W.D. Leukes

In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus

The selection of probiotics for aquaculture is usually based on their antagonism towards pathogens. However, other criteria such as growth, attachment to intestinal mucus and production of beneficial compounds should also be considered. We suggest a protocol for the isolation and selection of potential probiotic bacteria based on their in vitro growth characteristics and propose a ranking index (RI) to screen potential aquaculture probionts. We suggest that the lag period and doubling time are the most important criteria for the comparison of growth curves, hence the RI is based on the doubling time (t(d)) and lag period (lambda) obtained from the growth profile of each bacterium. Bacteria were isolated from the gut of the common clownfish, Amphiprion percula, and screened for antagonistic activity towards seven aquatic pathogens. All five candidate probiotics showed antagonism to various aquatic pathogens. When grown in intestinal fish mucus no probiotic had a RI higher than the two tested pathogens (Aeromonas hydrophila and Vibrio alginolyticus). However, candidate probiont AP1 had a faster specific growth rate (micro) (0.05) than the pathogens (0.049 and 0.047 respectively), while AP5 grown in marine broth had a shorter lag period than the pathogens. Strategies to increase probiotic concentration include the inoculation of high concentrations and the preconditioning of these bacteria to reduce the lag period. It should be tested whether or not such strategies will allow the probiotic bacteria to dominate initially and thereby gain a competitive advantage. This could become an important aspect under in vivo conditions where both attachment and nutrient supply differ from that found in in vitro studies.

A capillary membrane bioreactor using immobilized polyphenol oxidase for the removal of phenols from industrial effluents

Abstract A capillary membrane bioreactor has been developed and tested for the removal of phenolic compounds from synthetic and industrial effluents. Polyphenol oxidase was immobilized on single capillary membranes in a small-scale bioreactor using two morphologically different polymeric membranes. One has a novel structure with no external supporting skin layer. This membrane allows greater flux and was shown to facilitate high efficiency in removal of reaction products from the reactor. Using this high flux membrane, 949 μmol phenolics were removed from a solution containing 4 m m total phenolics using 45 U polyphenol oxidase in 8 h as compared with 120 μmol removed using non-immobilized enzyme which was inactivated (due to product inhibition) after 7 h. No precipitated melanoid products were observed in the permeate from the capillary membrane bioreactor, and in order to remove the colored quinone-type products of the reaction, a packed column containing chitosan was integrated into the system. Almost complete removal of the colored quinones and associated polymers from the permeate was observed.

Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation

Abstract Internally skinned polysulphone capillary membranes were coated with a viscous chitosan gel and used as an immobilization matrix for polyphenol oxidase. Bench-scale, single-capillary membrane bioreactors then were used to determine the influence of the chitosan coating on product removal after substrate conversion by immobilized polyphenol oxidase during the treatment of industrial phenolic effluents. The results indicate that greater efficiency was achieved in the removal of polyphenol oxidase-generated products by the chitosan membrane coating, as compared with chitosan flakes. This facilitated an increase in the productivity of the immobilized enzyme.

Fungal Bioremediation of Phenolic Wastewaters in an Airlift Reactor

Of the various types of industry‐generated effluents, those containing organic pollutants such as phenols are generally difficult to remediate. There is a need to develop new technologies that emphasize the destruction of these pollutants rather than their disposal. In this work the white rot fungus, Trametes pubescens, was demonstrated to be an effective bioremediation agent for the treatment of phenolic wastewaters. An airlift loop reactor was optimized, in terms of volumetric oxygen transfer rate (KLa = 0.45 s‐1), to provide an environment suited to rapid growth of T. pubescens (μ = 0.25 day‐1) and a particularly efficient growth yield on glucose of 0.87 g biomass·g glucose‐1. The phenolic effluent was shown to be a paramorphogen, influencing fungal pellet morphology in the reactor, as well as increasing laccase enzyme activity by a factor of 5 over the control, to a maximum of 11.8 U·mL‐1. This increased activity was aided by the feeding of nonrepressing amounts (0.5 g·L‐1) of glucose to the reactor culture. To our knowledge the degradation results represent the highest rate of removal (0.033 g phenol·g biomass‐1·day‐1) of phenolic compounds from water reported for white rot fungi.

Ultrafiltration of petrochemical industrial wastewater using immobilised manganese peroxidase and laccase: application in the defouling of polysulphone membranes

The application of laccase and manganese-dependant peroxidase (MnP) from Trametes versicolor to facilitate removal of aromatic hydrocarbons from a petrochemical industrial effluent was investigated. The enzymes were immobilised onto polysulphone ultrafiltration membranes thus facilitating enzyme-substrate contact and the activity and duration of activity was determined with respect to the enzymatic degradation of the aromatics within the effluent. The phenomenon of fouling typically associated with the operation of ultrafiltration systems was reduced by the action of the immobilised enzyme layer. To determine the different ‘defouling’ potential of MnP NaF and NaN3 were applied to the system as inhibitors of laccase. Differing ‘defouling’ efficiencies were then compared with standard inhibition kinetics associated with the non-immobilised enzyme suites. The system was also applied to an industrial petrochemical-based effluent and compared with the synthetic make-up effluent in terms of ‘defouling’ efficiency. The presence of high concentrations of fluoride at the membrane interface during the ultrafiltration of the petrochemical-based effluent contributed significantly to the inhibition of the immobilised enzyme suite and thus manifested as a significant decrease in ‘defouling’ potential in comparison with the system being operated using the synthetic make-up effluent.

Immobilisation of polyphenol oxidase on nylon and polyethersulphone membranes: Effect on product formation

Preliminary studies were made of the potential use of a membrane-immobilised enzymatic method for the treatment of p-cresol-containing wastewater. The enzyme polyphenol oxidase was immobilised onto hydrophobic polyethersulphone capillary membranes and hydrophilic nylon flat-sheet membranes, by adsorption, and by adsorption with glutaraldehyde cross-linking, respectively. It was found that the intermediate product of the polyphenol oxidase reaction, 4-methylcatechol, was detected when the enzyme was immobilised on the nylon membranes or was not immobilised, but only the o-quinone final product was detected when polyethersulphone was used as the immobilisation matrix.

Suitability of a modified capillary membrane for growth of fungal biofilms

A preliminary study was carried out to determine the feasibility of growing two model fungal organisms as biofilms in bioreactors in which the biofilm was supported on novel capillary membranes which have no external skin, and thus allow high transmembrane flux. The fungi used were Trametes versicolor and Neurospora crassa. Biofilm morphology and extracellular enzyme production were monitored. The removal from the bioreactor feed of a model pollutant, p-cresol, by T. versicolor was investigated, and results indicated that the cresol was removed from the solution.