Enriched Mixed Culture Research Articles

Compound-Specific Carbon, Nitrogen, and Hydrogen Isotope Analysis to Characterize Aerobic Biodegradation of 2,3-Dichloroaniline by a Mixed Enrichment Culture.

Compound-specific isotope analysis (CSIA) is an established tool to track the in situ transformation of organic chemicals at contaminated sites. In this work, we evaluated the potential of multi-element CSIA to assess biodegradation of 2,3-dichloroaniline (2,3-DCA), which is a major industrial feedstock. Using controlled laboratory experiments, we determined, for the first time, negligible carbon (<0.5‰) and hydrogen (<10‰) isotope fractionation and a significant inverse nitrogen isotope fractionation (>10‰) during aerobic 2,3-DCA biodegradation by a mixed enrichment culture. The tentative identification of a glutamate conjugate of 2,3-DCA as a reaction intermediate indicates that the initial multistep enzymatic reaction may be rate-limiting. The formation of the glutamate adduct would increase the bond energy at the N atom, thus likely explaining the observed inverse N isotope fractionation. The corresponding nitrogen enrichment factor was +6.8 ± 0.6‰. This value was applied to investigate the in situ 2,3-DCA biodegradation at a contaminated site where the carbon and nitrogen isotope signatures from field samples suggested similar aerobic processes by native microorganisms. Under the assumption of the applicability of the Rayleigh model in a pilot wetland treating contaminated groundwater, the extent of biodegradation was estimated to be up to 80-90%. This study proposes multi-element CSIA as a novel application to study 2,3-DCA fate in groundwater and surface water and provides insights into biodegradation pathways.

Investigating the efficiency of a two-stage anaerobic-aerobic process for the treatment of confectionery industry wastewaters with simultaneous production of biohydrogen and polyhydroxyalkanoates.

The scope of the current study was to investigate the efficiency of a two-stage anaerobic-aerobic process for the simultaneous treatment and valorization of selective wastewater streams from a confectionary industry. The specific wastewater (confectionary industry wastewater, CIW) was a mixture of the rinsing eluting during washing of the cauldrons in which jellies and syrups were produced, and contained mainly readily fermentable sugars, being thus of high organic load. The first stage of the process was the dark fermentation (DF) of the CIW in continuous, attached-biomass systems, in which the effect on hydrogen yields and distribution of metabolites were studied for different packing materials (ceramic or plastic), hydraulic retention times, HRTs (12 h–30 h) and feed substrate concentration (20 g COD/L- 50 g COD/L). In the second stage, the effectiveness of the aerobic treatment of the DF effluents was evaluated in terms of the reduction of the organic load and the production of polyhydroxyalkanoates (PHAs) through an enriched mixed microbial culture (MMC). The MMC was developed in a continuous draw and fill system, in which the accumulation potential of PHAs was studied. It was shown that the hydrogen production rates decreased for increasing substrate concentration and HRTs, with a maximum of 12.70 ± 0.35 m3 H2/m3 initial CIW achieved for the lowest HRT and feed concentration and using ceramic beads as packing material. Butyrate, acetate and lactate were the main metabolites generated in all cases, in different ratios. The distribution of metabolites during DF was shown to highly affect the efficiency of the second process in terms of both the reduction of organic load and the PHAs yields. The highest removal of organic load achieved after 48 h of aerobic treatment was 84.0 ± 0.9 %, whereas the maximum PHAs yield was 21.46 ± 0.13 kg PHAs/m3 initial CIW.

Microbiome enrichment from contaminated marine sediments unveils novel bacterial strains for petroleum hydrocarbon and heavy metal bioremediation

