Effect of micro-aeration on syntrophic and methanogenic activity in anaerobic sludge.

Micro-aeration was shown to improve anaerobic digestion (AD) processes, although oxygen is known to inhibit obligate anaerobes, such as syntrophic communities of bacteria and methanogens. The effect of micro-aeration on the activity and microbial interaction in syntrophic communities, as well as on the potential establishment of synergetic relationships with facultative anaerobic bacteria (FAB) or aerobic bacteria (AB), was investigated. Anaerobic sludge was incubated with ethanol and increasing oxygen concentrations (0-5% in the headspace). Assays with acetate or H2/CO2 (direct substrates for methanogens) were also performed. When compared with the controls (0% O2), oxygen significantly decreased substrate consumption and initial methane production rate (MPR) from acetate or H2/CO2. At 0.5% O2, MPR from these substrates was inhibited 30-40%, and close to 100% at 5% O2. With ethanol, significant inhibition (>36%) was only observed for oxygen concentrations higher than 2.5%. Oxygen was consumed in the assays, pointing to the stimulation of AB/FAB by ethanol, which helped to protect the syntrophic consortia under micro-aerobic conditions. This highlights the importance of AB/FAB in maintaining functional and resilient syntrophic communities, which is relevant for real AD systems (in which vestigial O2 amounts are frequently present), as well as for AD systems using micro-aeration as a process strategy. KEY POINTS: •Micro-aeration impacts syntrophic communities of bacteria and methanogens. •Oxygen stimulates AB/FAB, maintaining functional and resilient consortia. •Micro-aeration studies are critical for systems using micro-aeration as a process strategy.

Conversion of Cn-Unsaturated into Cn-2-Saturated LCFA Can Occur Uncoupled from Methanogenesis in Anaerobic Bioreactors.

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via ß-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA.

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment.

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.

Study of 16 Portuguese activated sludge systems based on filamentous bacteria populations and their relationships with environmental parameters.

A survey in 16 activated sludge wastewater treatment plants (WWTP) was conducted to contribute to the knowledge of the environmental parameters that determine the composition of the filamentous community. A total of 128 samples of mixed liquor from municipal WWTP were collected during 2 years, and 22 filamentous morphotypes were identified. The most frequent and abundant filamentous bacteria were, in both cases and by this order, type 0041/0675, type 0092, Microthrix parvicella and 1851, nocardioforms and Haliscomenobacter hydrossis. Concerning dominance, type 1851 was the most frequently dominant morphotype, followed by M. parvicella and types 0092 and 0041/0675. These were also, and by this order, the dominant morphotypes during bulking occurrences. Significant correlations were obtained between the abundance of filamentous bacteria and environmental parameters, but multivariate statistical analysis only confirmed the correlation between type 0092 and Sludge Volume Index (SVI), emphasizing the association of this filament with bulking. The discussion of the results in light of published works was complicated by the random use of terms such as frequency, abundance, and dominance with different and often unclear meanings. This reinforces the need of clarifying these terms when discussing the causes of filamentous overgrowth in WWTP.

Starvation combined with constant anode potential triggers intracellular electron storage in electro-active biofilms.

The accumulation of electrons in the form of Extracellular Polymeric Substances (EPS) and poly-hydroxyalkanoates (PHA) has been studied in anaerobic processes by adjusting the access of microorganisms to the electron donor and final electron acceptor. In Bio-electrochemical systems (BESs), intermittent anode potential regimes have also recently been used to study electron storage in anodic electro-active biofilms (EABfs), but the effect of electron donor feeding mode on electron storage has not been explored. Therefore, in this study, the accumulation of electrons in the form of EPS and PHA was studied as a function of the operating conditions. EABfs were grown under both constant and intermittent anode potential regimes and fed with acetate (electron donor) continuously or in batch. Confocal Laser Scanning Microscopy (CLSM) and Fourier-Transform Infrared Spectroscopy (FTIR) were used to assess electron storage. The range of Coulombic efficiencies, from 25 to 82%, and the biomass yields, between 10 and 20%, indicate that storage could have been an alternative electron consuming process. From image processing, a 0.92 pixel ratio of poly-hydroxybutyrate (PHB) and amount of cells was found in the batch fed EABf grown under a constant anode potential. This storage was linked to the presence of living Geobacter and shows that energy gain and carbon source starvation were the triggers for intracellular electron storage. The highest EPS content (extracellular storage) was observed in the continuously fed EABf under an intermittent anode potential, showing that constant access to electron donor and intermittent access to the electron acceptor leads to the formation of EPS from the excess energy gained. Tailoring operating conditions can thus steer the microbial community and result in a trained EABf to perform a desired biological conversion, which can be beneficial for a more efficient and optimized BES.

