A critical review on emerging industrial applications of chars from thermal treatment of biosolids.

Thermochemical treatment is rapidly emerging as an alternative method for the management of stabilised sewage sludges (biosolids) to effectively reduce waste volume, degrade contaminants, and generate valuable products, particularly biochar and hydrochar. Biosolids-derived char has a relatively high concentration of heavy metals compared with agricultural chars but is still applied to land due to its beneficial properties and ability to retain metals. However, non-agricultural applications can provide additional economic and environmental benefits, promote sustainability and support a circular economy. This review identifies extensive non-agricultural opportunity for biosolids biochar, including adsorption, catalysis, energy storage systems, biological process enhancement, and as additives for rubber compounding and construction. Biosolids chars have received limited attention vs agricultural char, and we draw on both areas of literature, as well as evaluating differences between agricultural and biosolids chars. A key opportunity for biosolids biochar in comparison with other materials and agricultural chars is its sustainable and low-cost nature, relatively high metals content, improving catalyst properties, and ability to modify in various stages to tune it to specific applications. The specific opportunities for hydrochar have only received limited attention. Research needs to include better understanding of the benefits and limitations for specific applications, as well as adjacent drivers, including society, regulation, and market and economics.

Substrate availability drives mixed culture fermentation of glucose to lactate at steady state.

Mixed-culture fermentation (MCF) enables carbon recycling from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, MCF systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive biochemical production changes under differing conditions are currently poorly understood. These mechanisms are critical to useful MCF production models. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by lactate, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this study, steady state lactate production was achieved by increasing the organic loading rate of a butyrate-producing system from limiting to nonlimiting conditions at pH 5.5. Crucially, butyrate production resumed upon return to substrate-limited conditions. 16S ribosomal DNA community profiling combined with metaproteomics demonstrated that the butyrate-producing lineage Megasphaera redirected carbon flow through the methylglyoxal bypass when substrate was nonlimiting, which altered the community structure and metabolic expression toward lactate production. This metabolic mechanism can be included in future MCF models to describe the changes in product generation in substrate nonlimiting conditions.

Uncertainty analysis of rising sewer models with respect to input parameters and model structure using Monte Carlo simulations and computational fluid dynamics.

Modelling conversion processes in sewers can help minimize odour and pipe corrosion issues, but model uncertainties and errors must be understood. In this study, the Wastewater Aerobic/Anaerobic Transformation in Sewers (WATS) model is implemented in two different frameworks; 1-D (CSTR-in-series) and computational fluid dynamics (CFD) to study the uncertainties due to model parameters and its mathematical form. The 1-D model is used to conduct uncertainty/sensitivity analysis using Monte Carlo simulations. Time-averaged outputs were represented using a general linearized model to quantify the importance of specific parameters. The sulfide formation rate per unit area of the biofilm is the most influential parameter. Parameters controlling anaerobic hydrolysis and fermentation are also significant. Uncertainty due to model structure is studied using CFD to explore the influences of non-homogeneous surface reactions and solids settling. These showed that the 1-D model provides a reasonable characterisation of the process for simple flows in pressure mains.

Self-Sustained Nitrite Accumulation at Low pH Greatly Enhances Volatile Solids Destruction and Nitrogen Removal in Aerobic Sludge Digestion.

Aerobic sludge digestion of waste activated sludge (WAS) is widely used as a stabilization option in small- and midsized wastewater treatment plants. However, the digestion process is often limited by low volatile solids (VS) destruction and poor pathogen removal efficiency. This study presents a novel operational strategy that achieves enhanced VS destruction and nitrogen removal by inducing sustained nitrite accumulation via a single spike of nitrite to aerobic digester operated at a natively low pH (<5.5). The strategy was demonstrated through the use of three laboratory aerobic sludge digesters, each continuously operated for over 300 days. Compared to control reactors, the strategy enhanced volatile solids destruction by 35.0-38.4%, nitrogen removal by 58.5-70.8%, and pathogen reduction by approximately 1 log. The standard oxygen uptake rate (SOUR) was reduced to 0.49 ± 0.03 mgO2/gVS/h, compared to 0.85 ± 0.01-1.68 ± 0.02 mgO2/gVS/h in the control, indicating enhanced stabilization. Free nitrous acid formed from nitrite at low pH, rather than nitrite itself, was identified to be the cause of improved digestion performance. Since the nitrite production is self-supporting, no additional ongoing costs are incurred.

