Nanoenvironmental Effects Dramatically Influence the Sensitivity of Immunoassays.

It is possible to improve the sensitivity of immunoassays by several orders of magnitude by exploiting nanoenvironmental effects. This approach can detect trace amounts of compounds and will better illuminate the presence of signal substances in biological systems. Here we describe a method for ultrasensitive immunoassays using 'normal' antibodies (Abs).

Chemically modified poly(2-hydroxyethyl methacrylate) cryogel for the adsorption of heparin.

Various clinical procedures, such as cardiovascular surgery or extracorporeal blood purification, involve systemic anticoagulation using heparin. High concentrations of circulating heparin require neutralization due to possible serious bleeding complications. The intravenous administration of the heparin antagonist protamine sulfate is routinely clinically performed, but is frequently associated with adverse reactions. Therefore, there is a need for a valid and safe alternative to achieve extracorporeal heparin removal from blood or plasma, such as a filter, a matrix, or an adsorbent. Here, we describe the development of a macroporous poly(2-hydroxyethyl methacrylate)-based monolithic cryogel functionalized with l-lysine (pHEMA-lys) and the characterization of its selective heparin adsorption. The maximum binding capacity was quantified in vitro using aqueous and serum solutions under static and dynamic conditions, and fresh human plasma under static conditions. The pHEMA-lys bound 40,500 IU and 32,500 IU heparin/g cryogel at the equilibrium in aqueous solution and 50% serum, respectively. In human plasma spiked with 100 IU/mL of heparin, the binding was still highly efficient (4330 IU/g cryogel after 30 min, i.e., 87% of the initial concentration). The cryogels showed good blood compatibility, as indicated by negligible adsorption of albumin, antithrombin III, and total protein, and may thus be suitable for extracorporeal heparin removal.

Polymer composite adsorbents using particles of molecularly imprinted polymers or aluminium oxide nanoparticles for treatment of arsenic contaminated waters.

Removal of As(V) by adsorption from water solutions was studied using three different synthetic adsorbents. The adsorbents, (a) aluminium nanoparticles (Alu-NPs, <50 nm) incorporated in amine rich cryogels (Alu-cryo), (b) molecular imprinted polymers (<38 µm) in polyacrylamide cryogels (MIP-cryo) and (c) thiol functionalised cryogels (SH-cryo) were evaluated regarding material characteristics and arsenic removal in batch test and continuous mode. Results revealed that a composite design with particles incorporated in cryogels was a successful means for applying small particles (nano- and micro- scale) in water solutions with maintained adsorption capacity and kinetics. Low capacity was obtained from SH-cryo and this adsorbent was hence excluded from the study. The adsorption capacities for the composites were 20.3 ± 0.8 mg/g adsorbent (Alu-cryo) and 7.9 ± 0.7 mg/g adsorbent (MIP-cryo) respectively. From SEM images it was seen that particles were homogeneously distributed in Alu-cryo and heterogeneously distributed in MIP-cryo. The particle incorporation increased the mechanical stability and the polymer backbones of pure polyacrylamide (MIP-cryo) were of better stability than the amine containing polymer backbone (Alu-cryo). Both composites worked well in the studied pH range of pH 2-8. Adsorption tested in real wastewater spiked with arsenic showed that co-ions (nitrate, sulphate and phosphate) affected arsenic removal for Alu-cryo more than for MIP-cryo. Both composites still adsorbed well in the presence of counter-ions (copper and zinc) present at low concentrations (µg/l). The unchanged and selective adsorption in realistic water observed for MIP-cryo was concluded to be due to a successful imprinting, here controlled using a non-imprinted polymer (NIP). A development of MIP-cryo is needed, considering its low adsorption capacity.

Immobilisation of TiO2 for combined photocatalytic-biological azo dye degradation.

The biodegradability of the azo dye Remazol Red RR (100 mg/l) was evaluated using unadapted activated sludge and the experiment confirmed the recalcitrance of the dye. Using a combination of photocatalysis and an aerobic biological step, the biodegradability was improved significantly and complete removal of both colour and COD were achieved. Furthermore, TiO2 was successfully immobilised on borosilicate glass slides by calcination, which facilitates reuse of the catalyst. The catalytic activity of the immobilised TiO2 was close to that of suspended TiO2. A reduced activity was however observed when the TiO2 slides were used repeatedly. When comparing NaOH, calcination and UV irradiation for regeneration of the TiO2 slides, immersion in NaOH was shown to be the most efficient method.

