Bioreactor Scalability: Laboratory-Scale Bioreactor Design Influences Performance, Ecology, and Community Physiology in Expanded Granular Sludge Bed Bioreactors.
ABSTRACT: Studies investigating the feasibility of new, or improved, biotechnologies, such as wastewater treatment digesters, inevitably start with laboratory-scale trials. However, it is rarely determined whether laboratory-scale results reflect full-scale performance or microbial ecology. The Expanded Granular Sludge Bed (EGSB) bioreactor, which is a high-rate anaerobic digester configuration, was used as a model to address that knowledge gap in this study. Two laboratory-scale idealizations of the EGSB-a one-dimensional and a three- dimensional scale-down of a full-scale design-were built and operated in triplicate under near-identical conditions to a full-scale EGSB. The laboratory-scale bioreactors were seeded using biomass obtained from the full-scale bioreactor, and, spent water from the distillation of whisky from maize was applied as substrate at both scales. Over 70 days, bioreactor performance, microbial ecology, and microbial community physiology were monitored at various depths in the sludge-beds using 16S rRNA gene sequencing (V4 region), specific methanogenic activity (SMA) assays, and a range of physical and chemical monitoring methods. SMA assays indicated dominance of the hydrogenotrophic pathway at full-scale whilst a more balanced activity profile developed during the laboratory-scale trials. At each scale, Methanobacterium was the dominant methanogenic genus present. Bioreactor performance overall was better at laboratory-scale than full-scale. We observed that bioreactor design at laboratory-scale significantly influenced spatial distribution of microbial community physiology and taxonomy in the bioreactor sludge-bed, with 1-D bioreactor types promoting stratification of each. In the 1-D laboratory bioreactors, increased abundance of Firmicutes was associated with both granule position in the sludge bed and increased activity against acetate and ethanol as substrates. We further observed that stratification in the sludge-bed in 1-D laboratory-scale bioreactors was associated with increased richness in the underlying microbial community at species (OTU) level and improved overall performance.
Project description:BACKGROUND: Biological activated sludge process must be functionally stable to continuously remove contaminants while relying upon the activity of complex microbial communities. However the dynamics of these communities are as yet poorly understood. A macroecology metric used to quantify community dynamic is the taxa-time relationship (TTR). Although the TTR of animal and plant species has been well documented, knowledge is still lacking in regard to TTR of microbial communities in activated sludge bioreactors. AIMS: 1) To characterize the temporal dynamics of bacterial taxa in activated sludge from two bioreactors of different scale and investigate factors affecting such dynamics; 2) to evaluate the TTRs of activated sludge microbial communities in two bioreactors of different scale. METHODS: Temporal variation of bacterial taxa in activated sludge collected from a full- and lab-scale activated sludge bioreactor was monitored over a one-year period using pyrosequencing of 16S rRNA genes. TTR was employed to quantify the bacterial taxa shifts based on the power law equation S?=?cTw. RESULTS: The power law exponent w for the full-scale bioreactor was 0.43 (R2?=?0.970), which is lower than that of the lab-scale bioreactor (w?=?0.55, R2?=?0.971). The exponents for the dominant phyla were generally higher than that of the rare phyla. Canonical correspondence analysis (CCA) result showed that the bacterial community variance was significantly associated with water temperature, influent (biochemical oxygen demand) BOD, bioreactor scale and dissolved oxygen (DO). Variance partitioning analyses suggested that wastewater characteristics had the greatest contribution to the bacterial community variance, explaining 20.3% of the variance of bacterial communities independently, followed by operational parameters (19.9%) and bioreactor scale (3.6%). CONCLUSIONS: Results of this study suggest bacterial community dynamics were likely driven partly by wastewater and operational parameters and provide evidence that the TTR may be a fundamental ecological pattern in macro- and microbial systems.
Project description:Membrane bioreactor (MBR) systems are typically known different from conventional activated sludge (CAS) systems in operational parameters, while current knowledge of their microbial differentiations is barely sufficient. To this end, the current study was launched to address the differences of the overall functional genes of an oxidation ditch (OD) and an MBR running parallelly at full-scale using a functional gene array-GeoChip 4.2. Two full-scale wastewater treatment systems applying the processes of oxidation ditch (OD) and membrane bioreactor (MBR) were investigated. They treated identical wastewater at the same scale. 12 mixed-liquor suspended sludge (MLSS) samples collected daily on 12 consecutive days from each system were analyzed by GeoChip 4.2.