Mainstream Ammonium Recovery to Advance Sustainable Urban Wastewater Management.

Throughout the 20th century, the prevailing approach toward nitrogen management in municipal wastewater treatment was to remove ammonium by transforming it into dinitrogen (N2) using biological processes such as conventional activated sludge. While this has been a very successful strategy for safeguarding human health and protecting aquatic ecosystems, the conversion of ammonium into its elemental form is incompatible with the developing circular economy of the 21st century. Equally important, the activated sludge process and other emerging ammonium removal pathways have several environmental and technological limitations. Here, we assess that the theoretical energy embedded in ammonium in domestic wastewater represents roughly 38-48% of the embedded chemical energy available in the whole of the discharged bodily waste. The current routes for ammonium removal not only neglect the energy embedded in ammonium, but they can also produce N2O, a very strong greenhouse gas, with such emissions comprising the equivalent of 14-26% of the overall carbon footprint of wastewater treatment plants. N2O emissions often exceed the carbon emissions related to the electricity consumption for the process requirements of WWTPs. Considering these limitations, there is a need to develop alternative ammonium management approaches that center around recovery of ammonium from domestic wastewater rather than deal with its "destruction" into elemental dinitrogen. Current ammonium recovery techniques are applicable only at orders of magnitude above domestic wastewater strength, and so new techniques based on physicochemical adsorption are of particular interest. A new pathway is proposed that allows for mainstream ammonium recovery from wastewater based on physicochemical adsorption through development of polymer-based adsorbents. Provided adequate adsorbents corresponding to characteristics outlined in this paper are designed and brought to industrial production, this adsorption-based approach opens perspectives for mainstream continuous adsorption coupled with side-stream recovery of ammonium with minimal chemical requirements. This proposed pathway can bring forward an effective resource-oriented approach to upgrade the fate of ammonium in urban water management without generating hidden externalized environmental costs.

Model-based analysis and optimization of a full-scale industrial high-rate anaerobic bioreactor.

The objective of this paper is to present the model-based optimization results of an anaerobic granular sludge internal circulation reactor. The International Water Association Anaerobic Digestion Model No. 1 extended with phosphorus (P), sulfur (S), and ethanol is used to describe the main biological and physico-chemical processes. The high-rate conditions within the reactor are simulated using a flow + reactor model comprised of a series of continuous stirred tank reactors followed by an ideal total suspended solids separation unit. Following parameter estimation by least squares on the measured data, the model had a relative mean error of 13 and 15% for data set #1 and data set #2, respectively. Response surfaces show that the reactor performance index (a metric combining energy recovery in the form of heat and electricity, as well as chemicals needed for pH control) could be improved by 45% when reactor pH is reduced down to 6.8. Model-based results reveal that influent S does not impose sufficient negative impacts on energy recovery (+5.7%, in MWh/day,+0.20 M€/year when influent S is removed) to warrant the cost of its removal (3.58 M€/year). In fact, the process could handle even higher S loads (ensuring the same degree of conversion) as long as the pH is maintained above 6.8. Nevertheless, a higher S load substantially increases the amount of added NaOH to maintain the desired operational pH (>25%) due to the acidic behavior of HS - . CO 2 stripping decreases the buffer capacity of the system and hence use of chemicals for pH control. Finally, the paper discusses the possibilities and limitations of the proposed approach, and how the results of this study will be put into practice.

Evaluation of anaerobic digestion processes for short sludge-age waste activated sludge combined with anammox treatment of digestate liquor.

