Roadside vegetated filter strips to simultaneously lower stormwater pollution loadings and improve economics of biorefinery feedstocks.

Roadside vegetated filters strips (VFSs) reduce roadway runoff pollution by intercepting stormwater and reducing pollutant loads. VFS maintenance and operating costs can be reduced by designing the VFSs to serve as sites for production of marketable biomass. This biomass can provide feedstock for the emerging bioeconomy producing renewable fuels and biobased chemicals and products. Economic evaluation is needed to quantify the benefit of combining VFS with bioenergy biomass production. This evaluation requires a place-based approach to quantify availability of land, transportation costs, and benefits to sensitive habitats. We evaluated roadside land, within the state right-of-way, in Western Washington, to determine the total area available for implementing VFSs. These data were then used to estimate the volume and cost, of biomass produced on the filter strips, and the resultant reduction in pollutants emitted through highway runoff. The analysis showed that up to 5600 ha were available for roadside VFSs that would be within transportation distance of the theoretical biorefinery location. This space could produce up to 97 dry Gg per year of poplar biomass. The resulting reduction in biorefinery feedstock cost was up to $24 per dry Mg compared to biomass from dedicated tree farms. The results showed that combining roadside poplar with traditional dedicated poplar feedstocks can reduce the feedstock cost of the biorefinery from $76 to $67 per Mg for a biorefinery processing 150 Gg biomass per year. Environmental impact analysis showed that within the study area half of urban roadways and one-third of rural roadways in highly sensitive aquatic areas were amenable to VFS. Construction of VFS in these amenable areas would reduce total loadings to sensitive aquatic areas in urban areas by 26% for TSS, copper, and zinc, and by 10% for phosphorus, and nitrogen and by 21% for lead. The impact for rural sensitive areas was even greater where the VFS had potential to reduce total loadings to sensitive aquatic areas by 38% for TSS, copper, and zinc, by 15% for phosphorus and nitrogen, and by 31% for lead. This research showed an approach combining geographic information system (GIS) mapping and economic analysis to document simultaneous evaluation of cost and environmental benefits when considering use of non-traditional land for bioenergy crop production.

Novel ethanol production using biomass preprocessing to increase ethanol yield and reduce overall costs.

BACKGROUND: Ethanol biorefineries need to lower their overall production costs to become economically feasible. Two strategies to achieve this are to reduce costs using cheaper feedstocks or to increase the ethanol production yield. Low-cost feedstocks usually have high non-structural components (NSC) content; therefore, a new process is necessary to accommodate these feedstocks and overcome the negative effects of NSC. This study developed a novel ethanol biorefinery process including a biomass preprocessing step that enabled the use of lower-cost feedstocks while improving ethanol production without detoxification (overliming). Two types of poplar feedstocks were used, low-quality whole-tree chips (WTC) and high-quality clean pulp chips (CPC), to determine if the proposed process is effective while using feedstocks with different NSC contents. RESULTS: Technical assessment showed that acidic preprocessing increased the monomeric sugar recovery of WTC from 73.2% (untreated) to 87.5% due to reduced buffering capacity of poplar, improved sugar solubilization during pretreatment, and better enzymatic hydrolysis conversion. Preprocessing alone significantly improved the fermentability of the liquid fraction from 1-2% to 49-56% for both feedstocks while overliming improved it to 45%. Consequently, it was proposed that preprocessing can substitute for the detoxification step. The economic assessment revealed that using poplar WTC via the new process increased annual ethanol production of 10.5 million liters when compared to using CPC via overliming (base case scenario). Also, savings in total operating costs were about $10 million per year when using cheaper poplar WTC instead of CPC, and using recycled water for preprocessing lowered its total operating costs by 45-fold. CONCLUSIONS: The novel process developed in this study was successful in increasing ethanol production while decreasing overall costs, thus facilitating the feasibility of lignocellulosic ethanol biorefineries. Key factors to achieving this outcome included substituting overliming by preprocessing, enabling the use of lower-quality feedstock, increasing monomeric sugar recovery and ethanol fermentation yield, and using recycled water for preprocessing. In addition, preprocessing enabled the implementation of an evaporator-combustor downstream design, resulting in a low-loading waste stream that can be treated in a wastewater treatment plant with a simple configuration.

