Bioaugmentation Culture Research Articles

From mec cassette to rdhA: a key Dehalobacter genomic neighborhood in a chloroform and dichloromethane-transforming microbial consortium.

Chloroform (CF) and dichloromethane (DCM) are regulated groundwater contaminants. A cost-effective approach to remove these pollutants from contaminated groundwater is to employ microbes that transform CF and DCM as part of their metabolism, thus depleting the contamination as the microbes continue to grow. In this work, we investigate bioaugmentation culture SC05, a mixed microbial consortium that effectively and simultaneously degrades both CF and DCM coupled to the growth of Dehalobacter. We identified the functional genes responsible for the transformation of CF and DCM in SC05. These genetic biomarkers provide a means to monitor the remediation process in the field.

Efficacy of bioaugmentation with nondomesticated mixed microbial consortia under ammonia inhibition in anaerobic digestion

Bioaugmentation shows promise in mitigating ammonia-induced microbial inhibition in anaerobic digestion processes. However, the advanced technical requirements and high costs associated with pure strain cultivation, as well as the time-consuming and labor-intensive process of domesticating consortia, present challenges for industrial applications. Herein, the efficacy of bioaugmentation with nondomesticated mixed microbial consortia was evaluated, which resulted in a significant methane production improvement of 5.6%–11.7% and 10.3%–13.5% under total ammonia nitrogen concentrations of 2.0 and 4.9 g-N/L, respectively. Microbial analysis revealed that at high ammonium levels, the bioaugmented culture facilitated a transition in the methanogenic pathway from acetoclastic to hydrogenotrophic by regulating symbiotic relationships between propionate- and acetate-oxidizing bacteria and methanogens. Consortium type and dose applied were identified as crucial factors determining bioaugmentation effectiveness. Overall, nondomesticated mixed microbial consortia demonstrate potential as cost-effective bioaugmentation agents for mitigating ammonia-induced inhibition.

Investigation of the Critical Biomass of Acclimated Microbial Communities to High Ammonia Concentrations for a Successful Bioaugmentation of Biogas Anaerobic Reactors with Ammonia Inhibition.

This study aimed to investigate the role of the bioaugmented critical biomass that should be injected for successful bioaugmentation for addressing ammonia inhibition in anaerobic reactors used for biogas production. Cattle manure was used as a feedstock for anaerobic digestion (AD). A mixed microbial culture was acclimated to high concentrations of ammonia and used as a bioaugmented culture. Different volumes of bioaugmented culture were injected in batch anaerobic reactors under ammonia toxicity levels i.e., 4 g of NH4+-N L-1. The results showed that injecting a volume equal to 65.62% of the total working reactor volume yielded the best methane production. Specifically, this volume of bioaugmented culture resulted in methane production rates of 196.18 mL g-1 Volatile Solids (VS) and 245.88 mL g-1 VS after 30 and 60 days of AD, respectively. These rates were not significantly different from the control reactors (30d: 205.94 mL CH4 g-1 VS and 60d: 230.26 mL CH4 g-1 VS) operating without ammonia toxicity. Analysis of the microbial community using 16S rRNA gene sequencing revealed the dominance of acetoclastic methanogen members from the genus Methanosaeta in all reactors.

Syntrophic Butyrate-Oxidizing Consortium Mitigates Acetate Inhibition through a Shift from Acetoclastic to Hydrogenotrophic Methanogenesis and Alleviates VFA Stress in Thermophilic Anaerobic Digestion