Petroleum hydrocarbons and heavy metals are some of the most widespread contaminants affecting marine ecosystems, urgently needing effective and sustainable remediation solutions. Microbial-based bioremediation is gaining increasing interest as an effective, economically and environmentally sustainable strategy. Here, we hypothesized that the heavily polluted coastal area facing the Sarno River mouth, which discharges >3 tons of polycyclic aromatic hydrocarbons (PAHs) and ∼15 tons of heavy metals (HMs) into the sea annually, hosts unique microbiomes including marine bacteria useful for PAHs and HMs bioremediation. We thus enriched the microbiome of marine sediments, contextually selecting for HM-resistant bacteria. The enriched mixed bacterial culture was subjected to whole-DNA sequencing, metagenome-assembled-genomes (MAGs) annotation, and further sub-culturing to obtain the major bacterial species as pure strains. We obtained two novel isolates corresponding to the two most abundant MAGs (Alcanivorax xenomutans strain-SRM1 and Halomonas alkaliantarctica strain-SRM2), and tested their ability to degrade PAHs and remove HMs. Both strains exhibited high PAHs degradation (60–100%) and HMs removal (21–100%) yield, and we described in detail >60 genes in their MAGs to unveil the possible genetic basis for such abilities. Most promising yields (∼100%) were obtained towards naphthalene, pyrene and lead. We propose these novel bacterial strains and related genetic repertoire to be further exploited for effective bioremediation of marine environments contaminated with both PAHs and HMs.

Expanding mechanistic models to represent purple phototrophic bacteria enriched cultures growing outdoors

The economic feasibility of purple phototrophic bacteria (PPB) for resource recovery relies on using enriched-mixed cultures and sunlight. This work presents an extended Photo-Anaerobic Model (ePAnM), considering: (i) the diverse metabolic capabilities of PPB, (ii) microbial clades interacting with PPB, and (iii) varying environmental conditions. Key kinetic and stoichiometric parameters were either determined experimentally (with dedicated tests), calculated, or gathered from literature. The model was calibrated and validated using different datasets from an outdoors demonstration-scale reactor, as well as results from aerobic and anaerobic batch tests. The ePAnM was able to predict the concentrations of key compounds/components (e.g., COD, volatile fatty acids, and nutrients), as well as microbial communities (with anaerobic systems dominated by fermenters and PPB). The results underlined the importance of considering other microbial clades and varying environmental conditions. The model predicted a minimum hydraulic retention time of 0.5 d−1. A maximum width of 10 cm in flat plate reactors should not be exceeded. Simulations showed the potential of a combined day-anaerobic/night-aerobic operational strategy to allow efficient continuous operation.

Salinity and hydraulic retention time induce membrane phospholipid acyl chain remodeling in Halanaerobium congolense WG10 and mixed cultures from hydraulically fractured shale wells.

Bacteria remodel their plasma membrane lipidome to maintain key biophysical attributes in response to ecological disturbances. For Halanaerobium and other anaerobic halotolerant taxa that persist in hydraulically fractured deep subsurface shale reservoirs, salinity, and hydraulic retention time (HRT) are important perturbants of cell membrane structure, yet their effects remain poorly understood. Membrane-linked activities underlie in situ microbial growth kinetics and physiologies which drive biogeochemical reactions in engineered subsurface systems. Hence, we used gas chromatography-mass spectrometry (GC-MS) to investigate the effects of salinity and HRT on the phospholipid fatty acid composition of H. congolense WG10 and mixed enrichment cultures from hydraulically fractured shale wells. We also coupled acyl chain remodeling to membrane mechanics by measuring bilayer elasticity using atomic force microscopy (AFM). For these experiments, cultures were grown in a chemostat vessel operated in continuous flow mode under strict anoxia and constant stirring. Our findings show that salinity and HRT induce significant changes in membrane fatty acid chemistry of H. congolense WG10 in distinct and complementary ways. Notably, under nonoptimal salt concentrations (7% and 20% NaCl), H. congolense WG10 elevates the portion of polyunsaturated fatty acids (PUFAs) in its membrane, and this results in an apparent increase in fluidity (homeoviscous adaptation principle) and thickness. Double bond index (DBI) and mean chain length (MCL) were used as proxies for membrane fluidity and thickness, respectively. These results provide new insight into our understanding of how environmental and engineered factors might disrupt the physical and biogeochemical equilibria of fractured shale by inducing physiologically relevant changes in the membrane fatty acid chemistry of persistent microbial taxa. GRAPHICAL ABSTRACTSalinity significantly alters membrane bilayer fluidity and thickness in Halanaerobium congolense WG10.

