Anaerobic digestion is the most successful bioenergy technology worldwide with, at its core, undefined microbial communities that have poorly understood dynamics. Here, we investigated the relationships of bacterial community structure (>400,000 16S rRNA gene sequences for 112 samples) with function (i.e., bioreactor performance) and environment (i.e., operating conditions) in a yearlong monthly time series of nine full-scale bioreactor facilities treating brewery wastewater (>20,000 measurements). Each of the nine facilities had a unique community structure with an unprecedented level of stability. Using machine learning, we identified a small subset of operational taxonomic units (OTUs; 145 out of 4,962), which predicted the location of the facility of origin for almost every sample (96.4% accuracy). Of these 145 OTUs, syntrophic bacteria were systematically overrepresented, demonstrating that syntrophs rebounded following disturbances. This indicates that resilience, rather than dynamic competition, played an important role in maintaining the necessary syntrophic populations. In addition, we explained the observed phylogenetic differences between all samples on the basis of a subset of environmental gradients (using constrained ordination) and found stronger relationships between community structure and its function rather than its environment. These relationships were strongest for two performance variables-methanogenic activity and substrate removal efficiency-both of which were also affected by microbial ecology because these variables were correlated with community evenness (at any given time) and variability in phylogenetic structure (over time), respectively. Thus, we quantified relationships between community structure and function, which opens the door to engineer communities with superior functions.T he production of bioenergy from wastes is an essential component in the global development of sustainable energy sources (1). Anaerobic digestion, which is the most prominent bioenergy technology worldwide, uses undefined microbial cultures to produce methane from organic substrates (2). Methanogenic bioreactors are maintained on the basis of decades of observed relationships between performance and operating parameters. However, differences underlying bioreactors that perform well and bioreactors that perform inadequately are often poorly understood (3). This has led to a general perception that methanogenic bioreactors are unreliable or unstable, inhibiting their wider adoption for bioenergy production (2). A deeper analysis of the structure and dynamics of bioreactor microbial communities as a function of performance and operating conditions has the potential to reveal important and unappreciated structure-function relationships.The efficient and stable operation of methanogenic bioreactors relies on syntrophic relationships among a community of microbes, including fermenting bacteria, specialized acidogenic and acetogenic syntrophs, and methanogenic archaea (4), with diverse and parallel pathways for...

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We determined the effect of different mixing intensities on the performance, methanogenic population dynamics, and juxtaposition of syntrophic microbes in anaerobic digesters treating cow manure from a dairy farm. Computer automated radioactive particle tracking in conjunction with computational fluid dynamics was performed to quantify the shear levels locally. Four continuously stirred anaerobic digesters were operated at different mixing intensities of 1,500, 500, 250, and 50 revolutions per min (RPM) over a 260-day period at a temperature of 34 +/- 1 degrees C. Animal manure at a volatile solids (VS) concentration of 50 g/L was fed into the digesters daily at five different organic loading rates between 0.6 and 3.5 g VS/L day. The different mixing intensities had no effect on the biogas production rates and yields at steady-state conditions. A methane yield of 0.241 +/- 0.007 L CH(4)/g VS fed was obtained by pooling the data of all four digesters during steady-state periods. However, digester performance was affected negatively by mixing intensity during startup of the digesters, with lower biogas production rates and higher volatile fatty acids concentrations observed for the 1,500-RPM digester. Despite similar methane production yields and rates, the acetoclastic methanogenic populations were different for the high- and low-intensity mixed digesters with Methanosarcina spp. and Methanosaeta concilii as the predominant methanogens, respectively. For all four digesters, epifluorescence microscopy revealed decreasing microbial floc sizes beginning at week 4 and continuing through week 26 after which no microbial flocs remained. This decrease in size, and subsequent loss of microbial flocs did not, however, produce any long-term upsets in digester performance.

g Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetatefed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.