Analysis of the microbial community of the biocathode of a hydrogen-producing microbial electrolysis cell.

The microbial electrolysis cell (MEC) is a promising system for hydrogen production. Still, expensive catalysts such as platinum are needed for efficient hydrogen evolution at the cathode. Recently, the possibility to use a biocathode as an alternative for platinum was shown. The microorganisms involved in hydrogen evolution in such systems are not yet identified. We analyzed the microbial community of a mixed culture biocathode that was enriched in an MEC bioanode. This biocathode produced 1.1 A m(-2) and 0.63 m3 H2 m(-3) cathode liquid volume per day. The bacterial population consisted of 46% Proteobacteria, 25% Firmicutes, 17% Bacteroidetes, and 12% related to other phyla. The dominant ribotype belonged to the species Desulfovibrio vulgaris. The second major ribotype cluster constituted a novel taxonomic group at the genus level, clustering within uncultured Firmicutes. The third cluster belonged to uncultured Bacteroidetes and grouped in a taxonomic group from which only clones were described before; most of these clones originated from soil samples. The identified novel taxonomic groups developed under environmentally unusual conditions, and this may point to properties that have not been considered before. A pure culture of Desulfovibrio strain G11 inoculated in a cathode of an MEC led to a current development from 0.17 to 0.76 A m(-2) in 9 days, and hydrogen gas formation was observed. On the basis of the known characteristics of Desulfovibrio spp., including its ability to produce hydrogen, we propose a mechanism for hydrogen evolution through Desulfovibrio spp. in a biocathode system.

Sequencing batch airlift reactors (SBAR): a suitable technology for treatment and valorization of mineral oil wastewaters towards lipids production.

Produced water (PW) and spent oil-based wastewaters are some of the largest mineral oil wastewaters produced. Due to the high toxicity of hydrocarbons, several countries set stringent discharge limits and its treatment is compulsory before discharge. In this work, biological treatment of mineral oil wastewaters coupled with the production of bacterial lipids is demonstrated in sequential batch airlift reactors (SBAR). Two SBAR (2 L working volume) were used for treatment of PW and lubricant-based wastewater (LW), inoculated with Alcanivorax borkumensis SK2 (SBARAb+PW) and Rhodococcus opacus B4 (SBARR.o+LW), respectively. A total petroleum hydrocarbon removal (TPH) efficiency up to 96% and 80% were achieved for SBARAb+PW and SBARR.o+LW, respectively. Intracellular lipids production in SBARAb+PW increased when lower TPH/N ratios and higher feast stage duration were applied (up to 0.74 g g-1 cell dry weight (CDW)), whereas in SBARR.o+LW higher lipids production was observed for higher TPH/N ratios (0.94 g g-1 in CDW). Triacylglycerols (TAG) were the main intracellular lipid accumulated in both SBARAb+PW and SBARR.o+LW operations, while wax ester (WE) production was only observed extracellularly in the SBARAb+PW.

In vitro assessment of prebiotic properties of xylooligosaccharides produced by Bacillus subtilis 3610.

Xylooligosaccharides (XOS) are emergent prebiotics exhibiting high potential as food ingredients. In this work, in vitro studies were performed using human fecal inocula from two healthy donors (D 1 and D2) to evaluate the prebiotic effect of commercial lactulose and XOS produced in a single-step by recombinant Bacillus subtilis 3610. The fermentation of lactulose led to the highest production of lactate (D1: 33.7 ±â€¯0.5 mM; D2:19.7 ±â€¯0.3 mM) and acetate (D1: 77.5 ±â€¯0.6 mM; D2: 81.0 ±â€¯0.7 mM), while XOS led to the highest production of butyrate (D1: 9.0 ±â€¯0.6 mM; D2: 10.5 ±â€¯0.8 mM) and CO2 (D1: 8.92 ±â€¯0.02 mM; D2: 11.4 ±â€¯0.3 mM). Microbiota analysis showed a significant decrease in the relative abundance of Proteobacteria for both substrates and an increase in Bifidobacterium and Lactobacillus for lactulose, and Bacteroides for XOS.

Tuning culturing conditions towards the production of neutral lipids from lubricant-based wastewater in open mixed bacterial communities.

