Electricity generation from cysteine in a microbial fuel cell

In a microbial fuel cell (MFC), power can be generated from the oxidation of organic matter by bacteria at the anode, with reduction of oxygen at the cathode. Proton exchange membranes used in MFCs are permeable to oxygen, resulting in the diffusion of oxygen into the anode chamber. This could either lower power generation by obligate anaerobes or result in the loss in electron donor from aerobic respiration by facultative or other aerobic bacteria. In order to maintain anaerobic conditions in conventional anaerobic laboratory cultures, chemical oxygen scavengers such as cysteine are commonly used. It is shown here that cysteine can serve as a substrate for electricity generation by bacteria in a MFC. A two-chamber MFC containing a proton exchange membrane was inoculated with an anaerobic marine sediment. Over a period of a few weeks, electricity generation gradually increased to a maximum power density of 19 mW/m(2) (700 or 1000 Omega resistor; 385 mg/L of cysteine). Power output increased to 39 mW/m(2) when cysteine concentrations were increased up to 770 mg/L (493 Omega resistor). The use of a more active cathode with Pt- or Pt-Ru, increased the maximum power from 19 to 33 mW/m(2) demonstrating that cathode efficiency limited power generation. Power was always immediately generated upon addition of fresh medium, but initial power levels consistently increased by ca. 30% during the first 24 h. Electron recovery as electricity was 14% based on complete cysteine oxidation, with an additional 14% (28% total) potentially lost to oxygen diffusion through the proton exchange membrane. 16S rRNA-based analysis of the biofilm on the anode of the MFC indicated that the predominant organisms were Shewanella spp. closely related to Shewanella affinis (37% of 16S rRNA gene sequences recovered in clone libraries).     

Electricity generation from swine wastewater using microbial fuel cells

Microbial fuel cells (MFCs) represent a new method for treating animal wastewaters and simultaneously producing electricity. Preliminary tests using a two-chambered MFC with an aqueous cathode indicated that electricity could be generated from swine wastewater containing 8320 +/- 190 mg/L of soluble chemical oxygen demand (SCOD) (maximum power density of 45 mW/m2). More extensive tests with a single-chambered air cathode MFC produced a maximum power density with the animal wastewater of 261 mW/m2 (200 omega resistor), which was 79% larger than that previously obtained with the same system using domestic wastewater (146 +/- 8 mW/m2) due to the higher concentration of organic matter in the swine wastewater. Power generation as a function of substrate concentration was modeled according to saturation kinetics, with a maximum power density of P(max) = 225 mW/m2 (fixed 1000 omega resistor) and half-saturation concentration of K(s) = 1512 mg/L (total COD). Ammonia was removed from 198 +/- 1 to 34 +/- 1 mg/L (83% removal). In order to try to increase power output and overall treatment efficiency, diluted (1:10) wastewater was sonicated and autoclaved. This pretreated wastewater generated 16% more power after treatment (110 +/- 4 mW/m2) than before treatment (96 +/- 4 mW/m2). SCOD removal was increased from 88% to 92% by stirring diluted wastewater, although power output slightly decreased. These results demonstrate that animal wastewaters such as this swine wastewater can be used for power generation in MFCs while at the same time achieving wastewater treatment.     

Hydrogen and electricity production from a food processing wastewater using fermentation and microbial fuel cell technologies