The need to reduce energy input and enhance energy recovery from wastewater is driving renewed interest in high-rate activated sludge treatment (i.e. short hydraulic and solids retention times (HRT and SRT, respectively)). This process generates short SRT activated sludge stream, which should be highly degradable. However, the evaluation of anaerobic digestion of short SRT sludge has been limited. This paper assesses anaerobic digestion of short SRT sludge digestion derived from meat processing wastewater under thermophilic and mesophilic conditions. The thermophilic digestion system (55°C) achieved 60 and 68% volatile solids destruction at 8 day and 10 day HRT, respectively, compared with 50% in the mesophilic digestion system (35°C, 10 day HRT). The digestion effluents from the thermophilic (8-10 day HRT) and mesophilic systems were stable, as assessed by residual methane potentials. The ammonia rich sludge dewatering liquor was effectively treated by a batch anammox process, which exhibited comparable nitrogen removal rate as the tests using a control synthetic ammonia solution, indicating that the dewatering liquor did not have inhibiting/toxic effects on the anammox activity.

Good modelling practice in applying computational fluid dynamics for WWTP modelling.

Computational fluid dynamics (CFD) modelling in the wastewater treatment (WWT) field is continuing to grow and be used to solve increasingly complex problems. However, the future of CFD models and their value to the wastewater field are a function of their proper application and knowledge of their limits. As has been established for other types of wastewater modelling (i.e. biokinetic models), it is timely to define a good modelling practice (GMP) for wastewater CFD applications. An International Water Association (IWA) working group has been formed to investigate a variety of issues and challenges related to CFD modelling in water and WWT. This paper summarizes the recommendations for GMP of the IWA working group on CFD. The paper provides an overview of GMP and, though it is written for the wastewater application, is based on general CFD procedures. A forthcoming companion paper to provide specific details on modelling of individual wastewater components forms the next step of the working group.

Microbial Internal Storage Alters the Carbon Transformation in Dynamic Anaerobic Fermentation.

Microbial internal storage processes have been demonstrated to occur and play an important role in activated sludge systems under both aerobic and anoxic conditions when operating under dynamic conditions. High-rate anaerobic reactors are often operated at a high volumetric organic loading and a relatively dynamic profile, with large amounts of fermentable substrates. These dynamic operating conditions and high catabolic energy availability might also facilitate the formation of internal storage polymers by anaerobic microorganisms. However, so far information about storage under anaerobic conditions (e.g., anaerobic fermentation) as well as its consideration in anaerobic process modeling (e.g., IWA Anaerobic Digestion Model No. 1, ADM1) is still sparse. In this work, the accumulation of storage polymers during anaerobic fermentation was evaluated by batch experiments using anaerobic methanogenic sludge and based on mass balance analysis of carbon transformation. A new mathematical model was developed to describe microbial storage in anaerobic systems. The model was calibrated and validated by using independent data sets from two different anaerobic systems, with significant storage observed, and effectively simulated in both systems. The inclusion of the new anaerobic storage processes in the developed model allows for more successful simulation of transients due to lower accumulation of volatile fatty acids (correction for the overestimation of volatile fatty acids), which mitigates pH fluctuations. Current models such as the ADM1 cannot effectively simulate these dynamics due to a lack of anaerobic storage mechanisms.

Can direct conversion of used nitrogen to new feed and protein help feed the world?

The increase in the world population, vulnerability of conventional crop production to climate change, and population shifts to megacities justify a re-examination of current methods of converting reactive nitrogen to dinitrogen gas in sewage and waste treatment plants. Indeed, by up-grading treatment plants to factories in which the incoming materials are first deconstructed to units such as ammonia, carbon dioxide and clean minerals, one can implement a highly intensive and efficient microbial resynthesis process in which the used nitrogen is harvested as microbial protein (at efficiencies close to 100%). This can be used for animal feed and food purposes. The technology for recovery of reactive nitrogen as microbial protein is available but a change of mindset needs to be achieved to make such recovery acceptable.

Variable cell morphology approach for individual-based modeling of microbial communities.

