Effects of oils and oil-related substrates on the synthetic activity of membrane-bound lipase from Rhizopus chinensis and optimization of the lipase fermentation media

The lipase from filamentous fungi Rhizopus chinensis, as a membrane-bound enzyme, possesses the excellent catalysis ability for esterification and transesterification reactions, and has a good potential in many industrial applications. In order to improve the synthetic activity of the lipase, the effects of oils and oil-related substrates on its production and the fermentation media optimization were investigated. Based on the results, it was suggested that oleic acid could be the important substrate for the lipase production. Among various oils and oil-related substrates, olive oil containing high content of oleic acid was the optimal one for the lipase production. Using orthogonal test and response surface methodology (RSM), the composition of fermentation media was further optimized. The optimized media for lipase synthetic activity and activity yield was composed of peptone 57.94 and 55.58 g L⁻¹, olive oil 21.94 and 22.99 g L⁻¹, maltose 12.91 and 14.34 g L⁻¹, respectively, with K₂HPO₄ 3 g L⁻¹, MgSO₄·7H₂O 5 g L⁻¹ and initial pH 6.0. Under the optimal conditions, the lipase activity and the activity yield were improved 61.5 and 93.4% comparing the results before optimization, respectively. The adequate models obtained had predicted the lipase production successfully.     

A novel photobioreactor with transparent rectangular chambers for cultivation of microalgae

An open tank photobioreactor containing transparent rectangular chambers (TRCs) was developed to improve the photosynthetic efficiency of microalgal cultivation. The TRCs, made of transparent acrylic, conducted light deep into the photobioreactor, especially at high cell concentrations. The average irradiance, I_av, was calculated by Lambert–Beer's law, and was used to determine the light conditions in the cultivation system. The photobioreactor provided large areas of illumination that improved the effective utilization of light energy for microalgae growth and created a good artificial environment for a high rate of cell growth, even at low I_av. The biomass concentration of Chlorella sp. reached 3.745 g L⁻¹ on the 13th day, with biomass productivity of 0.340 g L⁻¹ d⁻¹. The total biomass obtained was 56% more than that of similar culture systems without TRCs.     

Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor

The fresh water microalga Neochloris oleoabundans was investigated for its ability to accumulate lipids and especially triacylglycerols (TAG). A systematic study was conducted, from the determination of the growth medium to its characterization in an airlift photobioreactor. Without nutrient limitation, a maximal biomass areal productivity of 16.5 g m⁻² day⁻¹ was found. Effects of nitrogen starvation to induce lipids accumulation was next investigated. Due to initial N. oleoabundans total lipids high content (23% of dry weight), highest productivity was obtained without mineral limitation with a maximal total lipids productivity of 3.8 g m⁻² day⁻¹. Regarding TAG, an almost similar productivity was found whatever the protocol was: continuous production without mineral limitation (0.5 g m⁻² day⁻¹) or batch production with either sudden or progressive nitrogen deprivation (0.7 g m⁻² day⁻¹). The decrease in growth rate reduces the benefit of the important lipids and TAG accumulation as obtained in nitrogen starvation (37% and 18% of dry weight, respectively).     

Production and recovery of recombinant protease inhibitor α1-antitrypsin

Human α₁-antitrypsin (AAT) was produced in the recombinant yeast Saccharomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were 55 g·L⁻¹ and 1.23 g·L⁻¹, respectively, in the fed-batch. The maximum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L⁻¹h⁻¹, respectively, or substantially greater than the highest productivity values reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g·L⁻¹ biomass, 122 min of processing) by cross-flow microfiltration and the cells were disrupted by bead milling (3 passes of 3 min total retention time). The cell homogenate was treated with aluminum chloride or PBS (pH 7) to aid separation of the cell debris by flocculation and sedimentation. The clarified cell homogenate was subjected to ammonium sulfate fractionation to precipitate the recombinant antitrypsin. The AAT precipitated at 45–75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25°C), with a zero order deactivation rate of 1.815 × 10⁻³ ± 3.43 × 10⁻⁴ g AAT L⁻¹h⁻¹.     