Hydrogen can be produced from fermentation of sugars in wastewaters, but much of the organic matter remains in solution. We demonstrate here that hydrogen production from a food processing wastewater high in sugar can be linked to electricity generation using a microbial fuel cell (MFC) to achieve more effective wastewater treatment. Grab samples were taken from: plant effluent at two different times during the day (Effluents 1 and 2; 735+/-15 and 3250+/-90 mg-COD/L), an equalization tank (Lagoon; 1670+/-50mg-COD/L), and waste stream containing a high concentration of organic matter (Cereal; 8920+/-150 mg-COD/L). Hydrogen production from the Lagoon and effluent samples was low, with 64+/-16 mL of hydrogen per liter of wastewater (mL/L) for Effluent 1, 21+/-18 mL/L for Effluent 2, and 16+/-2 mL/L for the Lagoon sample. There was substantially greater hydrogen production using the Cereal wastewater (210+/-56 mL/L). Assuming a theoretical maximum yield of 4 mol of hydrogen per mol of glucose, hydrogen yields were 0.61-0.79 mol/mol for the Cereal wastewater, and ranged from 1 to 2.52 mol/mol for the other samples. This suggests a strategy for hydrogen recovery from wastewater based on targeting high-COD and high-sugar wastewaters, recognizing that sugar content alone is an insufficient predictor of hydrogen yields. Preliminary tests with the Cereal wastewater (diluted to 595 mg-COD/L) in a two-chambered MFC demonstrated a maximum of 81+/-7 mW/m(2) (normalized to the anode surface area), or 25+/-2 mA per liter of wastewater, and a final COD of <30 mg/L (95% removal). Using a one-chambered MFC and pre-fermented wastewater, the maximum power density was 371+/-10 mW/m(2) (53.5+/-1.4 mA per liter of wastewater). These results suggest that it is feasible to link biological hydrogen production and electricity producing using MFCs in order to achieve both wastewater treatment and bioenergy production.     

Fouling of proton exchange membrane (PEM) deteriorates the performance of microbial fuel cell

The fouling characteristics of proton exchange membrane (PEM) in microbial fuel cell (MFC) and the resulting deterioration of MFC performance were explored in this study. It was observed that the ion exchange capacity, conductivity and diffusion coefficients of cations of PEM were reduced significantly after fouling. Imaging analysis coupled with FTIR analysis indicated that the fouling layer attached on PEM consisted of microorganisms encased in extracellular polymers and inorganic salt precipitations. The results clearly demonstrate that PEM fouling deteriorated the performance of MFCs and led to a decrease in electricity generation. Cation transfer limitation might play an important role in the deterioration of MFC performance because of the membrane fouling. This was attributed to the physical blockage of charge transfer in the MFC resulted from the membrane fouling. With the experimental results, the effect of membrane fouling on the electrical generation of MFCs was evaluated. It was found that the decreased diffusion coefficients of cations and cathodic potential loss after membrane fouling contributed mainly to the deterioration of the MFC performance.     

Efficient electricity generation from sewage sludge using biocathode microbial fuel cell

Microbial fuel cells (MFCs) with abiotic cathodes require expensive catalyst (such as Pt) or catholyte (such as hexacynoferrate) to facilitate oxidation reactions. This study incorporated biocathodes into a three-chamber MFC to yield electricity from sewage sludge at maximum power output of 13.2 ± 1.7 W/m³ during polarization, much higher than those previously reported. After 15 d operation, the total chemical oxygen demand (TCOD) removal and coulombic efficiency (CE) of cell reached 40.8 ± 9.0% and 19.4 ± 4.3%, respectively. The anolyte comprised principally acetate and propionate (minor) as metabolites. The use of biocathodes produced an internal resistance of 36-46 Ω, lower than those reported in literature works, hence yielding higher maximum power density from MFC. The massively parallel sequencing technology, 454 pyrosequencing technique, was adopted to probe microbial community on anode biofilm, with dominant phyla belonging to Proteobacteria (45% of total bacteria), Bacteroidetes (19%), Uncultured bacteria (9%), Actinobacteria (7%), Firmicutes (7%), Chloroflex (7%). At genera level, Rhodoferax, Ferruginibacter, Propionibacterium, Rhodopseudomonas, Ferribacterium, Clostridium, Chlorobaculum, Rhodobacter, Bradyrhizobium were the abundant taxa (relative abundances > 2.0%).     