An individual-based, mass-spring modeling framework has been developed to investigate the effect of cell properties on the structure of biofilms and microbial aggregates through Lagrangian modeling. Key features that distinguish this model are variable cell morphology described by a collection of particles connected by springs and a mechanical representation of deformable intracellular, intercellular, and cell-substratum links. A first case study describes the colony formation of a rod-shaped species on a planar substratum. This case shows the importance of mechanical interactions in a community of growing and dividing rod-shaped cells (i.e., bacilli). Cell-substratum links promote formation of mounds as opposed to single-layer biofilms, whereas filial links affect the roundness of the biofilm. A second case study describes the formation of flocs and development of external filaments in a mixed-culture activated sludge community. It is shown by modeling that distinct cell-cell links, microbial morphology, and growth kinetics can lead to excessive filamentous proliferation and interfloc bridging, possible causes for detrimental sludge bulking. This methodology has been extended to more advanced microbial morphologies such as filament branching and proves to be a very powerful tool in determining how fundamental controlling mechanisms determine diverse microbial colony architectures.

Regulation mechanisms in mixed and pure culture microbial fermentation.

Mixed-culture fermentation is a key central process to enable next generation biofuels and biocommodity production due to economic and process advantages over application of pure cultures. However, a key limitation to the application of mixed-culture fermentation is predicting culture product response, related to metabolic regulation mechanisms. This is also a limitation in pure culture bacterial fermentation. This review evaluates recent literature in both pure and mixed culture studies with a focus on understanding how regulation and signaling mechanisms interact with metabolic routes and activity. In particular, we focus on how microorganisms balance electron sinking while maximizing catabolic energy generation. Analysis of these mechanisms and their effect on metabolism dynamics is absent in current models of mixed-culture fermentation. This limits process prediction and control, which in turn limits industrial application of mixed-culture fermentation. A key mechanism appears to be the role of internal electron mediating cofactors, and related regulatory signaling. This may determine direction of electrons towards either hydrogen or reduced organics as end-products and may form the basis for future mechanistic models.

Controlling mechanisms in directional growth of aggregated archaeal cells.

Members of the family Methanosarcinaceae are important archaeal representatives due to their broad functionality, ubiquitous presence, and functionality in harsh environments. A key characteristic is their multicellular (packet) morphology represented by aggregates of spatially confined cells. This morphology is driven by directed growth of cells in confinement with sequential variation in growth direction. To further understand why spatially confined Methanosarcina cells (and in general, confined prokaryotes) change their direction of growth during consecutive growth-division stages, and how a particular cell senses its wall topology and responds to changes on it a theoretical model for stress dependent growth of aggregated archaeal cells was developed. The model utilizes a confined elastic shell representation of aggregated archaeal cell and is derived based on a work-energy principle. The growth law takes into account the fine structure of archaeal cell wall, polymeric nature of methanochondroitin layer, molecular-biochemical processes and is based on thermodynamic laws. The developed model has been applied to three typical configurations of aggregated cell in 3D. The developed model predicted a geometry response with delayed growth of aggregated archaeal cells explained from mechanistic principles, as well as continuous changes in direction of growth during the consecutive growth-division stages. This means that cell wall topology sensing and growth anisotropy can be predicted using simple cellular mechanisms without the need for dedicated cellular machinery.

Effects of temperature and hydraulic retention time on acetotrophic pathways and performance in high-rate sludge digestion.

High-rate anaerobic digestion of organic solids requires rapid hydrolysis and enhanced methanogenic growth rates, which can be achieved through elevated temperature (>55 °C) at short hydraulic retention times (HRT). This study assesses the effect of temperatures between 55 °C and 65 °C and HRTs between 2 and 4 days on process performance, microbial community structure, microbial capability, and acetotrophic pathways in thermophilic anaerobic reactors. Increasing the temperature did not enhance volatile solids (VS) destruction above the base value of 37% achieved at 55 °C and 4 days HRT. Stable isotopic signatures (δ13C) revealed that elevated temperature promoted syntrophic acetate oxidation, which accounted for 60% of the methane formation at 55 °C, and increasing substantially to 100% at 65 °C. The acetate consumption capacity dropped with increasing temperature (from 0.69-0.81 gCOD gVS(-1) d(-1) at 55 °C to 0.21-0.35 gCOD gVS(-1) d(-1) at 65 °C), based on specific activity testing of reactor contents. Community analysis using 16S rRNA pyrosequencing revealed the dominance of Methanosarcina at 55-60 °C. However, a further increase to 65 °C resulted in loss of Methanosarcina, with an accumulation of organic acids and reduced methane production. Similar issues were observed when reducing the HRT to 2 days, indicating that temperature<60 °C and HRT>3 days are critical to operate these systems stably.