Integration of wastewater treatment into process design of lignocellulosic biorefineries for improved economic viability.

BACKGROUND: Production and use of bio-based products offer advantages over conventional petrochemicals, yet the relatively high cost of production has restricted their mainstream adoption. Optimization of wastewater treatment processes could reduce capital expenditures, lowering the barrier to market entry for lignocellulosic biorefineries. This paper characterizes wastewater associated with lignocellulosic ethanol production and evaluates potential wastewater treatment operations. RESULTS: It is found that organic material is intrinsic to bioconversion wastewater, representing up to 260 kg of biological oxygen demand per tonne of feedstock processed. Inorganics in the wastewater largely originate from additions during pretreatment and pH adjustments, which increase the inorganic loading by 44 kg per tonne of feedstock processed. Adjusting the ethanol production process to decrease addition of inorganic material could reduce the demands and therefore cost of waste treatment. Various waste treatment technologies-including those that take advantage of ecosystem services provided by feedstock production-were compared in terms of capital and operating costs, as well as technical feasibility. CONCLUSIONS: It is concluded that wastewater treatment technologies should be better integrated with conversion process design and feedstock production. Efforts to recycle resources throughout the biofuel supply chain through application of ecosystem services provided by adjacent feedstock plantations and recovery of resources from the waste stream to reduce overall capital and operating costs of bioconversion facilities.

Enhanced Biological Trace Organic Contaminant Removal: A Lab-Scale Demonstration with Bisphenol A-Degrading Bacteria Sphingobium sp. BiD32.

Discharge of trace organic contaminants (TOrCs) from wastewater treatment plants (WWTPs) may contribute to deleterious effects on aquatic life. Release to the environment occurs both through WWTP effluent discharge and runoff following land applications of biosolids. This study introduces Enhanced Biological TOrC Removal (EBTCR), which involves continuous bioaugmentation of TOrC-degrading bacteria for improved removal in WWTPs. Influence of bioaugmentation on enhanced degradation was investigated in two lab-scale sequencing batch reactors (SBRs), using bisphenol A (BPA) as the TOrC. The reactors were operated with 8 cycles per day and at two solids retention times (SRTs). Once each day, the test reactor was bioaugmented with Sphingobium sp. BiD32, a documented BPA-degrading culture. After bioaugmentation, BPA degradation (including both the dissolved and sorbed fractions) was 2-4 times higher in the test reactor than in a control reactor. Improved removal persisted for >5 cycles following bioaugmentation. By the last cycle of the day, enhanced BPA removal was lost, although it returned with the next bioaugmentation. A net loss of Sphingobium sp. BiD32 was observed in the reactors, supporting the original hypothesis that continuous bioaugmentation (rather than single-dose bioaugmentation) would be required to improve TOrCs removal during wastewater treatment. This study represents a first demonstration of a biologically based approach for enhanced TOrCs removal that both reduces concentrations in wastewater effluent and prevents transfer to biosolids.

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.

Identification of Putative Genes Involved in Bisphenol A Degradation Using Differential Protein Abundance Analysis of Sphingobium sp. BiD32.