Sub-attomolar detection of cholera toxin using a label-free capacitive immunosensor.

A label-free immunosensor for the direct detection of cholera toxin (CT) at sub-attomolar level has been developed based on potential-step capacitance measurements. Anti-CT antibody was adsorbed on gold nanoparticles (AuNPs) incorporated on a polytyramine-modified gold electrode. The concentration of CT was determined by detecting the change of capacitance caused by the formation of antibody-antigen complexes. By using AuNPs adsorbed to the sensing surface, the signal was dramatically increased leading to a significantly more sensitive assay. In fact, under optimum conditions the immunosensor could detect CT concentration with a limit of detection of 9 x 10(-20)M or 0.09 aM, with a dynamic range between 0.1 aM and 10 pM. Good analytical reproducibility could be obtained by injecting CT up to 36 times with an RSD of 2.5%. In addition, good performance of the developed immunosensor was achieved when applied to turbid water samples collected from a local stream that were spiked with CT.

Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1.

AIMS: Utilization of cheap and readily available agricultural residues as cheap carbon sources for poly(3-hydroxybutyrate) (PHB) production by Halomonas boliviensis. METHODS AND RESULTS: Wheat bran was hydrolysed by a crude enzyme preparation from Aspergillus oryzae NM1 to provide a mixture of reducing sugars composed mainly of glucose, mannose, xylose and arabinose. Growth of H. boliviensis using a mixture of glucose (0.75% w/v) and xylose (0.25% w/v) in the medium led to a PHB content and concentration of 45 wt% and 1 g l(-1), respectively, after 30 h. A similar PHB concentration was attained when H. boliviensis was grown on wheat bran hydrolysate but with a lower PHB content, 34 wt%. In a batch cultivation mode in a fermentor, using 1.8% (w/v) reducing sugars, the maximum PHB accumulation by H. boliviensis was attained in 20 h, but was reduced to about 30 wt%. By adding butyric acid (0.8% v/v), sodium acetate (0.8% w/v) and decreasing the reducing sugars concentration to 1 x 0% w/v in the medium, PHB accumulation and concentration were increased to 50 wt% and 4 g l(-1), respectively, after 20 h. Butyric acid and sodium acetate for PHB production could also be provided by anaerobic digestion of solid potato waste. CONCLUSIONS: Cheap and readily available agricultural residues can be used as substrates to produce PHB. The production of PHB by H. boliviensis using wheat bran hydrolysate as source of carbon is expected to reduce the production cost and motivates further studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Large-scale commercial utilization of PHB is mainly hampered by its high production cost. Carbon source for PHB production accounts up to 50% of the total production costs. Thus, the use of waste agricultural residues can substantially reduce the substrate cost (and in turn even provide value to the waste), and can downsize the production costs. This improves the market competitiveness. Studies on PHB production by moderate halophiles were recently initiated with H. boliviensis and findings show that it has potential for commercial exploitation. PHB production by H. boliviensis using wheat bran and potato waste is hence interesting.

Effect of temperature decrease on the microbial population and process performance of a mesophilic anaerobic bioreactor.