Nitrogen removal from wastewater using membrane aerated microbial fuel cell techniques

Nitrogen removal mainly relies on sequential nitrification and denitrification in wastewater treatment. Microbial fuel cells (MFCs) are innovative wastewater treatment techniques for pollution control and energy generation. In this study, bench-scale wastewater treatment systems using membrane-aerated MFC (MAMFC) and diffuser-aerated MFC (DAMFC) techniques were constructed for simultaneous removal of carbonaceous and nitrogenous pollutants and electricity production from wastewater. During 210 days of continuous flow operation, when the dissolved oxygen (DO) in the cathodic compartment was kept at 2 mg/L, both reactors demonstrated high COD removal (>99%) and high ammonia removal (>99%) but low nitrogen removal (<20%). When a lower DO (0.5 mg/L) was maintained after day 121, both the MFC-based reactors still had excellent COD removal (>97%). However, the nitrogen removal of MAMFC (52%) was 2-fold higher than that of DAMFC (24%), indicating an enhanced performance of denitrification after DO reduction in the cathodic compartment of the MAMFC. Meanwhile, terminal restriction fragment length polymorphism (T-RFLP) analysis of ammonia-oxidizing bacteria (AOB) population in the MAMFC indicated the diversity of AOB with equally important Nitrosospira and Nitrosomonas species present in the cathodic biofilm after DO reduction. The average voltage output in the MAMFC was significantly higher than that in DAMFC under both DO conditions. The results suggest that MAMFC systems have the potential for wastewater treatment with improved nitrogen removal and electricity production.     

Identifying the microbial communities and operational conditions for optimized wastewater treatment in microbial fuel cells

Microbial fuel cells (MFCs) are devices that exploit microorganisms as “biocatalysts” to recover energy from organic matter in the form of electricity. MFCs have been explored as possible energy neutral wastewater treatment systems; however, fundamental knowledge is still required about how MFC-associated microbial communities are affected by different operational conditions and can be optimized for accelerated wastewater treatment rates. In this study, we explored how electricity-generating microbial biofilms were established at MFC anodes and responded to three different operational conditions during wastewater treatment: 1) MFC operation using a 750 Ω external resistor (0.3 mA current production); 2) set-potential (SP) operation with the anode electrode potentiostatically controlled to +100 mV vs SHE (4.0 mA current production); and 3) open circuit (OC) operation (zero current generation). For all reactors, primary clarifier effluent collected from a municipal wastewater plant was used as the sole carbon and microbial source. Batch operation demonstrated nearly complete organic matter consumption after a residence time of 8–12 days for the MFC condition, 4–6 days for the SP condition, and 15–20 days for the OC condition. These results indicate that higher current generation accelerates organic matter degradation during MFC wastewater treatment. The microbial community analysis was conducted for the three reactors using 16S rRNA gene sequencing. Although the inoculated wastewater was dominated by members of Epsilonproteobacteria, Gammaproteobacteria, and Bacteroidetes species, the electricity-generating biofilms in MFC and SP reactors were dominated by Deltaproteobacteria and Bacteroidetes. Within Deltaproteobacteria, phylotypes classified to family Desulfobulbaceae and Geobacteraceae increased significantly under the SP condition with higher current generation; however those phylotypes were not found in the OC reactor. These analyses suggest that species related to family Desulfobulbaceae and Geobacteraceae are correlated with the electricity generation in the biofilm and may be key players for optimizing wastewater treatment rates and energy recovery in applied MFC systems.     

Simultaneous nitrification, denitrification and carbon removal in microbial fuel cells

Microbial fuel cells (MFCs) can use nitrate as a cathodic electron acceptor, allowing for simultaneous removal of carbon (at the anode) and nitrogen (at the cathode). In this study, we supplemented the cathodic process with in situ nitrification through specific aeration, and thus obtained simultaneous nitrification and denitrification (SND) in the one half-cell. Synthetic wastewater containing acetate and ammonium was supplied to the anode; the effluent was subsequently directed to the cathode. The influence of oxygen levels and carbon/nitrogen concentrations and ratios on the system performances was investigated. Denitrification occurred simultaneously with nitrification at the cathode, producing an effluent with levels of nitrate and ammonium as low as 1.0 ± 0.5 mg N L⁻¹ and 2.13 ± 0.05 mg N L⁻¹, respectively, resulting in a nitrogen removal efficiency of 94.1 ± 0.9%. The integration of the nitrification process into the cathode solves the drawback of ammonium losses due to diffusion between compartments in the MFC, as previously reported in a system operating with external nitrification stage. This work represents the first successful attempt to combine SND and organics oxidation while producing electricity in an MFC.     