In anaerobic digestion (AD), butyrate is degraded by syntrophic consortium, but can accumulate in highly loaded AD systems. The effect of butyrate on the AD process attracts much less attention than propionate or acetate. In this work, an enrichment culture of the thermophilic butyrate-oxidizing syntrophic consortium was obtained by gradually increasing the initial butyrate concentration from 20 to 170 mM. Surprisingly, even the highest butyrate concentration did not significantly inhibit the methanogenic community, and the stage of acetate degradation was the limiting overall rate of the process. At 170 mM butyrate, the bacterial community changed towards the dominance of syntrophic acetate-oxidizing (SAO) bacteria related to Syntrophaceticus (42.9%), Syntrophomonas (26.2%) and Firmicutes (26.2%), while the archaeal community experienced a sharp decrease in the abundance of Methanosarcina thermophila (from 86.0 to 25.0%) and increase in Methanothermobacter thermautotrophicus (from 3.2 to 53.1%) and Methanomassiliicoccus (from 3.2 to 21.9%). Thus, the shift from acetoclastic methanogenesis to SAO coupled to hydrogenotrophic methanogenesis occurred as an adaptive strategy to overcome high acetate (~200 mM) build-up. Bioaugmentation with the obtained enrichment culture was effective in mitigating the butyrate-dominated VFA build-up during the AD of readily biodegradable waste, increasing the methane production rate, methane yield and volatile solids removal by more than 3.5, 6.2 and 2.9 times, respectively. Our study revealed that the thermophilic butyrate-oxidizing consortia as bioaugmented culture could be the potential strategy to alleviate the high organic load and VFA stress of AD.

The importance of proper pH adjustment and control to achieve complete in situ enhanced reductive dechlorination.

In situ bioremediation of chlorinated compounds such as perchloroethylene (PCE) and trichloroethylene (TCE) through enhanced reductive dechlorination (ERD) requires appropriate growth conditions for organohalide-respiring bacteria (OHRB). One of the most important factors controlling OHRB metabolism is groundwater pH. Dehalococcoides spp. (DHC) growth may be inhibited when pH is lower than 6.0, which can lead to the accumulation of toxic daughter compounds including cis-dichloroethylene (cDCE) and vinyl chloride (VC). Aquifer pH may decline as HCl is released during reductive dechlorination and from substrate fermentation to fatty acids and carbonic acid. In this article, we demonstrate that using proper pH adjustment and control in situ is an appropriate strategy to achieve complete ERD (i.e., complete conversion of PCE and TCE to nontoxic ethylene) in remediation sites with inherently low pH values and/or low buffering capacity. To analyze the effectiveness of this approach, field monitoring results are presented for a challenging site containing high concentrations of PCE and TCE (>10 000 µg/L and >1000 µg/L, respectively) and low aquifer pH (~4.9). Addition of a bioaugmentation culture, emulsified vegetable oil (EVO), and a colloidal buffer (CoBupHTM ) to increase pH, stimulated rapid conversion of PCE and TCE to cDCE and VC. However, further conversion of cDCE and VC was very limited. To stimulate complete conversion to ethylene, additional CoBupHTM and nutrients were injected, resulting in a rapid increase in metabolic rates, and maintained the aquifer pH at ~6.5 for more than five years, thus demonstrating that complete ERD can be achieved in sites with similar characteristics. Proper pH adjustment and control is needed to limit the accumulation of toxic intermediates, maintaining in situ bioremediation as an efficient, affordable, and environmentally friendly option to treat chlorinated compounds. Integr Environ Assess Manag 2023;19:943-948. © 2022 SETAC.

First Full‐Scale In Situ Propane Biosparging for Co‐Metabolic Bioremediation of 1,4‐Dioxane

Abstract1,4‐Dioxane is an emerging contaminant of environmental concern, probable human carcinogen, and it can result in diffuse groundwater plumes and is not readily treated using common remedial techniques (e.g., via air stripping or granular activated carbon). In situ bioremediation of 1,4‐dioxane has been studied at the bench‐scale and pilot‐scale. This work documents the full‐scale application of in situ propane biosparging for the treatment of 1,4‐dioxane. The subject site is located at Vandenberg Space Force Base in California. Installation of the full‐scale treatment system came after several pilot tests (e.g., Lippincott et al. 2015), stable isotope testing (Bell et al. 2016), and rebound testing conducted from 2012 to 2016. The full‐scale propane biosparge system supplies air and propane at an average of 5 kg (11 pounds) of propane per day to a network of 97 biosparge wells, or 52 g (0.11 pounds) of propane per day per biosparge well. Additionally, a bioaugmentation culture and macronutrients were delivered to the subsurface. After approximately 6 months of operation, 1,4‐dioxane was reduced at up to 99.2%, with an average global reduction of 64.1% (excluding two anomalous monitoring wells) across a treatment area of approximately 30.5 by 61 m (100 by 200 ft).