Biodegradation of indole at high concentration by persolvent fermentation with the thermophilic fungus Sporotrichum thermophile

Indole and its derivatives form a class of toxic recalcitrant environmental pollulants. Sporotrichum thermophile was grown in a persolvent fermentation system containing a large amount of indole. The medium contained up to 20% by volume soybean oil and up to 2 g L⁻¹ indole. Most of the indole was partitioned in the organic solvent layer. When the organism was grown in the medium containing indole at 1 g L⁻¹, indole was totally consumed after 6 days. Under a fed–batch fermentation process where daily batches of indole (1 g L⁻¹) supplemented the microbial culture for 4 days, the biodegradation level was 3.0 g L⁻¹. These values make this process promising and worthy of further investigation for the microbial degradation of other toxic compounds.     

Entropy decrease associated to solute compartmentalization in the cell

We have deduced equations to quantify the entropy associated to the compartmentalization of components in eukaryotic cells as a function of cell and compartment volumes, and of the concentration of solutes. On the basis of known and plausible values of volume and solute concentrations and the deduced equations, we estimate that the contribution of compartmentalization to the decrease of entropy is approximately −14.4 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−0.7 J K⁻¹ L⁻¹) in the case of Saccharomyces cerevisiae, a typical eukaryotic cell, and approximately −49.6 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−1.0 J K⁻¹ L⁻¹) in the more complex Chlamydomonas reinhardtii. When compared with other potential contributing factors, such as the informational entropy of DNA and the conformational entropy of proteins, compartmentalization appears as an essential development that significantly decreased the entropy of living cells during biological evolution.     

Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in CO2 removal processes

In this paper the utilization of the cyanobacteria Anabaena sp. in carbon dioxide removal processes is evaluated. For this, continuous cultures of this strain were performed at different dilution rates; alternatives for the recovery of the organic matter produced being also studied. A maximum CO₂ fixation rate of 1.45 g CO₂ L⁻¹ day⁻¹ was measured experimentally, but it can be increased up to 3.0 g CO₂ L⁻¹ day⁻¹ outdoors. The CO₂ is mainly transformed into exopolysaccharides, biomass representing one third of the total organic matter produced. Organic matter can be recovered by sedimentation with efficiencies higher than 90%, the velocity of sedimentation being 2 · 10⁻⁴ s⁻¹. The major compounds were carbohydrates and proteins with productivities of 0.70 and 0.12 g L⁻¹ day⁻¹, respectively. The behaviour of the cultures of Anabaena sp. has been modelized, also the characteristics parameters requested to design separation units being reported. Finally, to valorizate the organic matter as biofertilizers and biofuels is proposed.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Red tide blooms of Cochlodinium polykrikoides in a coastal cove

Successive blooms of the dinoflagellate Cochlodinium polykrikoides occurred in Pettaquamscutt Cove, RI, persisting from September through December 1980 and again from April through October 1981. Cell densities varied from <100 cells L⁻¹ at the onset of the bloom and reached a maximum density exceeding 3.4 × 10⁶ cells L⁻¹ during the summer of 1981. The bloom was mainly restricted to the mid to inner region of this shallow cove with greatest concentrations localized in surface waters of the southwestern region during summer/fall periods of both years. Highly motile cells consisting of single, double and multiple cell zooids were found as chains of 4 and 8 cells restricted to the late August/September periods. The highest cell densities occurred during periods when annual temperatures were between 19 and 28 °C and salinities between 25 and 30. A major nutrient source for the cove was Crying Brook, located at the innermost region at the head of the cove. Inorganic nitrogen (NH₃ and NO₂ + NO₃) from the brook was continually detectable throughout the study with maximum values of 57.5 and 82.5 μmol L⁻¹, respectively. Phosphate (PO₄-P) was always present in the source waters and rarely <0.5 μmol L⁻¹; silicate always exceeded 30 μmol L⁻¹ with maximum concentrations reaching 226 μmol L⁻¹. Chlorophyll a and ATP concentrations during the blooms varied directly with cell densities. Maximum Chl a levels were 218 mg m⁻³ and ATP-carbon was >20 g C m⁻³. Primary production by the dinoflagellate-dominated community during the bloom varied between 4.3 and 0.07 g C m⁻³ d⁻¹. Percent carbon turnover calculated from primary production values and ATP-carbon varied from 6 to 129% d⁻¹. The dinoflagellates dominated the entire summer period; other flagellates and diatoms were present in lesser amounts. A combination of low washout rate due to the cove dynamics, active growth, and life cycles involving cysts allowed C. polykrikoides to maintain recurrent bloom populations in this area.     

Effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi

Effects of carbon concentration and carbon to nitrogen (C:N) ratio on six biocontrol fungal strains are reported in this paper. All fungal strains had extensive growth on the media supplemented with 6–12 g l⁻¹ carbon and C:N ratios from 10:1 to 80:1, and differed in nutrient requirements for sporulation. Except for the two strains of Paecilomyces lilacinus, all selected fungi attained the highest spore yields at a C:N ratio of 160:1 when the carbon concentration was 12 g l⁻¹ for Metarhizium anisopliae SQZ-1-21, 6 g l⁻¹ for M. anisopliae RS-4-1 and Trichoderma viride TV-1, and 8 g l⁻¹ for Lecanicillium lecanii CA-1-G. The optimal conditions for P. lilacinus sporulation were 8 g l⁻¹ carbon with a C:N ratio of 10:1 for M-14 and 12 g l⁻¹ carbon with a C:N ratio of 20:1 for IPC-P, respectively. The results indicated that the influence of carbon concentration and C:N ratio on fungal growth and sporulation is strain dependent; therefore, consideration for the complexity of nutrient requirements is essential for improving yields of fungal biocontrol agents.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1 μM of l-phenylalanine (6.2 g L⁻¹). Addition of 1 μM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7 μg L⁻¹), which was not detected in the untreated control culture. Supplementation with 100 μM of l-phenylalanine together with 100 μM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3 μg L⁻¹ (control untreated culture) to 568.2 μg L⁻¹, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8 μg L⁻¹) was observed when the medium was supplemented with 100 μM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.     

Evaluation of chromium(VI) removal behaviour by two isolates of Synechocystis sp. in terms of exopolysaccharide (EPS) production and monomer composition

Chromium(VI) removal and its association with exopolysaccharide (EPS) production in cyanobacteria were investigated. Synechocystis sp. BASO670 produced higher EPS (548 mg L⁻¹) than Synechocystis sp. BASO672 (356 mg L⁻¹). While the EC₅₀ of the Cr(VI) for Synechocystis sp. BASO670 and Synechocystis sp. BASO672 were determined as 11.5 mg L⁻¹, and 2.0 mg L⁻¹, respectively, there was no relation between Cr(VI) removal and EPS production. Synechocystis sp. BASO672, which has higher EPS value, removed (33%) more Cr(VI) than Synechocystis sp. BASO670. Monomer compositions of EPS of each of the isolates were determined differently. Synechocystis sp. BASO672 which removed higher Cr(VI), had higher values of uronic acid and glucuronic acid (192 μg/mg and 89%, respectively). Our results showed that EPS might play a role in Cr(VI) tolerance. Monomer composition, especially uronic acid and glucuronic acid content of EPS may have enhanced Cr(VI) removal.     

Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape

A strain of Bacillus subtilis (NJ-18) with broad antimicrobial activity was screened in the laboratory and in the field. NJ-18 inhibited the in vitro radial extension of hyphae of the phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. The bacterium apparently produced antifungal metabolites that diffused through the agar and caused abnormal swelling of hyphae. The in vitro data and observations indicated that one of the mechanisms of inhibition by NJ-18 is antibiosis. In field experiments for control of sheath blight of rice, fermentation of NJ-18 at 5.0 × 10⁷ cfu ml⁻¹ significantly reduced disease incidence and severity; NJ-18 alone or combined with 50% kresoxim-methyl treatment at 225 g ai ha⁻¹ provided better control than 50% kresoxim-methyl at 225 g ai ha⁻¹ or Jinggangmycin at 120 g ai ha⁻¹, and control by NJ-18 alone was as high as 100.0%. In field experiments for control of Sclerotinia stem rot of rape, fermentation of NJ-18 at 1.0 × 10⁷ cfu ml⁻¹ again significantly reduced disease incidence and severity; control by NJ-18 was as high as 77.1% and was comparable with control by 46% dimethachlon and better than control by 50% carbendazim at 750 g ai ha⁻¹. We conclude that strain NJ-18 of B. subtilis is a promising biological control agent and should be further studied and tested for control of sheath blight of rice, Sclerotinia stem rot of rape, and other diseases.     