Discharge of the endocrine disrupting compound bisphenol A (BPA) with wastewater treatment plant (WWTP) effluents into surface waters results in deleterious effects on aquatic life. Sphingobium sp. BiD32 was previously isolated from activated sludge based on its ability to degrade BPA. This study investigated BPA metabolism by Sphingobium sp. BiD32 using label-free quantitative proteomics. The genome of Sphingobium sp. BiD32 was sequenced to provide a species-specific platform for optimal protein identification. The bacterial proteomes of Sphingobium sp. BiD32 in the presence and absence of BPA were identified and quantified. A total of 2155 proteins were identified; 1174 of these proteins were quantified, and 184 of these proteins had a statistically significant change in abundance in response to the presence/absence of BPA (p ≤ 0.05). Proteins encoded by genes previously identified to be responsible for protocatechuate degradation were upregulated in the presence of BPA. The analysis of the metabolites from BPA degradation by Sphingobium sp. BiD32 detected a hydroxylated metabolite. A novel p-hydroxybenzoate hydroxylase enzyme detected by proteomics was implicated in the metabolic pathway associated with the detected metabolite. This enzyme is hypothesized to be involved in BPA degradation by Sphingobium sp. BiD32, and may serve as a future genetic marker for BPA degradation.

Monitoring the dynamics of syntrophic ß-oxidizing bacteria during anaerobic degradation of oleic acid by quantitative PCR.

The ecophysiology of long-chain fatty acid-degrading syntrophic ß-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic ß-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic ß-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic ß-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid ß-oxidization potential of anaerobic digester communities.

Kinetics modeling predicts bioaugmentation with Sphingomonad cultures as a viable technology for enhanced pharmaceutical and personal care products removal during wastewater treatment.

Pharmaceutical and personal care products (PPCPs) discharged with wastewater treatment effluents are a surface water quality concern. PPCPs are partially removed during wastewater treatment and biological transformation is an important removal mechanism. To investigate the potential for enhanced PPCP removal using bioaugmentation, bacteria were previously isolated from activated sludge capable of degrading PPCPs to ng/L concentrations. This study examined the degradation kinetics of triclosan and bisphenol A by five of these bacteria, both in pure culture and when augmented to activated sludge. Sorption coefficients were determined to account for the influence of partitioning during bioremoval. When the bacteria were added to activated sludge, degradation increased. Experimentally determined kinetic parameters were used to model a full-scale continuous treatment process, showing that low biomass could achieve reduced effluent PPCP concentrations. These results demonstrated that bioaugmentation may improve PPCP removal using established wastewater infrastructure under conditions of high solids partitioning.

Influence of bioselector processes on 17α-ethinylestradiol biodegradation in activated sludge wastewater treatment systems.

The removal of the potent endocrine-disrupting estrogen hormone, 17α-ethinylestradiol (EE2), in municipal wastewater treatment plant (WWTP) activated sludge (AS) processes can occur through biodegradation by heterotrophic bacteria growing on other organic wastewater substrates. Different kinetic and metabolic substrate utilization conditions created with AS bioselector processes can affect the heterotrophic population composition in AS. The primary goal of this research was to determine if these changes also affect specific EE2 biodegradation kinetics. A series of experiments were conducted with parallel bench-scale AS reactors treating municipal wastewater with estrogens at 100-300 ng/L concentrations to evaluate the effect of bioselector designs on pseudo first-order EE2 biodegradation kinetics normalized to mixed liquor volatile suspended solids (VSS). Kinetic rate coefficient (kb) values for EE2 biodegradation ranged from 5.0 to 18.9 L/g VSS/d at temperatures of 18 °C to 24 °C. EE2 kb values for aerobic biomass growth at low initial food to mass ratio feeding conditions (F/Mf) were 1.4 to 2.2 times greater than that from growth at high initial F/Mf. Anoxic/aerobic and anaerobic/aerobic metabolic bioselector reactors achieving biological nutrient removal had similar EE2 kb values, which were lower than that in aerobic AS reactors with biomass growth at low initial F/Mf. These results provide evidence that population selection with growth at low organic substrate concentrations can lead to improved EE2 biodegradation kinetics in AS treatment.

Characterization of persistent virus-like particles in two acetate-fed methanogenic reactors.