Bioaugmentation with syntrophic volatile fatty acids-oxidizing consortia to alleviate the ammonia inhibition in continuously anaerobic digestion of municipal sludge

Ammonia inhibition easily affects the performance of anaerobic digestion (AD) for municipal sludge and the oxidization of volatile fatty acids (VFAs) is the rate-limiting step of this process. Bioaugmentation is considered to be an effective method to alleviate ammonia inhibition of AD, but most study used the hydrogenotrophic methanogens as the bioaugmentation culture. In this study, bioaugmentation of mesophilic AD (MAD) and thermophilic AD (TAD) under ammonia inhibition with syntrophic acetate and propionate oxidizing consortia was investigated. The results showed that the bioaugmented reactors recovered earlier than control reactors with 20 (MAD) and 8 (TAD) days, respectively. The high-throughput 16S rRNA gene sequencing indicated that the relative abundance of carbohydrates fermenter (Lentimicrobium), syntrophic VFAs-oxidizing bacteria (Rikenellaceae_DMER64, Smithella and Syntrophobacter) and acetoclastic and hydrogenotrophic methanogens (Methanosaeta, Methanolinea and Methanospirillum) increased in MAD after bioaugmentation. However, part of the bioaugmentation culture could not adapt to the high free ammonia (FAN) concentration in MAD and the effect was weakened. In TAD, proteolytic bacteria (Keratinibaculum and Tepidimicrobium), syntrophic VFAs-oxidizing bacteria (Syntrophomonas) and hydrogenotrophic methanogen (Methanosarcina) were strengthened. The effect of bioaugmentation in TAD was durable even at higher organic loading rate (OLR), due to its positive influence on microbial community. These results suggested that the different bioaugmentation mechanism occurred in MAD and TAD, which are derived from the synergetic effects of ammonia tolerance and microbial interactions. Our study revealed the VFAs-oxidizing consortia as bioaugmented culture could be the potential strategy to alleviate the ammonia stress of AD.

Vinyl chloride and 1,4-dioxane metabolism by Pseudonocardia dioxanivorans CB1190

Vinyl chloride (VC) and 1,4-dioxane (DX) are carcinogens and co-occurring groundwater pollutants. Co-contaminants often affect the ability of microorganisms to biodegrade individual constituents. One of the mechanisms by which microbial cells overcome toxic inhibition is by transforming the inhibitory compounds. In this study, while VC inhibited DX biodegradation, it was surprisingly utilized as a growth substrate by Pseudonocardia dioxanivorans CB1190. Increasing concentrations of VC decreased DX biodegradation rates, whereas increasing DX did not have a strong effect on VC biodegradation. CB1190 was able to biodegrade both compounds simultaneously. The dxmB and aldH gene targets were down regulated during VC biodegradation and were likely not induced by VC. After incubating CB1190 with 13C-labeled VC, the detection of isotopic labeled metabolites engaged in lipid and protein synthesis (e.g., aspartate, alpha-ketoglutarate, acetyl-CoA) and energy production (e.g., ATP) provided direct evidence of CB1190’s ability to use VC as a source of carbon and energy. Collectively, these results confirm that CB1190 utilizes VC, a strong DX degradation inhibitor, as a growth substrate. This ability to aerobically degrade VC as well as DX would make CB1190 an attractive bioaugmentation culture at sites where enhanced reductive dechlorination stalls at VC due to oxygen intrusion or strain-specific limitations.

The influence of electrokinetic bioremediation on subsurface microbial communities at a perchloroethylene contaminated site.