The effect of a temperature decrease from 33 degrees C to 12 degrees C was investigated for anaerobic digestion of crop residues. A laboratory-scale reactor (R0) was inoculated with mesophilic sludge and operated as continuously stirred fed-batch system at temperatures of 12 degrees C, 18 degrees C and 33 degrees C. Changes in the microbial populations of the sludge were followed by means of fluorescence in situ hybridization analysis. Methane was produced in R0 at all temperatures. Stable long-term operation at 18 degress C was achieved yielding 151 mlCH4 gVS(added(-1) at a rate of 108 mlCH4 l(R)(-1)d(-1) once the microbial populations of the sludge had adapted to this temperature. After operation at 18 degrees C, the contents of R0 was mixed and distributed into three smaller reactors, which were operated at 18 degrees C (R18), 25 degrees C (R25) and 37 degrees C (R37), respectively. Methane production rates for R37 and R25 were 366 and 310 mlCH4 l(R)(-1)d(-1), respectively, which were higher than the 215 mlCH4 l(R)(-1)d(-1) obtained in R0 when this was operated at 33 degrees C. Hydrolysis was found to decrease when temperature was decreased and especially below 25 degrees C. At temperatures below 16 degrees C, acidogenesis and methanogenesis were the rate-limiting steps. Adaptation of the mesophilic sludge to 18 degrees C was indicated by an increase in the ratio of Bacteria to total prokaryotes (sum of Archaea and Bacteria). This was thought to be caused by enrichment of Bacteria in the sludge, which appeared to be an important adaptation mechanism. During the adaptation, the Methanomicrobiales and Methanosarcinaceae populations increased relative to the total Archaea population whereas the Methanosaeta population decreased. The population changes were reflected by reactor performance.

Polycyclic aromatic hydrocarbon biodegradation by a subtropical white rot fungus in packed bed and suspended carrier bioreactor systems.

Application of a biotreatment system utilising immobilised white rot fungi can become an alternative for treating water or effluent contaminated by organic pollutants such as polycyclic aromatic hydrocarbons (PAHs). The application of the packed bed and suspended carrier bioreactor systems for the degradation of PAHs in synthetic polluted media using a subtropical white rot fungal isolate DSPM95 was evaluated. The white rot fungal isolate, DSPM95 could reduce a mixture of selected PAHs namely; fluorene, phenanthrene, anthracene, pyrene and benzo(a)anthracene by 50 to 96% over the reactor operation time of 31 days and when the concentration of each PAH in the feed medium was 1 mg l(-1). High manganese peroxidase and laccase activities were detectable during PAH biodegradation in both the bioreactor systems, however the maximum enzyme activities could not be sustained in the bioreactors for extended periods of time. Varying concentrations of glucose to nutrient nitrogen in the feed medium could not help sustain high enzyme production in the bioreactor. It can be concluded that the white rot fungi used here could efficiently degrade the PAH compounds in both the packed bed and suspended carrier bioreactor system, which compares well with other studies as highlighted in this report.

A study of two-stage anaerobic digestion of solid potato waste using reactors under mesophilic and thermophilic conditions.

A two-stage anaerobic digestion process operated under mesophilic and thermophilic conditions was investigated for the treatment of solid potato waste to determine optimal methane yield, efficiency of operation and process stability. A solid-bed reactor was used for hydrolysis/acidification stage while an upflow anaerobic sludge blanket (UASB) reactor was used in the second stage, for methanogenesis. Three sets of conditions were investigated: (1) mesophilic + mesophilic, (II) mesophilic + thermophilic and (III) thermophilic + thermophilic in the hydrolysis/acidification and methanogenesis reactors, respectively. The methane yield was higher under mesophilic conditions (0.49 l CH4 g COD(-1)degraded) than thermophilic conditions (0.41 l CH4 g COD(-1)degraded) with reference to the methanogenic reactors. (COD)--chemical oxygen demand. However, the digestion period was shorter in systems II and III than in system I. Also, in system III the UASB reactor (thermophilic conditions) could handle a higher organic loading rate (OLR) (36 g COD 1(-1)d(-1)) than in system I (11 g COD 1(-1)d(-1)) (mesophilic conditions) with stable operation. Higher OLRs in the methanogenic reactors resulted in reactor failure due to increasing total volatile fatty acid levels. In all systems, the concentration of propionate was one of the highest, higher than acetic acid, among the volatile fatty acids in the effluent. The results show the feasibility of using a two-stage system to treat solid potato waste under both mesophilic and thermophilic conditions. If the aim is to treat solid potato waste completely within a short period of time thermophilic conditions are to be preferred, but to obtain higher methane yield mesophilic conditions are preferable and therefore there is a need to balance methane yield and complete digestion period when dealing with large quantities of solid potato waste.

Biological treatment of industrial wastes in a photobioreactor.

