer rate, k_La, at a superficial gas velocity of 0.7 cm s⁻¹ dropped by about 30% from 32 h⁻¹ in tap water to 22 h⁻¹ after a carrier load of 15% (v/v) was added. The measured k_La further dropped by about 20% to 18 h⁻¹ in the wastewater, a typical value of the bubbling fermenters with no stirring. Compared with the aerobic heterotrophs under optimum growth conditions, the microbes in this reactor which was fed with anaerobic effluent plus biomass behaved like oligotrophs and showed slow specific COD removal rates. This might be attributed to the presence of a significant amount of obligate anaerobes and facultative organisms in the aerobic reactor. This was confirmed by a relatively low intrinsic oxygen uptake rate of the microbial population in the reactor, 94 mg O₂ dm⁻³ h⁻¹ or 19 mg O₂g VS⁻¹ h⁻¹. © 1999 Society of Chemical Industry     

Yield and maintenance coefficients in S. cerevisiae cultures

Precise determination of yields deduced from measurements of metabolic rates needs an understanding of the physiology and energetics of growth of microorganisms. The linear growth equation derived from the simplified picture of microbial metabolism is generally accepted. However, the significance of the yields it gives is less well understood. The analysis of the theoretical and real observed and true yield coefficients for partly aerobic and anaerobic growth of S. cerevisiae is presented in this study. In the calculations of yields and maintenance coefficients, the pseudoconstants Y, P/O were used. A set of limitations imposed on true yields in the linear growth equation is established and the significance of the yields is also discussed. The experimental runs were performed in a laboratory-scale bioreactor (1·5 dm³ of operating capacity). The yeast Saccharomyces cerevisiae hybrid G-67 was cultured in synthetic Reader medium containing sucrose as the main carbon and energy source. The comparison between experimentally obtained yield coefficients relative to cell growth and ethanol formation and the real theoretical values is presented. The maintenance coefficients calculated for anaerobic and respiro-fermentative processes are 0·14 C-mole ATP (C-mole DM h)⁻¹. © 1998 SCI     

Respirometric estimation of the oxygen affinity constants for biological ammonium and nitrite oxidation

The nitrification process (ie biological ammonium oxidation to nitrate) is a two‐step process with nitrite as an intermediate product. As it is an aerobic process, its kinetics is highly dependent on the dissolved oxygen (DO) concentration in the medium. However, the influence of this limitation on the nitritation (first step) is shown to be less important than in the nitratation (second step). This dependence on DO concentration is generally described using a Monod‐type kinetics with K_O as the oxygen affinity constant. In this work, a procedure for the calculation of both affinity constants is presented. This procedure is based on monitoring the DO drop in the reactor when external aeration is stopped and the biomass is consuming without substrate (ammonium or nitrite) limitations. This methodology includes the contemplation of the oxygen transfer from the atmosphere, the response time of the DO probe and the inhibition of the nitratation step with sodium azide when estimating K_OA (nitritation oxygen affinity constant). The results obtained are K_OA = 0.74 ± 0.02 mg O₂ dm^?3 and K_ON = 1.75 ± 0.01 mg O₂ dm^?3. Moreover the influence of the aforementioned considerations on the estimated K_O values is also discussed. Copyright © 2005 Society of Chemical Industry     

Anaerobic and aerobic treatment of a simulated textile effluent

A simulated textile effluent (STE) was generated for use in laboratory biotreatment studies; this effluent contained one reactive azo dye, PROCION Red H‐E7B (1.5 g dm⁻³); sizing agent, Tissalys 150 (1.9 g dm⁻³); sodium chloride (1.5 g dm⁻³) and acetic acid (0.53 g dm⁻³) together with nutrients and trace elements, giving a mean COD of 3480 mg dm⁻³. An inclined tubular anaerobic digester (ITD) was operated for 9 months on the STE and a UASB reactor for 3 months. For a 57 day period anaerobic effluent from two reactors, a UASB and an ITD, was mixed and treated in an aerobic stage. In days 77–247 68% of the true colour of PROCION Red H‐E7B was removed by anaerobic treatment with no colour removal aerobically and up to 37% COD was removed anaerobically, with a corresponding BOD removal of 71%. For combined anaerobic and aerobic treatment a mean COD removal of 57% and BOD removal of 86% was achieved. Operation of the ITD at a 2.8 day HRT (volumetric loading rate (B _v) 1.24 g COD dm⁻³day⁻¹) and the UASB at a 2 day HRT (B _v 1.74 g COD dm⁻³day⁻¹) gave comparable COD removals but the UASB gave better true colour removal. Effluent from the combined process operating on this simulated waste still contained an average 1500 mg COD dm⁻³, and further treatment would be required to meet consent standards. © 1999 Society of Chemical Industry