Quadrupling the capacity of post aerobic digestion treating anaerobically digested sludge using a moving-bed biofilm (MBBR) configuration.

Wastewater treatment plants produce large amounts of sludge requiring stabilization before safe disposal. Traditional biological stabilization approaches are cost-effective but generally require either an extended retention time (10-40 days), or elevated temperatures (40-80 °C) for effective pathogens inactivation. This study overcomes these limitations via a novel acidic aerobic digestion process, leveraging an acid-tolerant ammonia-oxidizing bacterium (AOB) Candidatus Nitrosoglobus. To retain this novel but slowly growing AOB, we proposed the first-ever application of a classical wastewater configuration-moving bed biofilm reactor (MBBR)-for sludge treatment. The AOB in biofilm maintains acidic pH and high nitrite levels in sludge, generating free nitrous acid in situ to expedite sludge stabilization. This process was tested in two laboratory-scale aerobic digesters processing full-scale anaerobically digested sludge. At an ambient temperature of 20 °C, pathogens were reduced to levels well below the threshold specified for the highest stabilization level (Class A), within a retention time of 3.5 days. A high volatile solids reduction of 27.4 ± 5.2% was achieved. Through drastically accelerating stabilization and enhancing reduction, this process substantially saves capital and operational costs for sludge disposal.

Evaluating novel biodegradable polymer matrix fertilizers for nitrogen-efficient agriculture.

Enhanced efficiency fertilizers (EEFs) can reduce nitrogen (N) losses in temperate agriculture but are less effective in the tropics. We aimed to design a new EEF and evaluate their performance in simple-to-complex tests with tropical soils and crops. We melt-extruded urea at different loadings into biodegradable polymer matrix composites using biodegradable polyhydroxyalkanoate (PHA) or polybutylene adipate-co-terephthalate (PBAT) polymers with urea distributed throughout the pellet. These contrast with commercially coated EEF that have a polymer-coated urea core. We hypothesized that matrix fertilizers would have an intermediate N release rate compared to fast release from urea or slow release from coated EEF. Nitrogen release rates in water and sand-soil columns confirmed that the matrix fertilizer formulations had a more progressive N release than a coated EEF. A more complex picture emerged from testing sorghum [Sorghum bicolor (L.) Moench] grown to maturity in large soil pots, as the different formulations resulted in minor differences in plant N accumulation and grain production. This confirms the need to consider soil interactions, microbial processes, crop physiology, and phenology for evaluating fertilizer performance. Promisingly, crop δ15N signatures emerged as an integrated measure of efficacy, tracking likely N conversions and losses. The three complementary evaluations combine the advantages of standardized high-throughput screening and more resource-intensive and realistic testing in a plant-soil system. We conclude that melt-blended biodegradable polymer matrix fertilizers show promise as EEF because they can be designed toward more abiotically or more microbially driven N release by selecting biopolymer type and N loading rate.

Dead in the water - Role of relic DNA and primer choice for targeted sequencing surveys of anaerobic sewage sludge intended for biological monitoring.