The objective of this study was to characterize the morphology, size-distribution, concentration and genome size of virus-like particles (VLPs) in two acetate-fed Methanosaeta-dominated reactors to better understand the possible correlation between viruses and archaeal hosts. The study reactors were dominated by a single genus of acetoclastic methanogen, Methanosaeta, which was present at 6 to 13 times higher than the combined bacterial populations consisting of Proteobacteria, Firmicutes, and Bacteroidetes. Epifluorescent microscopy showed VLPs concentration of 7.1 ± 1.5 × 10(7) VLPs/ml and 8.4 ± 4.3 × 10(7) VLPs/ml in the two laboratory reactors. Observations of no detectable import of VLPs with the reactor feed combined long operational time since the last inocula were introduced suggests that the VLP populations were actively propagating in the reactors. Transmission electron microscopy images showed VLPs with morphology consistent with Siphoviridae in both reactors, and VLPs with morphologies consistent with Myoviridae in one of the reactors. The morphology, size-distribution and genome size of VLPs were distinct between reactors suggesting that unique viral populations inhabited each reactor, though the hosts of these VLPs remain unclear.

Functional gene array-based analysis of microbial communities in heavy metals-contaminated lake sediments.

Lake DePue (IL, USA) has been contaminated for > 80 years by an adjacent Zn-smelting facility. Previous work indicated that sulfate reduction increased and biomass declined as pore-water metal concentrations increased, while 16S rRNA gene profiles remained relatively stable. To better understand this phenomenon, the sediment microbial community structure and functional potential were investigated using a functional gene microarray (GeoChip) targeting > 10,000 functional genes. Nonmetric multidimensional scaling and clustering analyses showed that the overall community structure was similar across all sites based on the relative abundance of all detected genes, but some individual gene categories did show differences. A subset of sulfate reduction genes (dsr) and the most relevant metal resistance genes were more abundant than other categories and were highly correlated with metal contamination. The most significant correlations were between pore-water metal concentrations and dsr, with Zn, Cd, and Mn as the most predictive for the presence of dsr. These results suggest that metal contamination influences sediment microbial community structure and function by increasing the abundance of relevant metal-resistant and sulfate-reducing populations. These populations therefore appear to contribute significantly to the resistance and stability of the microbial communities throughout the gradient of metal contamination in Lake DePue.

Cultivation and characterization of bacterial isolates capable of degrading pharmaceutical and personal care products for improved removal in activated sludge wastewater treatment.

Pharmaceutical and personal care products (PPCPs) discharged with wastewater treatment plant (WWTP) effluents are an emerging surface water quality concern. Biological transformation has been identified as an important removal mechanism during wastewater treatment. The aim of this research was the identification of bacteria with characteristics for potential bioaugmentation to enhance PPCP removal. We report here the cultivation and characterization of bacteria capable of degrading PPCPs to ng/L concentrations. An isolation approach was developed using serial enrichment in mineral medium containing 1 mg/L of an individual PPCP as the sole organic carbon source available to heterotrophs until the original activated sludge inocula was diluted to ~10(-8) of its initial concentration, followed by colony growth on solid R2A agar. Eleven bacteria were isolated, eight that could remove triclosan, bisphenol A, ibuprofen, or 17ß-estradiol to below 10 ng/L, one that could remove gemfibrozil to below 60 ng/L, and two that could remove triclosan or E2, but not to ng/L concentrations. Most bacterial isolates degraded contaminants during early growth when grown utilizing rich carbon sources and were only able to degrade the PPCPs on which they were isolated. Seven of the bacterial isolates were sphingomonads, including all the triclosan and bisphenol A degraders and the ibuprofen degrader. The study results indicate that the isolated bacteria may have a positive influence on removal in WWTPs if present at sufficient concentrations and may be useful for bioaugmentation.

Enhanced methane generation during theromophilic co-digestion of confectionary waste and grease-trap fats and oils with municipal wastewater sludge.

