Peter Pollard

Measuring bacterial biomass-COD in wastewater containing particulate matter

Abstract The paper describes a method to determine bacterial biomass-COD in wastewater treatment systems in the presence of particulate matter. A quantitative measure of biomass-COD concentration could greatly enhance the accuracy and reliability of wastewater treatment models that rely on the component “biomass concentration”, which is usually expressed in terms of COD. Rarely is biomass-COD determined directly, despite the existence of microbiological methods for this measurement. Here, we have used a classic microbiological method, acridine orange stain direct counting (AODC), to count the number of bacteria. The average cell volume of wastewater bacteria was used to determine a conversion factor of 20 × 10−11 mg-COD/cell for calculating bacterial biomass-COD with a fast, reliable and simple method applied to wastewater samples in the presence of particulate substrate. Bacterial biomass-COD was measured in five different wastewater treatment systems and compared to the particulate COD.

Bacterial growth dynamics in activated sludge batch assays

Aerobic batch reactors are often used to estimate the maximum specific growth rates of bacteria in wastewater treatment environments. Results from these reactors vary, and they are sometimes confusing because they do not represent the bacterial populations in the original wastewater environment, i.e. in situ. For the first time in batch reactors, we present the in situ growth dynamics of these bacterial populations. We simultaneously measured bacterial numbers and growth rate, oxygen uptake rates (OUR), soluble COD and biomass COD (mg.l). With a substrate to biomass (S(o)/X(o),) ratio of 1.2, bacterial specific growth rates, measured using the thymidine growth assay, increased from 1 to 4.5 d. During the same period, the initial OUR remained relatively constant, with an OUR-based estimate of the maximum specific growth rate of 3.6 d. These observations challenge the fundamental theory of the batch assay that bacteria are growing at their maximum rate under substrate saturating conditions while the yield coefficient remains constant. Our results confirm that the batch reactor environment induces changes in the bacterial physiology and/or community structure compared to the original treatment environment, even at low S(o)/X(o), ratios. We discuss how, simultaneously using both OUR and thymidine assays, gives more information about how the cell allocates its energy resources between new cell synthesis and cell maintenance.Aerobic batch reactors are often used to estimate the maximum specific growth rates of bacteria in wastewater treatment environments. Results from these reactors vary, and they are sometimes confusing because they do not represent the bacterial populations in the original wastewater environment, i.e. in situ. For the first time in batch reactors, we present the in situ growth dynamics of these bacterial populations. We simultaneously measured bacterial numbers and growth rate, oxygen uptake rates (OUR), soluble COD and biomass COD (mg.l). With a substrate to biomass (S/X) ratio of 1.2, bacterial specific growth rates, measured using the thymidine growth assay, increased from 1 to 4.5 d. During the same period, the initial OUR remained relatively constant, with an OUR-based estimate of the maximum specific growth rate of 3.6 d. These observations challenge the fundamental theory of the batch assay that bacteria are growing at their maximum rate under substrate saturating conditions while the yield coefficient remains constant. Our results confirm that the batch reactor environment induces changes in the bacterial physiology and/or community structure compared to the original treatment environment, even at low S/X ratios. We discuss how, simultaneously using both OUR and thymidine assays, gives more information about how the cell allocates its energy resources between new cell synthesis and cell maintenance.

Measuring in situ bacterial specific growth rates and population dynamics in wastewater

Abstract Reliable design and operation of biological wastewater treatment systems demands robust modelling of the degradation processes. However, methods to directly measure key bacterial growth parameters have not been available to the wastewater industry. Here, we directly measure bacterial specific growth rates without changing the treatment environment. Bacterial growth (with the thymidine assay) and biomass (by direct counts) measurements were used to compare the dynamics of the microbial community in the different compartments of an activated sludge process. The aerobic compartment specific growth rate μ was 0.5 d−1. In this work, we explain the concept of the thymidine growth assay, the traps for the novice and how to apply the method to biological nutrient removal processes. These methods now allow us to distinguish bacterial growth from metabolic activity and observe how the cell allocates its resources between the two. This represents the first application of a very powerful tool to directly measure population dynamics and kinetics of bacteria in wastewater treatment processes.

Complementary independent molecular, radioisotopic and fluorogenic techniques to assess biofilm communities in two wastewater wetlands

Abstract Optimisation of nutrient removal processes requires a detailed understanding of the microbial physiologies actually occurring within the biomass of different treatment zones, and also knowledge of how these communities respond to environmental factors. This paper describes a suite of four independent and complementary, microbiological techniques, utilised to obtain detailed assessments of wastewater bacterial biomass. Examples of these techniques are shown as applied to biofilms from two surface-flow wetlands used for treating sewage effluent. Intact biofilms were prepared via freezing, followed by cryosectioning and direct microscopic observation. This approach was combined with prior, in situ incubation of biofilm with radiolabelled thymidine to assess in situ bacterial growth rates. Individual bacterial cells were assessed microscopically for in situ respiratory activity and phylogenetic identity, respectively through use of an inducibly-fluorescent redox stain (CTC), and fluorescent in situ hybridisation (FISH) of ribosomal RNA (rRNA). These complementary techniques enabled assessment of individual biofilm clusters according to their in situ status (growth, respiration, spatial location and phylogenetic affiliation). This approach detected significant spatial variations to btofilm; limited the bias inherent in data obtained using any one technique, and provided greater detail of subpopulations within the microbial community.

The direct measurement of bacterial growth in biofilms of emergent plants (schoenoplectus) of an artificial wetland

In the wastewater industry, artificial wetlands are used to improve water quality. Biofilms on these plant surfaces are thought to retain most of the active bacterial community that decomposes organic matter and aid in nutrient removal. Wetland design and operation could be enhanced with the in situ measurement of growth and dynamics of the biofilm-bacteria. This paper describes how to directly measure the rate of bacterial growth on the surface of submerged sections of emergent macrophytes, with the radioactively labelled DNA precursor [methyl-3H] thymidine. We found that the isotope was rapidly and efficiently incorporated into the bacteria growing on plant surfaces, without a lag phase. Isotope dilution was avoided by using a specific activity of 2 Ci.mmol−1. Highest growth rates appeared to be associated with the top 10 mm of submerged plant tissue. The method accommodated the natural heterogeneity of biofilms both between plants and along the stem of the same plant. These findings are important for future studies of biofilm dynamics.

The impact of microbiological tools on mathematical modelling of biological wastewater treatment

Biological systems are being used to treat an increasing range of complex wastes; domestic and industrial wastewaters containing nutrients and refractory organic compounds, soil sites and groundwater contaminated by organics, and organic solid residues. These treatment processes rely on micro-organisms and, more than ever before, must deliver higher quality outcomes at higher levels of reliability to protect the environment. At the same time, pressures to deliver cost-effective treatment have increased. The challenge for these biological treatment technologies and the associated engineering is to achieve the environmental and economic goals simultaneously. Mathematical modelling is an essential component in developing a detailed understanding of such processes, as well as design guidelines and suitable operating and control strategies. This paper provides a brief summary of the development of mathematical models for biological waste treatment systems, why they have become increasingly complex and how certain microbiological tools can provide the experimental means to validate more complex segregated and structured models of biological behaviour. With a number of specific modelling examples in the field of wastewater treatment, we illustrate the potential of these modern microbiological tools and their implications for gaining an improved understanding of biological waste treatment.