Efficient bioremediation of PAHs-contaminated soils by a methylotrophic enrichment culture

Bioaugmentation effectively enhances microbial bioremediation of hazardous polycyclic aromatic hydrocarbons (PAHs) from contaminated environments. While screening for pyrene-degrading bacteria from a former manufactured gas plant soil (MGPS), the mixed enrichment culture was found to be more efficient in PAHs biodegradation than the culturable pure strains. Interestingly, analysis of 16S rRNA sequences revealed that the culture was dominated by a previously uncultured member of the family Rhizobiaceae. The culture utilized C1 and other methylotrophic substrates, including dimethylformamide (DMF), which was used as a solvent for supplementing the culture medium with PAHs. In the liquid medium, the culture rapidly degraded phenanthrene, pyrene, and the carcinogenic benzo(a)pyrene (BaP), when provided as the sole carbon source or with DMF as a co-substrate. The efficiency of the culture in the bioremediation of PAHs from the MGPS and a laboratory waste soil (LWS) was evaluated in bench-scale slurry systems. After 28 days, 80% of Σ16 PAHs were efficiently removed from the inoculated MGPS. Notably, the bioaugmentation achieved 90% removal of four-ringed and 60% of highly recalcitrant five- and six-ringed PAHs from the MGPS. Likewise, almost all phenanthrene, pyrene, and 65% BaP were removed from the bioaugmented LWS. This study highlights the application of the methylotrophic enrichment culture dominated by an uncultured bacterium for the efficient bioremediation of PAHs.

Electricity-driven bioproduction from CO2 and N2 feedstocks using enriched mixed microbial culture

Microbial electrosynthesis (MES) is an emerging technology with the potential to reduce carbon emissions by converting CO2 and renewable power into chemicals. Here, MES of acetate using gaseous N2 as a nitrogen source is investigated as an approach to substitute fixed nitrogen (ammonia) in the process. At an applied cathode potential of − 0.995 V vs. SHE, the mixed microbial community with a few known N2 and CO2 fixing microorganisms produced 463.5 ± 14 mg/l acetate using gaseous N2 and CO2 feedstock along with traces of ammonia (0.80 ± 0.06 mg/l) confirming the simultaneous fixation of these gases. Up to 85%, 0.5%, and 4.9% electrons were recovered in acetate, ammonia, and biomass, respectively, with a maximum of 39% energetic efficiency. A preliminary analysis suggested the possibility of bringing down a considerable amount of CO2 from going into the environment both by fixing CO2 into acetate and avoiding the use of fixed ammonia, the production of which is associated with the CO2 emissions. Thus, the demonstrated process circumvents the conventional fixed nitrogen (ammonia) usage in MES while still being able to produce considerable acetate from CO2.

Caproate production from xylose via the fatty acid biosynthesis pathway by genus Caproiciproducens dominated mixed culture fermentation

Caproate production from organic wastes is deemed as a novel strategy in mixed culture fermtation (MCF). However, producing caproate from natural sugar of xylose by MCF is seldom reported and the metabolic pathway is still unclear. Thus, the caproate production from xylose was investigated in this study by mesophilic MCF. The results showed that the caproate concentration from xylose (10 g/L) was 1.2 ± 0.17 g/L (equal to 2.7 gCOD/L) under pH 5.0. Dosing extra ethanol of 5 g/L could slightly increase the caproate production by ∼ 30% (i.e., 1.6 g/L). While dosing extra acetate of 5 g/L negatively affected the caproate production, which was just 0.2 g/L. The microbial analysis illustrated that genus Caproiciproducens was a main identified caproate producer, occupying over 80% of enriched mixed culture. The fatty acid biosynthesis pathway was identified via metagenomic analysis. These unexpected differences extended the understanding of caproate production from organic wastes.