Hydrogen production using single-chamber membrane-free microbial electrolysis cells

Microbial electrohydrogenesis provides a new approach for hydrogen generation from renewable biomass. Membranes were used in all the reported microbial electrolysis cells (MECs) to separate the anode and cathode chambers. To reduce the potential losses associated with membrane and increase the energy recovery of this process, single-chamber membrane-free MECs were designed and used to investigate hydrogen production by one mixed culture and one pure culture: Shewanella oneidensis MR-1. At an applied voltage of 0.6V, this system with a mixed culture achieved a hydrogen production rate of 0.53m(3)/day/m(3) (0.11m(3)/day/m(2)) with a current density of 9.3A/m(2) at pH 7 and 0.69m(3)/day/m(3) (0.15m(3)/day/m(2)) with a current density of 14A/m(2) at pH 5.8. Stable hydrogen production from lactic acid by S. oneidensis was also observed. Methane was detected during the hydrogen production process with the mixed culture and negatively affected hydrogen production rate. However, by employing suitable approaches, such as exposure of cathodes to air, the hydrogenotrophic methanogens can be suppressed. The current density and volumetric hydrogen production rate of this system have potential to increase significantly by further reducing the electrode spacing and increasing the ratio of electrode surface area/cell volume.     

Further treatment of decolorization liquid of azo dye coupled with increased power production using microbial fuel cell equipped with an aerobic biocathode

A microbial fuel cell (MFC) incorporating a recently developed aerobic biocathode is designed and demonstrated. The aerobic biocathode MFC is able to further treat the liquid containing decolorization products of active brilliant red X-3B (ABRX3), a respective azo dye, and also provides increased power production. Batch test results showed that 24.8% of COD was removed from the decolorization liquid of ABRX3 (DL) by the biocathode within 12 h. Metabolism-dependent biodegradation of aniline-like compound might be mainly responsible for the decrease of overall COD. Glucose is not necessary in this process and contributes little to the COD removal of the DL. The similar COD removal rate observed under closed circuit condition (500 Ω) and opened circuit condition indicated that the current had an insignificant effect on the degradation of the DL. Addition of the DL to the biocathode resulted in an almost 150% increase in open cycle potential (OCP) of the cathode accompanied by a 73% increase in stable voltage output from 0.33 V to 0.57 V and a 300% increase in maximum power density from 50.74 mW/m² to 213.93 mW/m². Cyclic voltammetry indicated that the decolorization products of the ABRX3 contained in the DL play a role as redox mediator for facilitating electron transfer from the cathode to the oxygen. This study demonstrated for the first time that MFC equipped with an aerobic biocathode can be successfully applied to further treatment of effluent from an anaerobic system used to decolorize azo dye, providing both cost savings and high power output.     

Evaluating short-term changes in recreational water quality during a hydrograph event using a combination of microbial tracers, environmental microbiology, microbial source tracking and hydrological techniques: A case study in Southwest Wales, UK

Quantitative assessment of multiple sources to short-term variations in recreational water quality, as indexed by faecal indicator organism (FIO) concentrations, is becoming increasingly important with adoption of modern water quality standards and catchment-based water quality management requirements (e.g. the EU Water Framework Directive, Article 11 ‘Programmes of Measures’ and the US Clean Water Act, ‘Total Maximum Daily Loads’). This paper describes a study combining microbial tracers, intensive FIO measurement, open channel hydrology and molecular microbial source tracking (MST) to enhance understanding of recreational water quality at Amroth in southwest Wales, UK. Microbial tracers were released from four stream inputs during a moderate hydrograph event. Tracers from two local streams impacted simultaneously with a period of maximum FIO concentrations at the near-shore compliance monitoring site. Connection between these inputs and this site were rapid (9-33 min). Water quality impairment from a more remote stream input followed, 12.85 h after tracer release, sustaining FIO concentrations above desired compliance levels. MST analysis showed dominance of ruminant Bacteroidales genetic markers, associated with agricultural pollution. This integration of tracers and MST offers additional information on the movement and individual sources causing water quality impairment.     