There is an increased interest in finding remedies for contamination in low permeability and advection-limited aquifers. A technology applicable at these sites, electrokinetic-enhanced bioremediation (EK-BIO), combines traditional bioremediation and electrokinetic technologies by applying direct current to transport bioremediation amendments and microbes in situ. The effect of this technology on the native soil microbial community has only been previously investigated at the bench scale. This research explored the influence of EK-BIO on subsurface microbial communities at a field-scale demonstration site. The results showed that, similar to the findings in laboratory studies, alpha diversity decreased and beta diversity differed temporally, based on treatment phase. Enrichments in specific taxa were linked to the bioaugmentation culture and electron donor. Overall, findings from our study, one of the first field-scale investigations of the influence of electrokinetic bioremediation on subsurface microbial communities, are very similar to bench-scale studies on the topic, suggesting good correlation between laboratory and field experiments on EK-BIO and showing that lessons learned at the benchtop are important and relevant to field-scale implementation. KEY POINTS: • Microbial community analysis of field samples validates laboratory study results • Bioaugmentation cultures and electron donors have largest effect on microbial community.

Effects of organic loading rate and hydraulic retention time on bioaugmentation performance to tackle ammonia inhibition in anaerobic digestion

Three continuously stirred-tank reactors fed with manure operating under high ammonia levels (5.0 g NH3-N L−1) and with increased organic loading rate (OLR), (2.09 R1, 3.02 R2 and 4.0 R3 g VS L−1 d−1), achieved through glucose amendment in R2 and R3, were inoculated with an ammonia-acclimatized microbial culture. Successful bioaugmentation was endured only in R2 and R3, both reactors characterized by high OLR, resulting in 19.6 and 24.5% increase in methane production, respectively. The high OLRs in these reactors favored the co-occurrence of the hydrogenotrophic (Methanobacteriaceae), methylotrophic (Methanomethylophilaceae) and aceticlastic methanogenic pathways. The latter was supported by the successful establishment of ammonium-tolerant Methanosarcina, prevailing in the inoculum. Oppositely in R1, the low OLR prevented the establishment of Methanosarcina, leading to an exclusive hydrogenotrophic methanogenesis and reduced methane production. HRT shortening resulted in limited effect on biomethane performance, indicating a well establishment of the introduced bioaugmentation culture in the reactors.

The biomethanation of cow manure in a continuous anaerobic digester can be boosted via a bioaugmentation culture containing Bathyarchaeota

A bioaugmentation approach was used to enhance the performance of anaerobic digestion (AD) using cow manure (CM) as the substrate in a continuous system. To obtain the desirable microbial culture for bioaugmentation, a biochemical methane potential test (BMP) was used to evaluate three commonly used inocula namely (1) municipal solid waste (MSW), (2) wastewater treatment plant (WWTP), and (3) cow manure digester (CMMD) for their hydrolytic capacity. The highest lignocellulose removal (56% for cellulose and 50% for hemicellulose) and the most profusion of cellulolytic bacteria were obtained when CM was inoculated with CMMD. CMMD was thus used as the seed inoculum in a continuously operated reactor (Ra) with the fiber fraction of CM as the substrate to further enrich cellulolytic microbes. After 100 days (HRT: 30 days), the Bacteria fraction mainly contained Ruminofilibacter, norank_o_SBR1031, Treponema, Acetivibrio. Surprisingly, the Archaea fraction contained 97% ‘cellulolytic archaea’ norank_c_Bathyarchaeia (Phylum Bathyarchaeota). This enriched consortium was used in the bioaugmentation experiment. A positive effect of bioaugmentation was verified, with a substantial daily methane yield (DMY) enhancement (24.3%) obtained in the bioaugmented reactor (Rb) (179 mL CH4/gVS/d) than that of the control reactor (Rc) (144 mL CH4/gVS/d) (P < 0.05). Meanwhile, the effluent of Rb enjoyed an improved cellulose reduction (14.7%) than that of Rc, whereas the amount of hemicellulose remained similar in both reactors' effluent. When bioaugmentation stopped, its influence on the hydrolysis and methanogenesis sustained, reflected by an improved DMY (160 mL CH4/gVS/d) and lower cellulose content (53 mg/g TS) in Rb than those in Rc (DMY 144 mL/CH4/gVS/d and cellulose content 63 mg/g TS, respectively). The increased DMY of the continuous reactor seeded with a specifically enriched consortium able to degrade the fiber fraction in CM shows the feasibility of applying bioaugmentation in AD of CM.