Effect of the coexistence of endosulfan on the lindane biodegradation by Novosphingobium barchaimii and microbial enrichment cultures

Organochlorine pesticides, especially lindane and endosulfan, have been demonstrated to be both biodegradable and frequently coexistent, but their inhibitory effect has never been studied. In this study, we investigated the effect of endosulfan coexistence on lindane degradation to a lindane-degrading isolate, Novosphingobium barchaimii strain LL02, and mixed enrichment cultures from two different inocula. Our results of the lindane degradation batch experiments demonstrated that endosulfan concentration above 20 mg L−1 causes significant inhibition to the lindane degradation efficiency of the strain LL02. Besides, the acidic conditions at pH 5.0 to 6.0 further decreased its lindane degradation rate constants by 57% compared to the neutral and alkaline conditions. For the mixed microbial cultures, the lindane degradation efficiency in the lindane/endosulfan co-contamination conditions decreased by 35.7%–50.7% compared to the lindane alone conditions. From our 16S rRNA amplicon sequencing results through the PacBio platform, most of the predominant bacteria in the lindane-enriched cultures were depressed in the lindane/endosulfan-enriched cultures. Moreover, bacteria of Burkholderia australis, Chujaibacter soli, Flavitalea flava, and one Rhodanobacteraceae bacterium were relatively highly abundant in the co-contamination enrichment cultures, suggesting their potential for lindane degradation under the endosulfan stress. Our results demonstrated that endosulfan coexistence causes inhibitory impacts on lindane biodegradation toward both lindane-degrading bacteria and mixed microbial cultures. The coexistence of multiple organochlorine pesticides on the biodegradation efficiencies should be carefully considered when applying bioremediation to remove organochlorine pesticide contamination.

Impact of partially submersed iron scraps in simultaneously sulfate and nitrate removal using sulfate-reducing bacteria

In this study, partially submerged iron scraps were allowed to react to the contaminants at the headspace air and liquid interface for pollutants removal in the serum bottle reactors. The experiments were conducted using Box–Behnken design of Response Surface Methodology in anaerobic batch reactors (120 ml) at different concentrations of sulfate (0.5, 1.75, 3 g/L), nitrate (0.25, 0.375, 0.5 g/L) and iron scraps (0, 0.125, 0.250 g/50 ml). The sulfate reducer enriched mixed culture was used for simultaneous removal of SO42− and NO3−. The highest sulfate removal of 49.37 % was achieved at 0 g/50 ml ISs, 3 g/L SO4−2, and 0.375 g/L NO3−. Even at a higher concentration of both SO42− (3 g/L) and NO3−(0.5 g/L), a significant SO42− removal of 44.65% was achieved at an iron concentration of 125mg/50ml accompanied with NO3− conversion. Both the lower and higher concentrations of iron scraps were showing effective interaction at higher SO42− and NO3− levels. The results of the confirmatory experiments with iron scraps of 0.039 g/50 ml with the desirability of 0.92 showed a maximum SO42− removal of 50.62 % with complete conversion of nitrate.

Valorisation of waste cooking oil using mixed culture into short- and medium-chain length polyhydroxyalkanoates: Effect of concentration, temperature and ammonium

The production of polyhydroxyalkanoates (PHAs) from waste cooking oil (WCO) by a mixed culture was investigated in the present study at increasing WCO concentrations, temperature and ammonium availability. The PHA production was done in two steps: in the first step, a mixed culture was enriched in PHA-accumulating bacteria from activated sludge in a sequencing batch reactor operated in a feast-famine mode and in the second step the PHA accumulation by the enriched mixed culture was assessed in a batch reactor. In the enrichment step, two substrates, WCO and nonanoic acid were used for enrichment and in the PHA accumulation step only WCO was used. It was not possible to enrich a mixed culture in PHA-accumulating bacteria using WCO as substrate due to the development of filamentous bacteria causing foam formation and bulking in the reactor. However, our results showed that the mixed culture continuously fed with nonanoic acid was enriched in PHA-accumulating bacteria. This enriched culture accumulated both scl- and mcl-PHA using WCO as substrate. The maximum PHA accumulation capacity of this mixed culture from WCO was 38.2% cdw. Increasing the temperature (30–40 ℃) or WCO concentrations (5–20 g/l) increased the PHA accumulation capacity of the mixed culture and the ratios of scl-PHA to mcl-PHA. The presence of ammonium increased PHA accumulation (21.9% cdw) compared to the complete absence of ammonium (5.8% cdw). The thermal characterization of the PHA exhibited the advantageous properties of both scl- and mcl-PHA, i.e., higher melting temperature (152–172 ℃) similar to scl-PHA and a lower degree of crystallinity (12%) similar to mcl-PHA. This is the first study to report the potential of open mixed culture to produce scl- and mcl-PHA from WCO and thus contributing to the understanding of sustainable polymer production.