Production of bacterial lipid-based biofuels using inexpensive substrates, as wastes, is an emerging approach. In this work, a selective process using carbon feast-famine cycles was applied to obtain an indigenous microbial community of hydrocarbon-degrading and lipid-accumulating bacteria, using a real lubricant-based wastewater as carbon source. In the conditions applied, the enriched bacterial community, dominated by members of the genus Rhodococcus, Pseudomonas and Acinetobacter, was able to degrade almost all hydrocarbons present in the wastewater within 24 h' incubation and to accumulate, although in low levels, triacylglycerol (TAG) (<5% of cell dry weight (CDW)) and polyhydroxyalkanoates (PHA) (3.8% ±â€¯1.1% of the CDW) as well as an unknown lipid (29% ±â€¯6% of CDW), presumably a wax ester-like compound. The influence of culture conditions, namely carbon and nitrogen concentrations (and C/N ratio) and cultivation time, on the amount and profile of produced storage compounds was further assessed using a statistical approach based on a central composite circumscribed design and surface response methodology. The regression analysis of the experimental design revealed that only nitrogen concentration and C/N ratio are significant for neutral lipid biosynthesis (p < 0.05). Maximum neutral lipid content, i.e. 33% (CDW basis), was achieved for the lowest carbon and nitrogen concentrations evaluated (10 g COD L-1 and 0.02 g N L-1). PHA accounted for less than 5% of CDW. In these conditions, neutral lipid content was mainly composed by TAG, about 70% (w/w). TAG precursors, namely monoacylglycerols (MAG), diacylglycerols (DAG) and fatty acids (FA), accounted for 22% of total neutral lipids and WE for about 7%. Nevertheless, according to the applied response surface model, further improvement of neutral lipids content is still possible if even lower nitrogen concentrations are used. The fatty acids detected in TAG extracts ranged from myristic acid (C14:0) to linoleic acid (C18:2), being the most abundant palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1). This study shows the feasibility of combining treatment of hydrocarbon contaminated wastewater, herein demonstrated for lubricant-based wastewater, with the production of bacterial neutral lipids using open mixed bacterial communities. This approach can decrease the costs associated to both processes and contribute to a more sustainable waste management and production of lipid-based biofuels.

Harnessing the Power of PCR Molecular Fingerprinting Methods and Next Generation Sequencing for Understanding Structure and Function in Microbial Communities.

Polymerase chain reaction (PCR) is central to methods in molecular ecology. Here, we describe PCR-dependent approaches useful for investigating microbial diversity and its function in various natural, human-associated, and built environment ecosystems. Protocols routinely used for DNA extraction, purification, cloning, and sequencing are included along with various resources for the statistical analysis following gel electrophoresis-based methods (DGGE) and sequencing. We also provide insights into eukaryotic microbiome analysis, sample preservation techniques, PCR troubleshooting, DNA quantification methods, and commonly used ordination techniques.

Boosting dark fermentation with co-cultures of extreme thermophiles for biohythane production from garden waste.

Proof of principle of biohythane and potential energy production from garden waste (GW) is demonstrated in this study in a two-step process coupling dark fermentation and anaerobic digestion. The synergistic effect of using co-cultures of extreme thermophiles to intensify biohydrogen dark fermentation is demonstrated using xylose, cellobiose and GW. Co-culture of Caldicellulosiruptor saccharolyticus and Thermotoga maritima showed higher hydrogen production yields from xylose (2.7±0.1molmol(-1) total sugar) and cellobiose (4.8±0.3molmol(-1) total sugar) compared to individual cultures. Co-culture of extreme thermophiles C. saccharolyticus and Caldicellulosiruptor bescii increased synergistically the hydrogen production yield from GW (98.3±6.9Lkg(-1) (VS)) compared to individual cultures and co-culture of T. maritima and C. saccharolyticus. The biochemical methane potential of the fermentation end-products was 322±10Lkg(-1) (CODt). Biohythane, a biogas enriched with 15% hydrogen could be obtained from GW, yielding a potential energy generation of 22.2MJkg(-1) (VS).

Rhodococcus opacus B4: a promising bacterium for production of biofuels and biobased chemicals.