Recent interest in carbon-neutral biofuels has revived interest in co-digestion for methane generation. At wastewater treatment facilities, organic wastes may be co-digested with sludge using established anaerobic digesters. However, changes to organic loadings may induce digester instability, particularly for thermophilic digesters. To examine this problem, thermophilic (55 degrees C) co-digestion was studied for two food-industry wastes in semi-continuous laboratory digesters; in addition, the wastes' biochemical methane potentials were tested. Wastes with high chemical oxygen demand (COD) content were selected as feedstocks allowing increased input of potential energy to reactors without substantially altering volumetric loadings. Methane generation increased while reactor pH and volatile solids remained stable. Lag periods observed prior to methane stimulation suggested that acclimation of the microbial community may be critical to performance during co-digestion. Chemical oxygen demand mass balances in the experimental and control reactors indicated that all of the food industry waste COD was converted to methane.

Microbial community structures in anoxic freshwater lake sediment along a metal contamination gradient.

Contamination, such as by heavy metals, has frequently been implicated in altering microbial community structure. However, this association has not been extensively studied for anaerobic communities, or in freshwater lake sediments. We investigated microbial community structure in the metal-contaminated anoxic sediments of a eutrophic lake that were impacted over the course of 80 years by nearby zinc-smelting activities. Microbial community structure was inferred for bacterial, archaeal and eukaryotic populations by evaluating terminal restriction fragment length polymorphism (TRFLP) patterns in near-surface sediments collected in triplicate from five areas of the lake that had differing levels of metal contamination. The majority of the fragments in the bacterial and eukaryotic profiles showed no evidence of variation in association with metal contamination levels, and diversity revealed by these profiles remained consistent even as metal concentrations varied from 3000 to 27,000 mg kg(-1) total Zn, 0.125 to 11.2 µ pore water Zn and 0.023 to 5.40 µM pore water As. Although most archaeal fragments also showed no evidence of variation, the prevalence of a fragment associated with mesophilic Crenarchaeota showed significant positive correlation with total Zn concentrations. This Crenarchaeota fragment dominated the archaeal TRFLP profiles, representing between 35% and 79% of the total measured peak areas. Lake DePue 16S rRNA gene sequences corresponding to this TRFLP fragment clustered with anaerobic and soil mesophilic Crenarchaeota sequences. Although Crenarchaeota have been associated with metal-contaminated groundwater and soils, this is a first report (to our knowledge) documenting potential increased prevalence of Crenarchaeota associated with elevated levels of metal contamination.

Effect of anoxic selector configuration on sludge volume index control and bacterial population fingerprinting.

The effect of single-stage and 4-stage anoxic selectors and an anoxic/aerobic sequencing batch reactor (SBR) on the removal of readily biodegradable chemical oxygen demand (rbCOD) and slowly biodegradable COD (sbCOD) and on filamentous growth and sludge settleability was studied. Microbial community fingerprinting in the three selector configurations was done using automated ribosomal intergenic spacer analysis. Nostocoida limicola II and type 1851 filamentous bacteria were observed in all systems. The diluted sludge volume index (DSVI) decreased with increased selector staging. The rbCOD was almost completely removed in all selectors, and the lower DSVI values with increased selector staging were the result of greater sbCOD removal in the selector. The plug-flow kinetics in the SBR aerobic phase also improved DSVI. The bacteria community composition in the 4-stage selector system was found to be more similar to that for the SBR than for the single-stage selector.

Linkage of high rates of sulfate reduction in Yellowstone hot springs to unique sequence types in the dissimilatory sulfate respiration pathway.

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89 degrees C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.

Optimization of direct cell counting in sediment.

This study reports a method for optimizing direct counts of bacteria in sediment, designed to reduce the masking by sediment particles. The protocol was designed to determine appropriate dilution factors by incorporating counting statistics and was used to measure depth-associated changes in microbial abundance in metal-impacted freshwater sediments. We demonstrated a direct method to determine appropriate sample dilution for accurate counting by adding a known amount of cells to the sediment. For accurate counting in our sediment samples, we determined that the average number of bacteria per microscope ocular field must be between 8.5 and 10. This is well below the 30 bacteria/field previously suggested for accurate counting. These results indicate that an optimal dilution rate must be determined before accurate direct counts in sediment can be achieved.