Effect of mechanical agitation on the production of cellulases by Trichoderma reesei RUT-C30 in a draft-tube airlift bioreactor

With the aim to produce cellulases and to study the effect of mechanical agitation, a 35 L draft-tube airlift bioreactor equipped with a mechanical impeller was developed and validated to grow Trichoderma reesei RUT-C30 in a cellulose culture medium with lactose and lactobionic acid as fed batch. Cultures carried out without mechanical agitation resulted in higher volumetric enzyme productivity (200 U L⁻¹ h⁻¹), filter paper activity (17 U mL⁻¹), carboxymethyl cellulase activity (11.8 U mL⁻¹) and soluble proteins (3.2 mg mL⁻¹) when compared to those with agitation. Stereo and polarized light microscopy analyses reveal that mechanical agitation resulted in shorter mycelial hyphae and larger numbers of tips.     

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses; therefore, the knowledge of the volumetric mass transfer coefficient (k_La) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k_La values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium, in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2² full factorial design. Both variables showed statistically significant effects on k_La, and the highest values of this parameter in both media for simple fermentation (241 s⁻¹) and extractive fermentation with ATPS (70.3 s⁻¹) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k_La values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N³D²) and superficial gas velocity (V_s) determined in distilled water (α = 0.39 and β = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (α = 0.38 and β = 0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (α = 0.50 and β = 1.0). A reasonable agreement was found between the experimental data of k_La for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

The potential role of climate in the distribution and zonation of the introduced seagrass Zostera japonica in North America

The current distribution of the introduced seagrass Zostera japonica is restricted to the mid- to upper intertidal zone in the coastal Pacific Northwest region of North America. The climate in this region is cool and wet, becoming hotter and dryer with increasing distance southward. Since temperature is likely to be an important factor affecting distribution of this species, growth of two populations located near the northern and southern limits of its established range along the Pacific Coast of North America were measured in an experimental setting across a range of temperatures typical of those in the field during the growing season (10, 20, and 30 °C). The effects of temperature and population were both significant. Leaf elongation, growth, and areal productivity rates of the northern population were consistently lower than those of the southern population. Across the range of temperatures, mean leaf elongation rates ranged from 0.47 to 1.40 cm² shoot⁻¹ d⁻¹; mean growth rates ranged from 0.19 to 0.52 mg dry wt shoot⁻¹ d⁻¹. Mean areal productivity ranged from 0.54 to 1.92 g dry wt m⁻² d⁻¹. Maximum rates of leaf elongation, growth, and areal productivity for both populations were observed at 20 °C. However, leaf elongation, growth, and areal productivity of the northern population declined markedly at 30 °C, whereas no comparable declines were observed for the southern population. This suggests that Z. japonica populations near the southern limits of its established range may be better adapted to warmer temperatures than populations near the northern range limits and further range extensions southward along the California coast may be likely. These differences could be important in predicting the outcome of competitive interactions between native and introduced seagrass species, and in determining future patterns of distribution and zonation of Pacific Coast seagrasses.     

Two-stage biological treatment of olive mill wastewater with whey as co-substrate

Olive mill wastewater (OMW) is a highly polluting wastewater, caused by a high organic load and phenol content. These characteristics suggest that it may be suitable for aerobic treatment and anaerobic bacterial digestion. Aerobic treatment coupled with anaerobic bacterial digestion may be economically feasible as the methane produced is a valuable energy source while simultaneously purifying the OMW. In an attempt to improve the overall performance of the process, the addition of a co-substrate such as whey to the aerobic treatment pre-treatment of OMW by the yeast Candida tropicalis was studied.The two-stage system operated satisfactorily up to an organic loading rate (OLR) of 3.0 kg COD L⁻¹ day⁻¹ with a biogas production rate of 1.25 L_biogas L_reactor⁻¹ day⁻¹ and a total COD reduction in excess of 93% (62% COD reduction in aerobic pretreatment and 83% COD reduction in anaerobic digestion). Fifty-four percent of the phenol was biodegraded during the aerobic treatment stage, and biogas with between 68% and 75% methane was produced during anaerobic digestion.