Enhancing thermophilic dark fermentative hydrogen production at high glucose concentrations via bioaugmentation with Thermotoga neapolitana

The aim of the present study was to investigate the effect of gradually increasing glucose concentrations (from 5.6 to 111 mmol L−1) on the fermentative H2 production with and without bioaugmentation. A stirred tank reactor (STR) was operated at 70 °C and inoculated with a hyperthermophilic mixed culture or a hyperthermophilic mixed culture bioaugmented with Thermotoga neapolitana. With both the unaugmented (control) and augmented cultures, the H2 production rate was improved when the initial glucose concentration was increased. In contrast, the highest H2 yield (1.68 mol H2 mol−1 glucose consumed) was obtained with the augmented culture at the lowest glucose concentration of 5.6 mmol L−1 and was 37.5% higher than that obtained with the unaugmented culture at the same feed glucose concentration. Overall, H2 production rates and yields were higher in the bioaugmented cultures than in the unaugmented cultures whatever the glucose concentration. Quantitative polymerase chain reaction targeting T. neapolitana hydA gene and MiSeq sequencing proved that Thermotoga was not only present in the augmented cultures but also the most abundant at the highest glucose concentrations.

Enrichment Versus Bioaugmentation-Microbiological Production of Caproate from Mixed Carbon Sources by Mixed Bacterial Culture and Clostridium kluyveri.

Chain elongation is a process that produces medium chain fatty acids such as caproic acid, which is one of the promising products of the carboxylate platform. This study analyzed the impact of bioaugmentation of heat-treated anaerobic digester sludge with Clostridium kluyveri (AS + Ck) on caproic acid production from a mixed substrate (lactose, lactate, acetate, and ethanol). It was compared with processes initiated with non-augmented heat-treated anaerobic digester sludge (AS) and mono-culture of C. kluyveri (Ck). Moreover, stability of the chain elongation process was evaluated by performing repeated batch experiments. All bacterial cultures demonstrated efficient caproate production in the first batch cycle. After 18 days, caproate concentration reached 9.06 ± 0.43, 7.86 ± 0.38, and 7.67 ± 0.37 g/L for AS, Ck, and AS + Ck cultures, respectively. In the second cycle, AS microbiome was enriched toward caproate production and showed the highest caproate concentration of 11.44 ± 0.47 g/L. On the other hand, bioaugmented culture showed the lowest caproate production in the second cycle (4.10 ± 0.30 g/L). Microbiome analysis in both AS and AS + Ck culture samples indicated strong enrichment toward the anaerobic order of Clostridia. Strains belonging to genera Sporanaerobacter, Paraclostridium, Haloimpatiens, Clostridium, and Bacillus were dominating in the bioreactors.

In situ treatment of a dilute chlorinated solvent plume in an acidic aerobic aquifer

AbstractIn situ bioremediation was selected in the Record of Decision (ROD) as the remedial technology for a 29‐acre dilute, acidic and aerobic, chlorinated solvent plume (principally trichloroethylene [TCE] and 1,1‐dichloroethylene) for a Superfund site located in central New Jersey. Implementation of the remedy at full‐scale began in late 2010, using reductive dechlorination and bioaugmentation, and treatment has continued steadily over the last 9 years. The amendments injected include electron donor and alkaline (bicarbonate) buffer solution and, once anaerobic aquifer conditions became established, a bioaugmentation culture. Amendment injections occurred in multilevel injection wells (IWs), to maintain control over the vertical interval of amendment delivery. The areal coverage of the plume has been reduced by 59% based on the 10 µg/L TCE isocontour and the contaminant mass has been reduced by 79% through the 9 years of treatment. Lessons learned from this project include the need for bioaugmentation in the acidic aquifer and an efficient and effective manner of well construction and amendment injection using multiscreen single casing IWs and packer systems. Additional lessons learned include differences in longevity of the electron donor amendment versus the bicarbonate neutralization additive, and the need for varied amendment delivery techniques (IWs, direct injection, horizontal well installation) in selected lower permeable zones to attain treatment.