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.     

Optimization and modelling of volatile fatty acid generation in a leachate bed reactor for utilization in microbial fuel cells

Volatile fatty acid (VFA)-rich leachate generated from acidogenesis of kitchen waste in a leach bed reactor (LBR) was utilized in an earthen microbial fuel cell (EMFC) to generate electricity. Effects of organic loading rate (OLR, 5–10 g VS/L·day) and pH (5–7) on LBR enumerated optimized parameters of OLR (10 g VS/L·day) and pH (5.74) to obtain total VFA (TVFA) of 7.7 ± 0.3 g/L in the leachate, with maximum contribution from acetic acid. Leachate obtained from the LBR was fed to the EMFC with varying OLR (2–7 kg COD/m³·day). The highest power density of 0.76 W/m³ (at OLR 7 kg COD/m³·day) was obtained with higher VFA content in the leachate. A neural network based on the Levenberg–Marquard function effectively predicted chemical oxygen demand and TVFA removal. This study establishes LBR as a techno-economic method to obtain useful substrate for EMFC. Furthermore, the response modelling of EMFC demonstrates the potential of utilizing machine learning in biological treatment.     

Efficient electricity production and simultaneously wastewater treatment via a high-performance photocatalytic fuel cell

A great quantity of wastewater were discharged into water body, causing serious environmental pollution. Meanwhile, the organic compounds in wastewater are important sources of energy. In this work, a high-performance short TiO₂ nanotube array (STNA) electrode was applied as photoanode material in a novel photocatalytic fuel cell (PFC) system for electricity production and simultaneously wastewater treatment. The results of current work demonstrate that various model compounds as well as real wastewater samples can be used as substrates for the PFC system. As a representative of model compounds, the acetic acid solution produces the highest cell performance with short-circuit current density 1.42 mA cm⁻², open-circuit voltage 1.48 V and maximum power density output 0.67 mW cm⁻². The STNA photoanode reveals obviously enhanced cell performance compared with TiO₂ nanoparticulate film electrode or other long nanotubes electrode. Moreover, the photoanode material, electrolyte concentration, pH of the initial solution, and cathode material were found to be important factors influencing the system performance of PFC. Therefore, the proposed fuel cell system provides a novel way of energy conversion and effective disposal mode of organics and serves well as a promising technology for wastewater treatment.     

A comparative study on treatment of wastewaters with various biodegradability and various pH values using single‐chamber microbial fuel cells

This work assessed the performance of a single‐chamber microbial fuel cell (MFC) with various substrates. Primary settled domestic wastewaters were used to simulate wastewaters of high biodegradability; while phenol‐based wastewaters and benzene‐based wastewaters were used to simulate wastewaters of low biodegradability. Experiments were performed at initial pH values of 6, 7 and 8. The maximum voltage production, power density and removal of substrate were obtained using primary settled domestic wastewater, whereas the lowest values were obtained using phenol‐based wastewater. The maximum chemical oxygen demand removal efficiency, phenol removal efficiency and benzene removal efficiency were 80.8, 63.3 and 77.8%, respectively. The performance of the MFC was enhanced by increasing the influent pH. The lowest coulombic efficiencies were obtained from phenol‐based wastewater and benzene‐based wastewater, which indicated that electrogenic bacteria were not the primary microorganisms responsible for the biodegradation of low biodegradable wastewater.     