Microbe assisted depolymerization of lignin rich waste and its conversion to gaseous biofuel

Biomethanation potential of lignin rich residue (LRR) obtained from lignocellulosic ethanol fermentation was evaluated after subjecting to microbe assisted pretreatment using selectively enriched lignin depolymerizing consortia (LDC). The efficiency of LDC in lignin depolymerization was established using alkali and dealkali lignins (AL and DL) along with LRR as feedstocks. Microbial growth on media having lignin as sole carbon source, activity of lignin depolymerizing enzymes, viz., lignin peroxidase and laccase, ability of culture to decolorize the lignin mimicking dyes like methylene blue and ramezol brilliant blue, were considered to confirm the efficiency of enriched mixed culture. Microbial treatment using LDC showed significant positive impact on lignin breakdown irrespective of the substrate (LRR, 46.33%; AL, 31.37%; DL, 34.20%). The hydrolysate of LRR obtained from microbial pretreatment showed higher biogas yield (424 ml/g VS) owing to the efficiency of lignin depolymerization and availability of readily available biodegradable components in residual lignin from previous processing. Depolymerization of commercial lignins also produced a good amount of biogas (302–324 ml/g VS) after pretreatment with LDC. Overall, an additional energy conversion efficiency of about 11.75 kJ/g VS was obtained by valorizing the residual lignin through integrating biomethanation technology to ethanol fermentation. Outcome of this study indicated the feasibility of using lignin rich residue generated from the second generation cellulosic bioethanol plants as a potential feedstock to meet the current gaseous fuel demands. This integration also helps in closing the biomass based biorefinery loop and also promotes the circular economy.

Carbon Source Applied in Enrichment Stage of Mixed Microbial Cultures Limits the Substrate Adaptability for PHA Fermentation Using the Renewable Carbon.

Suitability of different substrates for enriched mixed microbial cultures (MMCs) is of importance to the polyhydroxyalkanoate (PHA) fermentation using renewable carbon. In this study, three enriched MMCs were evaluated for their fermentation features and kinetics with different carbon sources (sodium acetate, glucose, or starch). The results showed that the highly specific bacterial community composition was developed depending on the applied carbon source. Correspondence analysis suggested that the genus affiliated in Gammaproteobacteria_unclassified was related to 3-hydroxybutyrate (HB) synthesis in acetate-fed MMC (relative abundance of 38%) and glucose-fed MMC (relative abundance of 76.7%), whereas Vibrio genus was related to 3-hydroxyvalerate (HV) production in glucose-fed MMC (relative abundance of 0.4%) and starch-fed MMC (relative abundance of 94.6%). The acetate-fed MMC could not use glucose and starch as fermentation carbon sources, showing the limitation of microbial species developed with the specific metabolic substrate. Glucose-fed MMC produced the highest PHA cell content of 64.2% cell dry weight when using sodium acetate as the fermentation carbon. Glucose-fed MMC showed wide resilience and adaptation to various carbon sources. When actual landfill leachate was used for fermentation by glucose-fed MMC, maximum PHA cell content of 45.5% cell dry weight and the PHA volumetric productivity of 0.265 g PHA/(L·h) were obtained. This study suggested carbon sources applied in the MMC enrichment stage had a significant influence on utilization of carbon in the fermentation stage.