Project description:Graphical abstract Highlights • MBR and moving bed-MBR processes were compared in textile wastewater treatment.• Almost equal COD and color removal efficiencies were found in both operations.• Physical and chemical membrane cleaning were required throughout the MBR operation.• No physical or chemical membrane cleaning was required during MB-MBR operation.• Lower polysaccharide and floc sizes, and higher zeta potential were found in MB-MBR. In this study, conventional membrane bioreactor (MBR) and moving bed-membrane bioreactor (MB-MBR) processes were compared in synthetic textile wastewater treatment. For this purpose, the bioreactors were operated as a conventional MBR, an MB-MBR with a biocarrier filling ratio of 20 % and an MB-MBR with a biocarrier filling ratio of 10 %, respectively. In the conventional MBR operation, 93.1 % chemical oxygen demand (COD) and 87.1 % color (Reactive Red 390) removal efficiencies were obtained. In both MB-MBR operations, almost equal COD and color removal efficiencies were found as 98.5 % and 89.5 %, respectively. Moreover, offline physical and chemical membrane cleaning processes were applied every other day and every 15 days throughout the conventional MBR operation, respectively, while no physical or chemical membrane cleaning was required during both MB-MBR operations. Furthermore, lower polysaccharide concentrations of extracellular polymeric substances (EPS) and floc sizes of sludge and higher zeta potential of sludge were determined in MB-MBR. Considering the obtained results, it may be stated that the MB-MBR process is an attractive treatment technology for reducing membrane fouling propensity for treatment of textile wastewater.
Project description:The massive production and improper disposal of organohalides resulted in worldwide contamination in soil and water. However, their environmental survey based on chromatographic methods was hindered by challenges in testing the extremely wide variety of organohalides. Dehalococcoides as obligate organohalide-respiring bacteria exclusively use organohalides as electron acceptors to support their growth, of which the presence could be coupled with organohalides and, therefore, could be employed as a biomarker of the organohalide pollution. In this study, Dehalococcoides was screened in various samples of bioreactors and subsurface environments, showing the wide distribution of Dehalococcoides in sludge and sediment. Further laboratory cultivation confirmed the dechlorination activities of those Dehalococcoides. Among those samples, Dehalococcoides accounting for 1.8% of the total microbial community was found in an anaerobic granular sludge sample collected from a full-scale bioreactor treating petroleum wastewater. Experimental evidence suggested that the influent wastewater in the bioreactor contained bromomethane which support the growth of Dehalococcoides. This study demonstrated that Dehalococcoides could be employed as a promising biomarker to test the present of organohalides in wastestreams or other environmental samples.
Project description:Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO2- entering the reactor from an upstream trickling filter (Wells et al 2009). Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO2- production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct ‘Nitrosomonas-like’ lineage dominated in activated sludge. Prior time series indicated that this ‘Nitrosomonas-like’ lineage was dominant when NO2- levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO2- levels were high. This is consistent with the hypothesis that NO2- production may co-occur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors. Overall design: Samples described in this study were obtained at the Palo Alto Regional Water Quality Control Plant (PARWQCP) in Palo Alto, CA on 12 November 2008. Sampling along a transect within the PARWQCP occurred on 12 November 2008. Upon temporary shutdown of the rotating distributor arm in one of the trickling filters, a surface PVC media module (0.9 m deep) was removed from the reactor, and 1-3 g biofilm samples were obtained with a sterile spatula, placed in sterile 1.5 ml tubes, weighed, and stored at -20oC. Concurrently, samples of influent, trickling filter effluent, and activated sludge were obtained. Activated sludge samples represented a 24-h composite from the combined outlet of all four aeration basins using a Sigma 900 refrigerated automatic sampler (Hach, Loveland, CO). Eight 1.5 ml activated sludge samples were centrifuged onsite for 5 min at 5000 g, decanted, and stored at -20oC. Raw influent and trickling filter effluent samples were transported on ice to the laboratory, vacuum filtered in 10 ml aliquots onto 0.22 μm Hydrophilic Durapore PVDF filters (25 mm diameter, Millipore, Billerica, MA), and archived at -20oC. PhyloChip analyses were performed on biological triplicate samples from trickling filter biofilm, trickling filter effluent, and activated sludge, and biological duplicate samples (due to biomass limitations) from the plant influent.
Project description:Methanogenic sludge granules are densely packed, small, spherical biofilms found in anaerobic digesters used to treat industrial wastewaters, where they underpin efficient organic waste conversion and biogas production. Each granule theoretically houses representative microorganisms from all of the trophic groups implicated in the successive and interdependent reactions of the anaerobic digestion (AD) process. Information on exactly how methanogenic granules develop, and their eventual fate will be important for precision management of environmental biotechnologies. Granules from a full-scale bioreactor were size-separated into small (0.6-1 mm), medium (1-1.4 mm), and large (1.4-1.8 mm) size fractions. Twelve laboratory-scale bioreactors were operated using either small, medium, or large granules, or unfractionated sludge. After >50 days of operation, the granule size distribution in each of the small, medium, and large bioreactor sets had diversified beyond-to both bigger and smaller than-the size fraction used for inoculation. Interestingly, extra-small (XS; <0.6 mm) granules were observed, and retained in all of the bioreactors, suggesting the continuous nature of granulation, and/or the breakage of larger granules into XS bits. Moreover, evidence suggested that even granules with small diameters could break. "New" granules from each emerging size were analyzed by studying community structure based on high-throughput 16S rRNA gene sequencing. Methanobacterium, Aminobacterium, Propionibacteriaceae, and Desulfovibrio represented the majority of the community in new granules. H2-using, and not acetoclastic, methanogens appeared more important, and were associated with abundant syntrophic bacteria. Multivariate integration (MINT) analyses identified distinct discriminant taxa responsible for shaping the microbial communities in different-sized granules.