An algal-bacterial consortium was tested for the treatment from a coke factory. A Chlorella vulgaris strain and a phenol-degrading Alcaligenes sp. were first isolated from the wastewater treatment plant to serve as inocula in the subsequent biodegradation tests. Batch tests were then conducted with samples from the real wastewater or using a synthetic wastewater containing 325 mg phenol/l and 500 mg NH4+/l as target pollutants. Direct biological treatment of the real wastewater was not possible due to the toxicity of organic compounds. Activated carbon adsorption and UV(A-B)-irradiation were efficient in detoxifying the effluent for subsequent biological treatment as inoculation of pretreated samples with the algal-bacterial consortium was followed by complete phenol removal and NH4+ removal of 45%. Complete phenol removal and 33% NH4+ removal were achieved during the fed-batch treatment of artificial wastewater at 6 d hydraulic retention time (HRT). Under continuous feeding at 3.6 d HRT, phenol and NH4+ removal dropped to 58 and 18%, respectively. However, complete phenol removal and 29% NH4+ removal were achieved when 8 g NaHCO3/l was added to the artificial wastewater to enhance algal growth. This study confirms the potential of solar-based industrial wastewater treatment based on solar-based UV pretreatment followed by algal-bacterial biodegradation.

Removal of trace contaminants using molecularly imprinted polymers.

This work was conducted to study the potential of molecularly imprinted polymers (MIPs) for the removal of oestradiol at trace concentrations (1 ppm-1 ppb). An MIP synthesised with 17beta-oestradiol as template was compared to non-imprinted polymers (NIP) synthesised under the same conditions but without template, a commercial C18 extraction phase and granulated activated carbon. At 1 ppb oestradiol was recovered by 98 +/- 2% when using the MIP, compared to 90 +/- 1, 79 +/- 1, and 84 +/- 2% when using the NIP, a C18 phase, or granulated activated carbon, respectively. According to these levels, the MIP was capable of producing an effluent with a quality 5-10 times higher than the other materials. The same levels of oestradiol recovery were achieved with the MIP when supplying 17beta-oestradiol at 0.1 ppm. Phenolic compounds added as interferences bound less to the MIP than to the NIP, confirming the selectivity of the MIP. Oestradiol biodegradation was also demonstrated at high concentrations (50 ppm), showing the pollutants can be safely destructed after being enriched by molecular extraction. This study demonstrates the potential of molecular imprinted polymers as a highly efficient specific adsorbent for the removal of trace contaminants.

Extremum-seeking with variable gain control for intensifying biogas production in anaerobic fermentation.

A state-dependent variable-gain control system is implemented to follow the characteristics of a laboratory-scale up-flow anaerobic fixed-bed reactor dynamically. The transition from one state to another is determined on an hourly basis, depending on difference between the setpoint of the reactor pH and its true value. Considerable improvement of the process stability--reduction of oscillation in both the reactor pH and biogas production rate during high-rate operation, has been achieved, although the control structure is simple and intuitive.

Influence of temperature on process efficiency and microbial community response during the biological removal of chlorophenols in a packed-bed bioreactor.

Two reactors, initially operated at 14 and 23+/-1 degrees C (RA and RB, respectively), were inoculated with a bacterial consortium enriched and acclimatized to the respective temperatures over 4 months. The biofilms, formed in the reactors, were studied using scanning electron microscopy, cultivation of the biofilm microflora, and physiological analysis of the isolates. Two bacteria able to mineralize chlorophenols under a large range of temperature (10-30 degrees C) were isolated from the biofilm communities of reactors RA and RB and characterized as Alcaligenaceae bacterium R14C4 and Cupriavidus basilensis R25C6, respectively. When temperature was decreased by 10 degrees C, the chlorophenols removal capacity was reduced from 51.6 to 22.8 mg l(-1) h(-1) in bioreactor RA (from 14 to 4 degrees C) and from 59.3 to 34.7 mg l(-1) h(-1) in bioreactor RB (from 23+/-1 to 14 degrees C). Fluorescence in situ hybridization (FISH) of the biofilm communities showed that, in all temperatures tested, the beta-proteobacteria were the major bacterial community (35-47%) followed by the gamma-proteobacteria (12.0-6.5%). When the temperature was decreased by 10 degrees C, the proportions of gamma-proteobacteria and Pseudomonas species increased significantly in both microbial communities.

Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.

Aerobic phenanthrene biodegradation in a two-phase partitioning bioreactor.

The aerobic degradation of phenanthrene by a Pseudomonas migulae strain under classical mechanical aeration and under photosynthetic oxygenation (using a Chlorella sorokiniana strain) in a two-phase partitioning bioreactor (TPPB) constructed with silicone oil as organic phase was investigated. When traditional mechanical aeration was used, an increase in the aeration and/or in the agitation rate enhanced phenanthrene biodegradation. Thus, phenanthrene removal rates (based on the total liquid volume of cultivation) ranged from 22 +/- 1 to 36 +/- 2 mg/l h at 100 rpm and 1 vvm and 400 rpm and 3 vvm, respectively. On the other hand, during phenanthrene biodegradation using the algal-bacterial microcosm a maximum rate of 8.1 +/- 1.2 mg/l h at 200 rpm and 8000 lux of illuminance was achieved.

Functional activity of insulinoma cells (INS-1E) and pancreatic islets cultured in agarose cryogel sponges.

Here, we describe the preparation, structure, and properties of cryogel sponges, which represent a new type of macroporous biomaterial for tissue engineering. Cryogels were produced through freeze-thawing techniques, either from agarose alone or from agarose with grafted gelatin. The aim of this study was to evaluate agarose cryogel sponges as scaffolds for culturing both isolated pancreatic islets and insulinoma cells (INS-1E). In order to evaluate the effect of cell entrapment in artificial scaffolds, cell function reflected by insulin secretion and content was studied in cells cultivated for a 2-week period either in culture plastic plates or in cryogel sponge disks. Our results show that tumor-derived INS-1E cells grown either on plastic or on cryogels do not differ in their proliferation, morphology, insulin release, and intracellular insulin content. However, isolated pancreatic islets cultivated on cryogels sponge show 15-fold higher basal insulin secretion at 3.0 mM glucose than islets cultivated on plastic plates and fail to respond to stimulation with 16.7 mM glucose. In addition, these islets have about 2-fold lower insulin content compared to those grown in plastic plates. It is possible that the cell dysfunction noted in these in vitro experiments is due to the effect of the limited oxygen supply to the islets cultivated in cryogel sponge. Further in vivo studies are needed to clarify the nature of such an observation since according to previous reports, agarose and gelatin induce new vessel formation supporting enhanced oxygen supply.

Photosynthetically oxygenated acetonitrile biodegradation by an algal-bacterial microcosm: a pilot-scale study.

A 43-L column photobioreactor was tested for the treatment of acetonitrile using a symbiotic consortium consisting of a Chlorella sorokiniana strain and a Comamonas strain. Complete biodegradation of 1 g acetonitrile/l was achieved in 79 hours under continuous illumination at 500 microE/m(2)s and 26 degrees C. When the photobioreactor was operated at 26 degrees C under a 14/10 hours light/dark illumination regime at 500 microE/m(2)s, complete mineralization of 1 g acetonitrile/l was achieved in 111 hours. However, when acetonitrile was supplied at 2 g/l, the biodegradation process was severely inhibited by the increase of pH and NH4+ concentration during cultivation. In addition to saving energy for aeration, the microalgae assimilated 33% of the NH4+ released during acetonitrile biodegradation, which significantly reduces the need for subsequent nitrogen removal.

Poly(beta-hydroxybutyrate) production by a moderate halophile, Halomonas boliviensis LC1 using starch hydrolysate as substrate.