Optimization of the operational parameters for mesophilic biohydrogen production from palm oil mill effluent using enriched mixed culture

Improvements in biohydrogen production from palm oil mill effluent (POME) has been achieved by optimizing the physicochemical conditions, using an enriched sludge as inoculum. Enrichment of biohydrogen-producing communities in the sludge was first carried out by heat treatment and acclimatization to POME. Response surface methodology (RSM) was then used for optimizing the operational parameters. The factors investigated were temperature, initial pH, and inoculum concentration. The output responses were evaluated based on the biohydrogen yield (YH2), biohydrogen production rate (RH2), and COD removal efficiency. Microbial analysis of the enriched sludge at 100% POME concentration revealed the abundance of the Firmicutes phylum (98%) among the Bacteria population, with Clostridiaceae as the most abundant family. Archaea were inhibited at both 30% and 100% POME concentration. The optimum operational parameters generated by the RSM model were 34.7 °C, pH 5.81, and inoculum size of 14.22% (v/v), with the following predicted outcomes: YH2 of 42.29 mL H2 gCOD−1, RH2 of 106.09 mL L−1 h−1, and COD removal efficiency of 23.66%. Meanwhile, the experimental data generated were as follows: YH2 of 41.18 mL H2 gCOD−1, RH2 of 109 mL L−1 h−1, and COD removal efficiency of 25.96%. The process reliability was further demonstrated in a larger (2.5 L) continuously stirred-tank reactor, with the maximum biohydrogen yield obtained of 282 mL H2 gCOD−1 and COD removal efficiency of 35%. Butyric and acetic acids were the dominant soluble metabolites produced. These findings suggest that the RSM model generated was successfully used to optimize biohydrogen production from POME using enriched anaerobic digested sludge.

Self-flocculation of enriched mixed microalgae culture in a sequencing batch reactor.

Microalgae-based biodiesel has gained widespread interest as an alternative energy source. Low-cost microalgae harvesting technologies are important for economically feasible biodiesel production. This study investigated, for the first time, the impact of adaptation period and height to diameter (H/D) ratio of a reactor on the growth and self-flocculation of microalgae, without the addition of bacteria. Six reactors were grouped into three sets of experiments, and each reactor was operated for 30 days at similar operating conditions (volume exchange ratio = 25% and settling time = 30 min). In set 1, two 8-L reactors, H5a (H/D ratio: 5) and H8a (H/D ratio: 8), were operated under batch operation. In set 2, reactors H5b and H8b were operated as sequential batch reactors (SBRs) without an adaptation period. In set 3, the reactors H5c and H8c were operated as SBRs with an adaptation period. The findings showed a threefold improvement in biomass productivity for the higher H/D ratio (H8c) and a reduction in biomass loss for microalgae. The H8c reactor exhibited 95% settling efficiency within 5 days, in comparison to 30 days for the H5c reactor. This study demonstrated that a higher H/D ratio and the introduction of an adaptation period in SBR operation positively influences growth and self-flocculation of enriched mixed microalgae culture.

Recovery of Polyhydroxyalkanoates from Cooked Mussel Processing Wastewater at High Salinity and Acidic Conditions

Polyhydroxyalkanoates (PHA) are biodegradable polymers that can be intracellularly produced by microorganisms valorizing organic-rich wastes. In the present study, a PHA production system was fed with mussel cooker wastewater after acidogenic fermentation. Besides low pH (4.0 ± 0.3) and high salt (21.7 ± 2.9 g NaCl/L) concentrations, this wastewater also contained nitrogen concentrations (0.8 ± 0.1 g N/L), which were previously reported to be a challenge to the PHA accumulating bacteria enrichment. Bacteria with a PHA storage capacity were selected in an enrichment sequencing batch reactor (SBR) after 60 days of operation. The enriched mixed microbial culture (MMC) was mainly formed by microorganisms from phylum Bacteroidetes, and genera Azoarcus, Comamonas and Thauera from phylum Proteobacteria. The MMC was able to accumulate up to 25 wt% of PHA that was mainly limited by the wastewater nitrogen content, which promoted biomass growth instead of PHA accumulation. Indeed, when the presence of nutrient was limited, PHA stored in the accumulation reactor increased to up to 40.9 wt%. This work demonstrated the feasibility of the enrichment of a MMC with a PHA storage ability valorizing the fish-canning industrial wastewater at low pH, which is generally difficult to treat in wastewater treatment plants.