Project description:Seasonal community structure and regionally synchronous population dynamics have been observed in natural microbial ecosystems, but have not been well documented in wastewater treatment bioreactors. Few studies of community dynamics in full-scale activated sludge systems facing similar meteorological conditions have been done to compare the importance of deterministic and neutral community assembly mechanisms. We subjected weekly activated sludge samples from six regional full-scale bioreactors at four wastewater treatment plants obtained over 1 year to Illumina sequencing of 16S ribosomal RNA genes, resulting in a library of over 17 million sequences. All samples derived from reactors treating primarily municipal wastewater. Despite variation in operational characteristics and location, communities displayed temporal synchrony at the individual operational taxonomic unit (OTU), broad phylogenetic affiliation and community-wide scale. Bioreactor communities were dominated by 134 abundant and highly regionally synchronized OTU populations that accounted for over 50% of the total reads. Non-core OTUs displayed abundance-dependent population synchrony. Alpha diversity varied by reactor, but showed a highly reproducible and synchronous seasonal fluctuation. Community similarity was dominated by seasonal changes, but individual reactors maintained minor stable differences after 1 year. Finally, the impacts of mass migration driven by direct biomass transfers between reactors was investigated, but had no significant effect on community similarity or diversity in the sink community. Our results show that population dynamics in activated sludge bioreactors are consistent with niche-driven assembly guided by seasonal temperature fluctuations.
Project description:BACKGROUND:Anaerobic ammonium oxidation (anammox) is a biological process employed to remove reactive nitrogen from wastewater. While a substantial body of literature describes the performance of anammox bioreactors under various operational conditions and perturbations, few studies have resolved the metabolic roles of their core microbial community members. RESULTS:Here, we used metagenomics to study the microbial community of a laboratory-scale anammox bioreactor from inoculation, through a performance destabilization event, to robust steady-state performance. Metabolic analyses revealed that nutrient acquisition from the environment is selected for in the anammox community. Dissimilatory nitrate reduction to ammonium (DNRA) was the primary nitrogen removal pathway that competed with anammox. Increased replication of bacteria capable of DNRA led to the out-competition of anammox bacteria, and the loss of the bioreactor's nitrogen removal capacity. These bacteria were highly associated with the anammox bacterium and considered part of the core microbial community. CONCLUSIONS:Our findings highlight the importance of metabolic interdependencies related to nitrogen- and carbon-cycling within anammox bioreactors and the potentially detrimental effects of bacteria that are otherwise considered core microbial community members.
Project description:The microbial community of a laboratory-scale bioreactor based on the anammox process was investigated by using metagenomic approaches and fluorescent in situ hybridization (FISH). The bioreactor was initially inoculated with activated sludge from the denitrifying bioreactor of a municipal wastewater treatment station. By constantly increasing the ammonium and nitrite load, a microbial community containing the novel species of anammox bacteria "Candidatus Jettenia ecosi" developed in the bioreactor after 5 years when the maximal daily nitrogen removal rate reached 8.5 g/L. Sequencing of the metagenome of anammox granules and the binning of the contigs obtained, allowed a high quality draft genome of the dominant anammox bacterium, "Candidatus Jettenia ecosi" to be assembled. Annotation of the 3.9 Mbp long genome revealed 3970 putative protein-coding genes, 45 tRNA genes, and genes for 16S/23S rRNAs. Analysis of the genome of "Candidatus Jettenia ecosi" revealed genes involved in anammox metabolism, including nitrite and ammonium transporters, copper-containing nitrite reductase, a nitrate reductase complex, hydrazine synthase, and hydrazine dehydrogenase. Autotrophic carbon fixation could be accomplished through the Wood Ljungdahl pathway. The composition of the community was investigated through a search of 16S rRNA sequences in the metagenome and FISH analysis of the anammox granules. The presence of the members of Ignavibacteriae, Betaproteobacteria, Chloroflexi and other microbial lineages reflected the complexity of the microbial processes in the studied bioreactor performed by anammox Planctomycetes, fermentative bacteria, and denitrifiers.
Project description:Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500?m<sup>2</sup>, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67-4?m<sup>2</sup>) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.