The use of packed-bed reactors for biohydrogen production often results in performance losses in the short term because of the negative effects of operational factors, such as biomass accumulation and inadequate pH conditions. Because packed-bed systems constitute a promising technology for biohydrogen production, studies on continuous hydrogen production over the long term must be carefully conducted by applying proper operational strategies. Therefore, this study assessed continuous biohydrogen production in a packed-bed reactor operated under thermophilic conditions (55 C) using sugarcane stillage as the substrate. The results indicated that the acidogenic reactor presented a capacity for recovering from performance losses, regardless of their cause, and maintaining continuous hydrogen production rates under long-term operation (240 days). pH proved to be a key factor for obtaining continuous hydrogen production, and the optimal results were observed in a pH range from 5.1 to 5.2. Furthermore, an optimal specific organic loading rate of 6.3e6.4 g carbohydrates g À1 volatile suspended solids d À1 was observed, and this value is consistent with the results of previous studies focused on hydrogen production from fermentative systems.

h i g h l i g h t s An innovative fixed-film anaerobic reactor was applied to sugarcane vinasse. Stable operation was observed for OLRs as high as 30 kg COD m À3 day À1. Propionate buildup did not impact the stability of the structured-bed reactor. Enhanced bioenergy recovery was estimated from biodigestion with phase separation. Energy extraction was over 20% higher compared to single-phase systems.

Conventional studies of the optimum growth conditions for methanogens (methane-producing, obligate anaerobic archaea) are typically conducted with serum bottles or bioreactors. The use of microfluidics to culture methanogens allows direct microscopic observations of the time-integrated response of growth. Here, we developed a microbioreactor (BR) with ϳ1-l microchannels to study some optimum growth conditions for the methanogen Methanosaeta concilii. The BR is contained in an anaerobic chamber specifically designed to place it directly onto an inverted light microscope stage while maintaining a N 2 -CO 2 environment. The methanogen was cultured for months inside microchannels of different widths. Channel width was manipulated to create various fluid velocities, allowing the direct study of the behavior and responses of M. concilii to various shear stresses and revealing an optimum shear level of ϳ20 to 35 Pa. Gradients in a single microchannel were then used to find an optimum pH level of 7.6 and an optimum total NH 4 -N concentration of less than 1,100 mg/liter (<47 mg/liter as free NH 3 -N) for M. concilii under conditions of the previously determined ideal shear stress and pH and at a temperature of 35°C.Microfluidic networks have recently gained importance for their wide variety of microbial applications. For example, microchannels were used by DiLuzio et al. (11) to examine the swimming behavior of Escherichia coli. DiLuzio et al. showed that E. coli sensed the presence of channel walls at distances of up to 10 m. Balagaddé et al. (5) built a microfluidic bioreactor containing a feedback control loop, which was able to correlate sustained oscillation in the cellular density of planktonic E. coli with morphological changes. The microfluidic networks used in these studies allowed researchers to directly observe the responses of microbial cells to various stimuli and provided new and unique insights into the growth and behavior of these cells. The use of microfluidics has become practicable because of the development of an inexpensive, biocompatible, and transparent but readily diffusive polymeric material (i.e., polydimethylsiloxane [PDMS]), which is used to construct micron-scale fluid networks in virtually any two-dimensional configuration (12, 23). Due to the extensive gas permeability of PDMS and the elevated cost of nondiffusive materials to construct microchannels, the application of microfluidics to the study of the growth and behavior of anaerobic microorganisms has been hindered.Conventional studies of the behavior of anaerobes, their responses to various stimuli, and their attachment have been performed with medium bottles or bioreactors ranging anywhere from several milliliters to several liters in size (2, 3, 6, 24). These systems serve to provide the anaerobic conditions necessary for growth. They do not, however, allow for any type of direct observation (without sampling disturbance) of microbe behavior, morphology, or the ability to attach to a matrix during growth. By utilizing microfluidics, ...

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