Elsevier

Water Research

Volume 37, Issue 9, May 2003, Pages 2162-2172
Water Research

Assessment of activated sludge microbial community analysis in full-scale biological wastewater treatment plants using patterns of fatty acid isopropyl esters (FAPEs)

https://doi.org/10.1016/S0043-1354(02)00625-5Get rights and content

Abstract

This investigation introduces the application of a relatively rapid technique to obtain information about the dynamic nature of microbial communities in activated sludge. The objective has been to consider variability due to measurement errors and protocol changes within the same quantitative framework as the analysis of systematic differences in microbial communities in large-scale aerobic activated sludge secondary wastewater treatment systems. Adjustments to the methodology were considered due to their potential for simplifying and shortening the analysis procedure. All modifications to the protocols used to assay the composition of microbial fatty acids (MFAs) of activated sludge imposed some bias to the chromatographic data. This methodological bias was similar in magnitude to the level of discrimination between activated sludge microbial community structures that were considered as part of the present study. MFA analysis supported the expectations of subtle but systematic community structure differences and shifts in activated sludge based on the current understanding of these wastewater treatment systems. A standardized MFA methodology was shown to be sensitive to minor systematic changes in activated sludge communities due the anticipated underlying factors of selective pressures from the process configuration, history, operational conditions and/or nutrient status. The chemometric approach of fatty acid isopropyl ester analysis of activated sludge can provide a routine tool for meaningful and quantitative information of changes in activated sludge quality in full-scale treatment systems.

Introduction

Fatty acid analysis for the estimation of microbial biomass or for the examination of community structure has been effectively used to study microbial communities in soil environments [1], [2], [3], composts [4], biofilters [5] and peatlands [6]. It has also been shown that this same technique has the potential to be applied to biological wastewater treatment systems [7], [8], and that fatty acid compositions can be used as a parameter for estimating microbial growth kinetics associated with biological wastewater treatment [9], [10]. The composition or profile of microbial fatty acids (MFAs) from a biomass sample represents a weighted average of the unique MFA profiles of the constituent populations of species [11] convoluted with changes in the physiological state of individual organisms [12], [13]. However, the potential applications of MFA analysis as a tool of applied microbial ecology in biological wastewater treatment optimization and control is still at an early stage of development [14]. Advancements in the interpretation of the potential wealth of diagnostic information of community structure and/or physiological state contained in the dynamic patterns of fatty acids extracted from microbial communities [15], [16] requires that a wider field of experience be established between the changing patterns and treatment process performance. To this end, further simplification and streamlining of the analytical procedures would help to make MFA analysis become more widely chosen as an informative, albeit currently explorative, monitoring tool in wastewater treatment research. At the same time establishing a quantitative framework to judge the significance of relative differences in observed MFA patterns would provide researchers with a basis with which to compare results and judge community structure stability both within and between experiments and laboratories.

The objective of the present investigation has been to juxtapose the influence of changes to the MFA analysis methodology with respect to the method sensitivity in detecting subtle differences in MFA community structure in samples of activated sludge at full scale. The interest in exploring a new methodology has been to reduce the effort of routine sample preparation and analysis while working to use a minimal number of relatively non-hazardous reagents. To provide important perspective to this method development activity, the relative significance of measurement errors and sampling representativeness of activated sludge were considered concurrently. The goal has been to set a quantitative foundation for interpreting observed differences in activated sludge microbial community structures (or MFA compositions) in time or space within large-scale biological wastewater treatment systems. In this part of the investigation the relative effects of activated sludge reactor configuration and nutrient status on microbial community structure were examined.

There exist two distinct protocols for the extraction of fatty acids from a biomass sample, namely phospholipid and whole cell fatty acid analysis [11]. Phospholipid fatty acid (PLFA) analysis, by definition, considers only those fatty acids linked to membrane phospholipids. In contrast, whole cell fatty acid (WCFA) analysis extracts cellular fatty acids from all membrane sources. The PLFA profile has been shown to be a subset of the WCFA profile [9]. WCFA analysis is more commonly referred to as fatty acid methyl ester (FAME) analysis. However, with either of the common PLFA [17] or WCFA [18] protocols, the enumerated analytes of interest are FAMEs. Therefore, PLFA and WCFA have been adopted as more informative acronyms. The loss in analytical specificity with WCFA analysis is accompanied by a significant decrease in sample preparation time. Because of these logistical considerations, WCFA was our preferred method.

Measuring biomass and fingerprinting the microbial community structure of activated sludge by MFA analysis involves steps of (1) cell harvesting or biomass sample collection, (2) biomass solubilization to release the cell lipid fraction, (3) release and derivatization of the lipid fatty acids, (4) extraction of the fatty acid esters, and (5) capillary gas chromatographic (GC) quantification and identification of the fatty acid composition [19] with either a flame ionization detector (FID) or mass spectrometric detector (MSD).

Commonly, the MIDI [18] method is used in the preparation of whole cell methyl esters for analysis by gas chromatography. A variation of the MIDI WCFA procedure has also been successfully applied for investigations on contaminant fate in biological treatment of pulp mill wastewater [7]. The MIDI protocol involves a saponification step to disrupt cell membranes and cleave the fatty acids from the released microbial lipids. Saponification can be followed by (1) acid catalysed methylation and solvent extraction [18] or (2) acidification and solvent extraction followed by diazomethane derivatization [7]. The FAMEs can then be enumerated by GC/FID based on equivalent chain length calibration [20], [21]. The MIDI-FAME protocol therefore requires two distinct and sequential preparation stages, namely lipid and MFA solubilization followed by derivatization.

The step of cell solubilization can be readily combined with derivatization if isopropyl esters are formed instead of methyl esters of the MFAs. Peuchant et al. [22] have applied a much less labour and reagent intensive protocol with whole red blood cell fatty acid analysis. Red blood cells were solubilized and lipid bound fatty acids transesterified in an acidic isopropyl alcohol (IPA) matrix. By this protocol, fatty acid isopropyl esters (FAPEs) are enumerated rather than FAMEs and our interest was to consider WCFA-FAPE analysis of activated sludge samples. Although much more detailed individual lipid and fatty acid analysis is undertaken in microbial ecology [23], our objective is to examine the MFA pattern and to begin to explore the information content and potential engineering applications associated with routinely monitored systematic changes in this pattern [15], [16]. The protocol used in the present investigation was similar to the one applied by Peuchant et al. [22]. An added advantage to the reduced analytical burden found with this FAPE protocol is that it allowed more flexibility and convenience with the selection of any one of a number of readily available commercially prepared FAMEs as non-interfering internal standards in the chromatography. The FAPE protocol also avoids the use of diazomethane [7], the necessity of a base wash after solvent extraction [18], and chloroform [23] or MTBE [7] as solvents. The first research question addressed for the present investigation was if WCFA-FAPE analysis provides the same kind of chromatographic information of microbial community structure as MIDI-FAME analysis.

Isopropyl esters of free and lipid bound fatty acids are formed by acid catalysed derivatization at an elevated temperature. The acid catalysed reaction of IPA and fatty acids results in an equilibrium with water as an esterification by-product, and so the presence of water in the sample can shift the equilibrium, preventing total esterification of the sample fatty acids. Water must be removed from the biomass sample before derivatization. Drying can be accomplished using toluene [24] or anhydrous sodium sulphate [22]. Alternatively, thermal drying to remove the excess biomass sample moisture is advantageous as it avoids the use of additional chemicals that are an added complication and a potential source for contamination. A drying oven can be used to this end, but oven drying can be time consuming, taking in excess of 1 h [25]. However, a microwave oven can be used in place of a drying oven [26]. Microwave drying would significantly reduce the sample preparation time and would, therefore, be attractive if the resolved biomass MFA composition is not somehow adversely effected. Therefore, experiments were conducted to examine if microwave drying significantly influenced the WCFA-FAPE profile or the measurement errors of activated sludge samples as compared to oven drying.

Biomass can be harvested (isolated and concentrated) from an activated sludge sample by centrifugation or filtration. With centrifugation there exists the possibility that some organisms will not sediment. With filtration some microorganisms may be small enough to pass through the filter pores. In principle, filtration is more rapidly applied but we wanted to have the flexibility to harvest biomass by either approach. Filtering is more conveniently applied in the field and centrifuging is simpler to apply in the laboratory. Therefore, the influences of these two biomass collection methods on the resulting WCFA-FAPE profile were considered.

Following Peuchant et al. [22], extraction of FAPEs from an acidic alcoholic aqueous matrix is performed using an organic solvent such as hexane for liquid sample injection in GC/FID or GC/MSD analysis. Another approach that avoids the need for solvent extraction altogether is solid phase micro-extraction (SPME). SPME involves exposing a polymer-coated fibre to the sample matrix and allowing the analytes to partition between the sample matrix and the fibre coating [27]. The feasibility of eliminating the need for solvent extraction by using SPME was investigated due to the reported success of SPME fatty acid extraction from other aqueous alcoholic mixtures [27].

Finally, it was of important practical interest to place these questions of methodological variation in the context of random and systematic variability of activated sludge community structure observed from temporally and spatially distinct grab samples from the aeration basins of a full-scale biological wastewater treatment process. To what extent would measurement or replicate variability hinder the ability to resolve systematic spatial or temporal differences in MFA community structure? Therefore, a complementary set of field samples were obtained to assess the sensitivity of WCFA-FAPE analysis in discriminating between expected shifts in activated sludge community structure as a function of location or time. Recognizing that there is still research advancement that is required in order to establish structure function relationships with MFA analysis and activated sludge treatment, the present objective was to see if MFA fingerprinting using a WCFA-FAPE approach would support the expectations based on the current understanding of these systems.

Section snippets

Biomass collection by filtration

In cell harvesting for FAPE analysis, 47 mm ∅ Whatman 934AH organic free and tared glass fibre filters were used. Aliquots of 5 mL well-mixed activated sludge mixed liquor were filtered by vacuum. Filters were first rinsed with 5 mL aliquots of deionized water (18.2  cm resistivity at 25°C—Millipore Alpha Q Ultra-pure Water System). The filters were dried at 105°C for 1.5 h. After drying, the filters were cooled and weighed, and the total suspended solids determined [25]. The filters were then

Results and discussion

This investigation sought to address both random and systematic measurement variability of activated sludge MFA patterns as key elements requiring characterization before conducting more in-depth studies on the dynamic nature of MFA microbial community structure within large-scale biological treatment systems. The sensitivity of the results to analysis methodology were examined by repeated analyses with methodological variations on the same mixed liquor grab sample (Waterloo WWTP). This

Conclusions

WCFA-FAPE analysis yields information about microbial community structure that is similar but distinct from MIDI-FAME analysis. The differences between the FAPE and FAME data limit the ability to make direct comparison between MFA profiles generated by these two protocols without correction factors. Steps to shorten the sample preparation (microwave versus oven drying), and to suit needs or laboratory resources (filtration versus centrifuging) exhibited a much reduced impact on the resolved MFA

Acknowledgments

The authors are grateful to the City of Hamilton (Ontario, Canada) Water Quality Section in providing an essential practical component and financial support in the development of techniques in applied microbial ecology for improved control of wastewater treatment systems. Financial support has been provided by an NSERC (Natural Sciences and Engineering Research Council of Canada) operating grant and the Department of Civil Engineering at the University of Waterloo. Jennifer Becker and Carly

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