Direct utilization of industrial carbon dioxide with low impurities for acetate production via microbial electrosynthesis

The present study aimed to demonstrate the utilization of unpurified industrial CO2 with low impurities for acetate production via microbial electrosynthesis (MES) for the first time. In MES experiments with CO2-rich brewery gas, the enriched mixed culture dominated by Acetobacterium produced 1.8 ± 0.2 g/L acetic acid at 0.26 ± 0.03 g/Lcatholyte/d rate and outperformed a pure culture of Clostridium ljungdahlii (1.1 ± 0.02 g/L; 0.138 ± 0.004 g/Lcatholyte/d). The electron recovery in acetic acid was also more for mixed culture (84 ± 13%) than C. ljungdahlii (42 ± 14%). Electrochemical analysis of biocathodes suggested the role of microbial biofilm in improved hydrogen electrocatalysis. In comparative gas fermentation tests, the mixed culture outperformed C. ljungdahlii and produced acetic acid at a similar level with both industrial and pure CO2 feedstocks. These results suggest the robustness and capability of the mixed microbial community for utilizing slightly impure industrial CO2 for bioproduction and presents a major advancement in MES technology.

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H2 concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H2/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H2 concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H2/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.

Optimization of an enriched mixed culture to increase PHA accumulation using industrial saline complex wastewater as a substrate

Polyhydroxyalkanoates (PHA) appear as good candidates to substitute conventional petroleum-based plastics since they have similar properties but with the advantage of being biodegradable. Wastewater streams with high organic content are feasible substrates for PHA production resulting in an opportunity for waste recovery. One of the main challenges is the optimization of the selection of microorganisms with high PHA storage capacity. This microbial selection is performed in sequencing batch reactors (SBR) operated under an aerobic feast/famine (F/F) regime. In the present study, a settling stage was added at the end of the feast phase of the enrichment cycle of a SBR fed with pre-acidified cooked mussel processing wastewater (containing up to 12 g NaCl/L). Settling and subsequent supernatant discharge favoured the wash-out of non-accumulating microorganisms as well as the removal of substances that enhanced their undesired development (proteins and carbohydrates). Microbial analysis performed by fluorescence in situ hybridization (FISH) technique showed shifts in the microbial community; the presence of genus Paracoccus increased whereas genera Comamonas decreased. Moreover, the process efficiency was improved with the increase of the PHA production yield (YPHA) and the maximum PHA storage capacity (max. PHA) from 0.48 to 0.72 CmmolPHA/CmmolVFA and from 40 to 60 wt%, respectively. The polymer composition also changed, its HB:HV ratio varied from 83:17 to 70:30. Results obtained in the present study showed that settling after the feast phase promoted the removal of carbon sources that did not contribute to PHA production and the washout of non-storing bacteria, which favoured the culture enrichment.

Continuous cultivation strategy for yeast industrial wastewater-based polyhydroxyalkanoate production

Polyhydroxyalkanoates (PHAs) present an eco-friendly alternative for conventional plastics. Industrial wastewater from the food industry is a copious source of organic carbon that can be recovered in the form of PHA. However, the wastewater composition varies considerably among the different industries demanding for an industry-specific investigation of the PHA production process. Wastewater from the yeast industry, besides its high concentration in organic carbon, also contains a high ammonium concentration which might decrease the PHA production. Thus, this study aims to investigate PHA production using yeast industry wastewater via an enriched mixed microbial culture (MMC). A less explored cultivation strategy, i.e., continuous cultivation for PHA production was evaluated and compared to the widely used batch cultivation. PHA accumulating MMC was enriched using a sequencing batch reactor (SBR) operated under aerobic dynamic feeding. An MMC dominated by the Thauera species was successfully enriched in the SBR. Experimental results showed that ammonium is indeed required for PHA accumulation and the complete absence of ammonium negatively affects the accumulation process. Using wastewater, batch and continuous feeding strategies, respectively, yielded the PHA accumulation of 72 % and 65 % per dry cell weight. Despite the slightly lower PHA accumulation with continuous cultivation, four times more biomass growth was produced. Consequently, higher theoretical PHA production (270 t/year) can be expected using continuous cultivation in half of the reactor volume (45m3). Therefore, this study asserts the viability of continuous cultivation as a feasible investigatory tool and PHA production strategy.