Estimating true human and animal host source contribution in quantitative microbial source tracking using the Monte Carlo method

Cultivation- and library-independent, quantitative PCR-based methods have become the method of choice in microbial source tracking. However, these qPCR assays are not 100% specific and sensitive for the target sequence in their respective hosts’ genome. The factors that can lead to false positive and false negative information in qPCR results are well defined. It is highly desirable to have a way of removing such false information to estimate the true concentration of host-specific genetic markers and help guide the interpretation of environmental monitoring studies. Here we propose a statistical model based on the Law of Total Probability to predict the true concentration of these markers. The distributions of the probabilities of obtaining false information are estimated from representative fecal samples of known origin. Measurement error is derived from the sample precision error of replicated qPCR reactions. Then, the Monte Carlo method is applied to sample from these distributions of probabilities and measurement error. The set of equations given by the Law of Total Probability allows one to calculate the distribution of true concentrations, from which their expected value, confidence interval and other statistical characteristics can be easily evaluated. The output distributions of predicted true concentrations can then be used as input to watershed-wide total maximum daily load determinations, quantitative microbial risk assessment and other environmental models. This model was validated by both statistical simulations and real world samples. It was able to correct the intrinsic false information associated with qPCR assays and output the distribution of true concentrations of Bacteroidales for each animal host group. Model performance was strongly affected by the precision error. It could perform reliably and precisely when the standard deviation of the precision error was small (≤0.1). Further improvement on the precision of sample processing and qPCR reaction would greatly improve the performance of the model. This methodology, built upon Bacteroidales assays, is readily transferable to any other microbial source indicator where a universal assay for fecal sources of that indicator exists.     

Reducing CO2 emission from exhaust gases using molten carbonate fuel cells: a new approach

The aim of this study is to couple molten carbonate fuel cell (MCFC) stack with integrated gasification combined cycle fed by refinery residues, to remove CO₂ from gas turbine exhaust gases that have CO₂ emission rate of 14,200 ton/year. By applying multi-objective optimisation (MOO) using genetic algorithm, the optimal values of operating load and the corresponding values of objective functions are obtained. The MOO of the MCFC system regarding two scenarios is performed. The first scenario is minimisation of cost of electricity (COE) and CO₂ emission rate. Objective functions of the second scenario are the same as in the first scenario while CO₂ tax is taken into account. Results show that the second scenario has 29.5% lower average optimal COE and 2.5% lower average emission rate in comparison with the first scenario. A sensitivity analysis is also performed to study the effect of fuel price and CO₂ tax variations on optimal solutions.     

Method for rapid and sensitive detection of Enterococcus sp. and Enterococcus faecalis/faecium cells in potable water samples

We have developed a rapid and robust technological solution including a membrane filtration and dissolution method followed by a molecular enrichment and a real-time PCR assay, for detecting the presence of Enterococcus sp. or Enterococcus faecalis/faecium per 100 mL of water in less than 5 h and we compared it to Method 1600 on mEI agar in terms of specificity, sensitivity, and limit of detection. The mEI and the Enterococcus sp.-specific assay detected respectively 73 (64.0%) and 114 (100%) of the 114 enterococcal strains tested. None of the 150 non-enterococcal strains tested was detected by both methods with the exception of Tetragenococcus solitarius for the Enterococcus sp. assay. The multiplexed E. faecalis/faecium assay efficiently amplified DNA from 47 of 47 (100%) E. faecalis and 27 of 27 (100%) E. faecium strains tested respectively, whereas none of the 191 non-E. faecalis/faecium strains tested was detected. By simultaneously detecting the predominant fecal enterococcal species, the E. faecalis/faecium-specific assay allows a better distinction between enterococcal strains of fecal origin and those provided by the environment than Method 1600. Our procedure allows the detection of 4.5 enterococcal colony forming units (CFU) per 100 mL in less than 5 h, whereas the mEI method detected 2.3 CFU/100 mL in 24 h (95% confidence). Thus, our innovative and highly effective method provides a rapid and easy approach to concentrate very low numbers of enterococcal cells present in a 100 mL water sample and allows a better distinction between fecal and environmental enterococcal cells than Method 1600.     

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections—augmented with local observations and land-use patterns—indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination.