Cellulases (CMCases) and polyphenol oxidases from thermophilic Bacillus spp

In composting, organic matter is degraded by cellulases and ligninolytic enzymes at temperatures typically above 50 °C. This study isolated thermophilic microorganisms from a compost system that were then screened for cellulase and polyphenol oxidase activity. Temperature optima for the cellulases and polyphenol oxidases were determined as 70 and 40 °C, respectively. Maximal cellulase activity was determined as 1.333 mg glucose released ml[superscript −1] min[superscript −1]. Maximal polyphenol oxidase activity attained was 5.111 nmol phenol ml[superscript −1] min[superscript −1]. Cellulases were found to be stable over a period of 1 h. The isolated compost microorganisms were identified as strains of Bacillus using 16S ribosomal DNA sequence analysis

ATP-sulphurylase: An enzymatic marker for biological sulphate reduction

Adenosine triphosphate-sulphurylase (ATPS) plays a major role in dissimilatory sulphate reduction. In this study, the level of ATPS activity was monitored in a time course study using a biosulphidogenic batch bioreactor system. A coincident decrease in ATPS activity with a decline in sulphate concentration and an increase in sulphide concentration as biosulphidogenesis proceeded was observed. Flask studies further showed sulphate to be stimulatory to ATPS, while sulphide proved to be inhibitory. The effect of ions (Ca^(2+), Cl^(−), Fe^(2+) and Zn^(2+)) on the ATPS activity was also investigated. Most of the ions studied (Ca^(2+), Cl^(−) and Fe^(2+)) were stimulatory at lower concentrations (40–120 mg/l) but proved toxic at higher concentrations (>120 mg/l). In contrast, Zn^(2+) was inhibitory even at low concentrations ( 40 mg/l). ATPS may potentially be used as an enzymatic marker for biological sulphate reduction in sulphate-rich wastewaters and natural environments (anaerobic systems such as soils and sediments found in freshwater and marine systems), providing all residual sulphide and interfering ions are removed using a simple preparative step

The use of liver histopathology, lipid peroxidation and acetylcholinesterase assays as biomarkers of contaminant-induced stress in the Cape stumpnose, Rhabdosargus holubi (Teleostei: Sparidae), from selected South African estuaries

Three biomarkers of contaminant-induced stress (liver histopathology, the lipid peroxidation (LPx) assay and the acetylcholinesterase (AChE) assay) were adapted for application to the estuarine-dependent marine fish Rhabdosargus holubi (Steindachner, 1881). Specimens of R. holubi were collected using a seine net from 3 temporarily open/closed estuaries in the Eastern Cape, South Africa, each impacted by different anthropogenic activities. The East Kleinemonde estuary has a housing settlement on the banks in the lower reaches and some agriculture in its catchment. The Old Woman’s estuary has a golf course adjacent to its lower and middle reaches and is crossed by a national road in its upper reaches. The Mtana estuary is virtually pristine, with limited cattle grazing occurring along the banks of the estuary and some subsistence agriculture in the catchment. According to the biomarker results from this study, R. holubi from the East Kleinemonde were in good health, as reflected by low LPx and high AChE levels. The liver histopathology did, however, suggest possible previous exposure to stress (increased melanomacrophage centres, increased perivascular connective tissue and severe vacuolation). Overall, liver histopathology results did not differ significantly between estuaries. Fish from the Old Womans recorded significantly inhibited AChE and increased LPx, while fish from the Mtana exhibited significantly increased LPx only, suggesting possible exposure to anticholinesterase contaminants in the former estuary and some form of oxidative stress in the latter. Although water samples were collected from each of the 3 estuaries and analysed for polychlorinated biphenyls, organochlorines, organophosphorous pesticides and pyrethroids, none of these chemicals were detected. Aspesticide residues in water are highly variable, both temporally and spatially, future studies should focus on measuring tissue burdens of organisms in order to identify the contaminant stressor. This study has shown that while chemical analyses of water provide a ‘snap-shot’ of water quality at the time of sampling, biomonitoring can integrate past exposures to stress and is thus useful for identifying potential situations of concern that require further detailed investigation.Keywords: biomonitoring, pollution, estuaries, AChE, LPx, histopatholog

Comparison of the direct enzyme assay method with the membrane filtration technique in the quantification and monitoring of microbial indicator organisms - seasonal variations in the activities of coliforms and E.coli, temperature and pH

The aim of this project was to monitor variations and relationships between coliform and E. coli counts, the activities of their marker enzymes GAL and GUD, and temperature and pH over a period of 12 months in river samples obtained from the Eastern Cape, South Africa. Several polluted water samples were collected for direct coliform β-D-galactosidase (B-GAL) and Escherichia coli β-D-glucuronidase (B-GUD) assays and the membrane filtration technique. While all the samples showed enzyme activities, not all exhibited growth on CM1046 media. Variation in B-GAL activity (40%) was observed between November (highest activity month) and May (lowest activity month). The highest and lowest B-GUD activities were observed in the months of September and May/June, respectively. The sensitivity of the spectrophotometric assay method was indicated by a limit of detection (LOD) of 1 coliform forming unit (CFU)/100 mℓ and 2 CFU/100 mℓ for coliforms and E. coli, respectively. There was a significant (P < 0.05) positive correlation between E. coli counts and GUD activity (R2 = 0.8909). A correlation of R2 = 0.9151 was also observed between total coliforms and B-GAL activity, even though the CFUs were not evenly distributed. Direct enzyme assays were also shown to be more sensitive than the membrane filtration (MF) technique

Hydrolytic enzymes in sewage sludge treatment: a mini-review

Biological wastewater treatment processes can be classified as either aerobic or anaerobic. These two biological treatment processes are each characterised by groups of micro-organisms and their associated enzymes. Hydrolytic enzymes secreted by these micro-organisms are vital for the rate-limiting step of hydrolysis in the treatment of highly polymeric substrates present in sewage sludge. In this mini-review, the effects of mass transfer limitation, metabolic intermediates, extracellular polymeric substances (EPS), electron acceptor conditions and pH and temperature on the activity of these enzymes are summarised. The most salient and current perspectives of the significance and the role that hydrolytic enzymes play in sewage sludge treatment are highlighted

A novel biosensor for the detection and monitoring of -d-galactosidase of faecal origin in water

A voltammetric sensor prepared by the immobilization of metallophthalocyanine complexes onto a glassy carbon electrode has been developed for the detection of β-d-galactosidase (B-GAL) of faecal origin in water. Electrooxidation of chlorophenol red, a breakdown product of the chromogenic substrate chlorophenol red β-d-galactopyranoside, was used as a measure of β-d-galactosidase activity. At metallophthalocyanine modified electrodes, in particular copper(II) phthalocyanine, a decrease in electrode fouling was observed. The sensor was sensitive to fluctuations in pH, not significantly affected by temperature variations and could detect one colony forming unit/100 mL in 15 min. Loss of 40% sensitivity was observed over a period of 30 days. A strong correlation between sensor sensitivity and colony forming units was observed. The sensor is capable of detecting viable but nonculturable bacteria, overcoming this drawback of the use of culture media for detection of coliforms

Cleaning fouled membranes using sludge enzymes

Maintenance of membrane performance requires inevitable cleaning or "defouling" of fouled membranes. Membrane cleaning using sludge enzymes, was investigated by first characterising ostrich abattoir effluent for potential foulants, such as lipids, proteins and polysaccharides. Static fouling of polysulphone membranes using abattoir effluent was also performed. Biochemical analysis was performed using quantitative and qualitative methods for detection of proteins on fouled and defouled membranes. The ability of sulphidogenic proteases to remove proteins adsorbed on polysulphone membranes and capillary ultrafiltration membranes after static fouling, and ability to restore permeate fluxes and transmembrane pressure after dynamic fouling was also investigated. Permeate volumes were analysed for protein and amino acids concentrations. The abattoir effluent contained 553 μg/ml of lipid, 301 μg/ml of protein, 141 μg/ml of total carbohydrate, and 0.63 μg/ml of total reducing sugars. Static fouled membranes removed 23.4percent of proteins. Defouling of dynamically fouled capillary ultrafiltration membranes using sulphidogenic proteases was successful at pH 10, 37°C, within 1 h. Sulphidogenic protease activity was 2.1 U/ml and Flux Recovery (FR percent) was 64 percent

Effect of phenolic compounds on the rapid direct enzymatic detection of β-D-galactosidase and β-D-glucuronidase

For the detection of indicator bacteria such as total coliforms or Escherichia coli (E. coli), microbial enzyme profiles are a preferred option compared to classical methods, because the reactions are more sensitive and rapid to perform and bacteria can be detected and enumerated through specific enzyme activities (Romprẻ et al., 2002). Chromogenic or fluorogenic enzyme substrates are used to detect the enzymes b-D-galactosidase (&beta;-GAL) and b-D-glucuronidase (&beta;-GUD). The chromogenic enzyme substrates are phenol-based, for example the enzyme substrates for &beta;-GUD are p-nitrophenyl-b-D-glucuronide (PNPG) and 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (XGLU), where PNPG produces &rho;-nitrophenol (a yellow colour) and XGLU produces a blue indoxyl product. The fluorogenic substrate used to detect &beta;-GUD is 4-methylumbelliferyl-b- D-glucuronide (MUG) (Manafi, 1996; 2000). &beta;-GAL is detected by using the chromogenic substrates chlorophenol red- &beta;-galactopyranoside (CPRG) and o-nitrophenyl-b-D-galactopyranoside (ONPG) (Edberg et al., 1988; Cheng et al., 2002). As phenolic compounds have extensive industrial applications, they are often found to be present in the aquatic environment (Llompart et al., 2002; Asan and Isildak, 2003). Severalmajor sources are responsible for the presence of these phenolic compounds in the environment, e.g. pesticides, bactericides, wood preservatives and dyes. Phenolic compounds are also present in pulp processing, petroleum refining, leather tannery, textiles and plastics (Lee et al., 1996; Angelino and Gennaro, 1997; Pẽnalver et al., 2002; Asan and Isildak, 2003; Lupetti et al., 2004). Furthermore, phenols may also be found in fertilizers and explosives (Aktas et al., 2006).Keywords: coliforms, E. coli, faecal, b-D-galactosidase, b-D-glucuronidase, inhibition, wastewate