DNA-based monitoring of microbial communities that are responsible for the performance of anaerobic digestion of sewage wastes has the potential to improve resource recoveries for wastewater treatment facilities. By treating sludge with propidium monoazide (PMA) prior to amplicon sequencing, this study explored how the presence of DNA from dead microbial biomass carried over with feed sludge may mislead process-relevant biomarkers, and whether primer choice impacts such assessments. Four common primers were selected for amplicon preparation, also to determine if universal primers have sufficient taxonomic or functional coverage for monitoring ecological performance; or whether two domain-specific primers for Bacteria and Archaea are necessary. Anaerobic sludges of three municipal continuously stirred-tank reactors in Victoria, Australia, were sampled at one time-point. A total of 240 amplicon libraries were sequenced on a Miseq using two universal and two domain-specific primer pairs. Untargeted metabolomics was chosen to complement biological interpretation of amplicon gene-based functional predictions. Diversity, taxonomy, phylogeny and functional potentials were systematically assessed using PICRUSt2, which can predict community wide pathway abundance. The two chosen universal primers provided similar diversity profiles of abundant Bacteria and Archaea, compared to the domain-specific primers. About 16 % of all detected prokaryotic genera covering 30 % of total abundances and 6 % of PICRUSt2-estimated pathway abundances were affected by PMA. This showed that dead biomass in the anaerobic digesters impacted DNA-based assessments, with implications for predicting active processes, such as methanogenesis, denitrification or the identification of organisms associated with biological foams. Hence, instead of running two sequencing runs with two different domain-specific primers, we propose conducting PMA-seq with universal primer pairs for routine performance monitoring. However, dead sludge biomass may have some predictive value. In principal component analysis the compositional variation of 239 sludge metabolites resembled that of 'dead-plus-alive' biomass, suggesting that dead organisms contributed to the potentially process-relevant sludge metabolome.

MiDAS 5: Global diversity of bacteria and archaea in anaerobic digesters.

Anaerobic digestion of organic waste into methane and carbon dioxide (biogas) is carried out by complex microbial communities. Here, we use full-length 16S rRNA gene sequencing of 285 full-scale anaerobic digesters (ADs) to expand our knowledge about diversity and function of the bacteria and archaea in ADs worldwide. The sequences are processed into full-length 16S rRNA amplicon sequence variants (FL-ASVs) and are used to expand the MiDAS 4 database for bacteria and archaea in wastewater treatment systems, creating MiDAS 5. The expansion of the MiDAS database increases the coverage for bacteria and archaea in ADs worldwide, leading to improved genus- and species-level classification. Using MiDAS 5, we carry out an amplicon-based, global-scale microbial community profiling of the sampled ADs using three common sets of primers targeting different regions of the 16S rRNA gene in bacteria and/or archaea. We reveal how environmental conditions and biogeography shape the AD microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 692 genera and 1013 species. These represent 84-99% and 18-61% of the accumulated read abundance, respectively, across samples depending on the amplicon primers used. Finally, we examine the global diversity of functional groups with known importance for the anaerobic digestion process.

Methanosarcinaceae and acetate-oxidizing pathways dominate in high-rate thermophilic anaerobic digestion of waste-activated sludge.

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH4/kg of VS(added). According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the Methanosarcinaceae, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.

Shearing of biofilms enables selective layer based microbial sampling and analysis.

Granules are large, self-supporting biofilms that form naturally in high-rate anaerobic treatment systems and are extremely important to reactor functionality. Granules exhibit functional and phylogenetic layering, interesting to both scientists and technologists. Until now, it has only been possible to analyze layering through sectioning and microscopic analysis with fluorescent in situ hybridization, or to analyze the whole granule through DNA extraction and microbial community profiling methods. This means different functional and spatial layers cannot be analyzed separately, including next generation sequencing techniques, such as pyrotag sequencing. In this work, we describe a method to remove microbes selectively from successive spatial layers through hydraulic shearing and demonstrate its application on anaerobic granules of three different types (VFA-, carbohydrate-, protein-fed) in size ranges 0.6-2 mm. Outer layers in particular could be selectively sheared as confirmed by FISH. TRFLP was used as an example bulk DNA method on selectively sheared fractions. A shift in dominant population was found from presumptive acidogens (such as Bacteroidetes and Anaerolinea) in outer layers to syntrophs (such as Syntrophomonas and Geobacter) in inner layers, with progressive changes through the depth. The strength of the shear-bulk molecular method over FISH was that a deeper phylogenetic profile could be obtained, even with TRFLP, and that prior knowledge of the community is not required.