Sustained Dechlorination of Vinyl Chloride to Ethene in Dehalococcoides-Enriched Cultures Grown without Addition of Exogenous Vitamins and at Low pH.

Trichloroethene (TCE) bioremediation has been demonstrated at field sites using microbial cultures harboring TCE-respiring Dehalococcoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of vinyl chloride (VC). Here, we report growth of the Dehalococcoides-containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In these cultures, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are dominant acetogens. At neutral pH, Acetobacterium supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in cobalamin (∼20-fold). Sustained dechlorination of VC to ethene was achieved at pH as low as 5.5. Below pH 5.0, dechlorination was not stimulated by DMB supplementation but was restored by raising pH to neutral. Cell-extract assays revealed that vinyl chloride reductase activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the importance of cobamide-producing populations and pH in microbial dechlorinating communities for successful bioremediation at field sites.

Detection of Organohalide-Respiring Enzyme Biomarkers at a Bioaugmented TCE-Contaminated Field Site.

RNA-based biomarkers have been successfully detected at field sites undergoing in situ bioremediation, but the detection of expressed enzymes is a more direct way to prove activity for a particular biocatalytic process of interest since they provide evidence of potential in situ activity rather than simply confirming presence and abundance of genes in a given population by measurement of DNA copies using qPCR. Here we successfully applied shotgun proteomics to field samples from a trichloroethene (TCE)-contaminated industrial site in southern Ontario, Canada that had been bio-augmented with the commercially available KB-1TM microbial culture. The KB-1TM culture contains multiple strains of Dehalococcoides mccartyi (D. mccartyi) as well as an organohalide respiring Geobacter species. The relative abundances of specific enzymatic proteins were subsequently compared to corresponding qPCR-derived levels of DNA and RNA biomarkers in the same samples. Samples were obtained from two wells with high hydraulic connectivity to the KB-1TM-bioaugemented enhanced in situ bioremediation system, and two control wells that showed evidence of low levels of native organohalide respiring bacteria (OHRB), Dehalococcoides and Geobacter. Enzymes involved in organohalide respiration were detected in the metaproteomes of all four field samples, as were chaperonins of D. mccartyi, chemotaxis proteins, and ATPases. The most highly expressed RDase in the bioaugmentation culture (VcrA) was the most highly detected enzyme overall in the bioaugmented groundwater samples. In one background groundwater well, we found high expression of the Geobacter pceA RDase. The DNA and RNA biomarkers detected using qPCR-based assays were a set of orthologs of Dehalococcoides reductive dehalogenases (VcrA, TceA, BvcA, dehalogenase “DET1545”), and the Ni-Fe uptake hydrogenase, HupL. Within a sample, RNA levels for key enzymes correlated with relative protein abundance. These results indicate that laboratory observations of TCE-bioremediation biomarker protein expression are recapitulated in field environmental systems and that both RNA and protein biomarker monitoring hold promise for activity monitoring of in situ populations of OHRB.

Bioaugmentation of anaerobic digesters treating lignocellulosic feedstock by enriched microbial consortia.

Three different bioaugmentation cultures enriched from natural and engineered cellulolytic environments (cow and goat rumen, a biogas reactor digesting sorghum biomass) were compared for their enhancement potential on the anaerobic digestion of wheat straw. Methane yields were determined in batch tests using the Automatic Methane Potential Test System operated for 30 days under mesophilic conditions. All cultures had positive effects on substrate degradation, and higher methane yields were observed in the bioaugmented reactors compared to control reactors set up with standard inoculum. However, the level of enhancement differed according to the type of the enrichment culture. Methane yield in batch reactors augmented with 2% cow rumen derived enrichment culture was increased by only 6%. In contrast, reactors amended with 2% goat rumen derived enrichment culture or with the bioaugmentation culture obtained from the biogas reactor digesting sorghum biomass produced 27 and 20% more methane, respectively. The highest methane yield was recorded in reactors amended with 6% goat rumen derived enrichment culture, which represented an increase by 36%. The microbial communities were quite similar at the end of the batch tests independently of the bioaugmentation sources, indicating that the introduced microbial communities of the enrichment cultures did not dominate the reactors.