AIM: The objective of the present work was to enable the use of starch hydrolysate, generated by the action of a recombinant maltooligosaccharide forming amylase from Bacillus halodurans LBK 34, as the carbon source for the production of poly-beta-hydroxybutyrate (PHB) by Halomonas boliviensis LC1. METHODS AND RESULTS: In this work, different amounts of the alpha-amylase (Amy 34) were utilized for starch hydrolysis, resulting in the production of mixtures of maltooligosaccharides (G1-G6) at varying ratios. The highest PHB accumulation (56 wt%) by H. boliviensis cultivated in shake flasks (with agitation at 160 rev min(-1)) was obtained when 6.4 U ml(-1) of the amylase was used for starch hydrolysis. When H. boliviensis cells were grown in a fermentor with no oxygen limitation the accumulation of PHB was decreased to 35 wt%. Although some improvements in PHB accumulation and cell mass concentration were reached by the addition of peptone and phosphate, respectively, major enhancements were attained when oxygen limitation was induced in the fermentor. CONCLUSIONS: Halomonas boliviensis uses preferentially maltose for PHB formation from starch hydrolysate. It is also able to hydrolyse higher sugars if no other simpler carbon source is available but with a significantly lower polymer yield. Furthermore, H. boliviensis is able to adjust its metabolism to oxygen limitation, most probably by directing the excess NAD(P)H to PHB accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: There have been no reports related to PHB production amongst the members of the genus Halomonas. The use of a maltooligosaccharide forming alpha-amylase, which is active at a temperature and pH close to that required for growth of H. boliviensis, and the versatility of this bacterium in the selection of the carbon source may provide an attractive alternative for the utilization of starch-derived raw materials.

Polycyclic aromatic hydrocarbon biodegradation in extracellular fluids and static batch cultures of selected sub-tropical white rot fungi.

Four sub-tropical white rot fungi, Trametes versicolor, Trametes pocas, Trametes cingulata and isolate DSPM95 were studied alongside the well studied white rot fungus, Phanerochaete chrysosporium, for their ability to remove polycyclic aromatic hydrocarbons (PAHs) from culture media. Both static shallow cultures and extracellular fluids were studied using media contaminated with a defined mixture of the PAHs; fluorene, phenanthrene, anthracene, pyrene and benzo(a)anthracene. With all isolates, the total loss of the parent compound in 31 days was high for fluorene, at +60%, phenanthrene at +40% and anthracene at +42%. Biotransformation of pyrene and benzo(a)anthracene by all the isolates was low, with the highest reduction of pyrene of 15.2% and benzo(a)anthracene of 15.8% being achieved with P. chrysosporium. Disappearance of the more condensed PAHs, pyrene and benzo(a)anthracene, increased in shallow static cultures with the addition of glucose and glucose oxidase as a source of additional H2O2. The addition of Mn2+ and ABTS (2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) to culture supernatants was associated with higher levels of biotransformation. Comparison of the isolates T. versicolor, T. pocas, T. cingulata and isolate DSPM95 with P. chrysosporium showed that these strains were competitive in the reduction of the PAHs, reducing the PAHs by more or less the same magnitude. Also these sub-tropical isolates did not accumulate a lot of HPLC detectable metabolites as much as P. chrysosporium.

Influence of recirculation flow rate on the performance of anaerobic packed-bed bioreactors treating potato-waste leachate.

The performance of anaerobic, packed-bed bioreactors treating leachate from potato waste was evaluated in terms of organic loading rate (OLR) as well as the recirculation flow rate. Two 1 litre bioreactors, filled with porous glass beads as biofilm carriers and with continuous recirculation flow rates of 10 ml min(-1) and 20 ml min(-1) respectively, were used in the experiment. The OLR applied to each bioreactor was increased stepwise from 4 to 12 kg chemical oxygen demand (COD) m(-3)d(-1) by increasing feed flow rate. The methane yields decreased with increasing OLR in both bioreactors. The methane yield for the bioreactor with the lower recirculation flow rate ranged between 0.10 and 0.14 m3 CH4 kg COD(-1) removed, while for the other bioreactor it was 0.14-0.20 m3 CH4 kg COD(-1) removed. Both bioreactors demonstrated stable COD removal which was around 95% for the bioreactor with lower flow of recirculation while for the other it was 92%, over a range of OLRs of 4-8 kg COD m(-3)d(-1). The bioreactor with the lower recirculation flow rate showed operational stability when a high OLR, namely 12 kg COD m(-3)d(-1), was applied, while the other one became overloaded. There was an accumulation of volatile fatty acids which gave a corresponding drop in pH because the system had a low buffering capacity and this thus ultimately led to process failure. This study demonstrated the suitability of a packed bed bioreactor operated at lower recirculation flow rate for treating leachate from potato waste.