Biomarkers' Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM.

Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10−12 to 5.9 × 10−10 microelectron equivalents per cell per hour (μeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R2 = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration rates to decline immediately (>95% decline within one hour). Although transcript levels did decline, they did so more slowly than the respiration rate observed (transcript decay rates between 0.02 and 0.03 per hour). Data from strain-specific probes on the pangenome array strains suggest that a minor DMC strain in KB-1™ that harbors a bvcA homolog preferentially recovered following oxygen stress relative to the dominant, vcrA-containing strain.

Rumen bacteria at work: bioaugmentation strategies to enhance biogas production from cow manure.

To investigate the effects of different bioaugmentation strategies for enhancing the biogas production from cow manure and evaluate microbial community patterns. Co-inoculation with cow rumen fluid and cow rumen-derived enriched microbial consortia was evaluated in anaerobic batch tests at 36°C and 41°C. Singular addition of both rumen fluid and enriched bioaugmentation culture had a promising enhancement on methane yields; however, the highest methane yield (311mlCH4 per gram VS at 41°C) was achieved when the anaerobic seed sludge was co-inoculated together with rumen fluid and enriched bioaugmentation culture. Bacterial community profiles were investigated by Ion PGM Platform, and specific lignocellulolytic bacteria dynamics in batch tests were assessed by qPCR. The temperature had minor effects on the abundance of bacterial community; in which Bacteroidetes and Firmicutes were the most abundant phyla in all digesters. Furthermore, Rikenellaceae, Clostridiaceae, Porphyromonadaceae, Bacteroidaceae and Ruminococcaceae played a crucial role during the anaerobic degradation of cow manure. There was an important impact of Firmicutes flavefaciens and Ruminococcus albus at 41°C, which in turn positively affected the methane production. The degree of enhancement in biogas production can be upgraded by the co-inoculation of rumen-derived bioaugmentation culture with anaerobic seed sludge with high methanogenic activity. A close look at the biotic interactions and their associations with abiotic factors might be valuable for evaluating rumen-related bioaugmentation applications.

Short chain and medium chain fatty acids production using food waste under non-augmented and bio-augmented conditions

Bioprocess for conversion of ethanol and short chain fatty acids (SCFAs) into medium chain fatty acids (MCFAs) is termed as microbial chain elongation. MCFAs are saturated monocarboxylic acids with applications as antimicrobials, corrosion inhibitors and are precursors for the production of biodiesel and bioplastics. In the present study, a two-stage mixed culture fermentation was employed for the production of SCFAs and MCFAs from food waste (FW). In the first stage, FW was hydrolyzed into leachate (contains SCFAs) by the addition of anaerobic bacteria and in the second stage SCFAs present in leachate were converted in to MCFAs by the chain elongation bioprocess. The production of MCFAs such as caproic, heptanoic, caprylic and decanoic acids were demonstrated for 91 days in four different experimental conditions, i.e., control (no bacteria), pure culture (Clostridium kluyveri), non-augmentation (only enriched mixed culture), and bio-augmentation (C. kluyveri bio-augmented with enriched mixed culture). Produced SCFAs, MCFAs and biogas were analysed at different time intervals. Bio-augmented culture showed highest SCFAs (8.9 g/l of butyric acid) and MCFAs (8.1 g/l of caproic acid) production than other conditions. Microbial population was characterized by using denaturing gradient gel electrophoresis (DGGE) analysis with the highest MCFAs producing culture. The bacterial diversity observed contained predominantly Clostridia, Sphingobacteriales, Desulfobacteraceae and Bacillus groups in the bioaugmented mixed culture. Results have depicted the potential of employing the bio-augmentation strategy for the valorization of food waste into useful products like SCFAs and MCFAs.