Bacterial lipids have relevant applications in the production of renewable fuels and biobased oleochemicals. The genus Rhodococcus is one of the most relevant lipid producers due to its capability to accumulate those compounds, mainly triacylglycerols (TAG), when cultivated on different defined substrates, namely sugars, organic acids and hydrocarbons but also on complex carbon sources present in industrial wastes. In this work, the production of storage lipids by Rhodococcus opacus B4 using glucose, acetate and hexadecane is reported for the first time and its productivity compared with Rhodococcus opacus PD630, the best TAG producer bacterium reported. Both strains accumulated mainly TAG from all carbon sources, being influenced by the carbon source itself and by the duration of the accumulation period. R. opacus B4 produced 0.09 and 0.14 g L(-1) at 24 and 72 h, with hexadecane as carbon source, which was 2 and 3.3 fold higher than the volumetric production obtained by R. opacus PD630. Both strains presented similar fatty acids (FA) profiles in intact cells while in TAG produced fraction, R. opacus B4 revealed a higher variability in fatty acid composition than R. opacus PD630, when both strains were cultivated on hexadecane. The obtained results open new perspectives for the use of R. opacus B4 to produce TAG, in particular using oily (alkane-contaminated) waste and wastewater as cheap raw-materials. Combining TAG production with hydrocarbons degradation is a promising strategy to achieve environmental remediation while producing added value compounds.

Valorization of lubricant-based wastewater for bacterial neutral lipids production: Growth-linked biosynthesis.

Lipids produced by microorganisms are currently of great interest as raw material for either biofuels or oleochemicals production. Significant biosynthesis of neutral lipids, such as triacylglycerol (TAG) and wax esters (WE) are thought to be limited to a few strains. Hydrocarbonoclastic bacteria (HCB), key players in bioremediation of hydrocarbon contaminated ecosystems, are among this group of strains. Hydrocarbon rich wastewaters have been overlooked concerning their potential as raw material for microbial lipids production. In this study, lubricant-based wastewater was fed, as sole carbon source, to two HCB representative wild strains: Alcanivorax borkumensis SK2, and Rhodococcus opacus PD630. Neutral lipid production was observed with both strains cultivated under uncontrolled conditions of pH and dissolved oxygen. A. borkumensis SK2 was further investigated in a pH- and OD-controlled fermenter. Different phases were assessed separately in terms of lipids production and alkanes removal. The maximum TAG production rate occurred during stationary phase (4 mg-TAG/L h). The maximum production rate of WE-like compounds was 15 mg/L h, and was observed during exponential growth phase. Hydrocarbons removal was 97% of the gas chromatography (GC) resolved straight-chain alkanes. The maximum removal rate was observed during exponential growth phase (6 mg-alkanes/L h). This investigation proposes a novel approach for the management of lubricant waste oil, aiming at its conversion into valuable lipids. The feasibility of the concept is demonstrated under low salt (0.3%) and saline (3.3%) conditions, and presents clues for its technological development, since growth associated oil production opens the possibility for establishing continuous fermentation processes.

Ultrasound intensification suppresses the need of methanol excess during the biodiesel production with Lipozyme TL-IM.

The synthesis of biodiesel from sunflower oil and methanol based on transesterification using the immobilized lipase from Thermomyces lanuginosus (Lipozyme TL-IM) has been investigated under silent conditions and under an ultrasound field. Ultrasound assisted process led to reduced processing time and requirement of lower enzyme dosage. We found for the first time that oil to methanol ratio of 1:3 was favored for the ultrasound assisted enzymatic process which is lower than that observed for the case of conventional stirring based approach (ratio of 1.4). Our results indicate that intensification provided by ultrasound suppresses the need of the excess of the methanol reactant during the enzymatic biodiesel production. Ultrasound assisted enzymatic biodiesel production is therefore a faster and a cleaner processes.

Production of fermented cheese whey-based beverage using kefir grains as starter culture: evaluation of morphological and microbial variations.

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grain's matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.

Molecular monitoring of microbial diversity in expanded granular sludge bed (EGSB) reactors treating oleic acid.

Abstract A molecular approach was used to evaluate the microbial diversity of bacteria and archaea in two expanded granular sludge bed (EGSB) reactors fed with increasing oleic acid loading rates up to 8 kg of chemical oxygen demand (COD) m(-3) day(-1) as the sole carbon source. One of the reactors was inoculated with granular sludge (RI) and the other with suspended sludge (RII). During operation, the sludge in both reactors was segregated in two layers: a bottom settled one and a top floating one. The composition of the bacterial community, based on 16S rDNA sequence diversity, was affected most during the oleate loading process in the two reactors. The archaeal consortium remained rather stable over operation in RI, whereas in RII the relative abundance of Methanosaeta-like organisms became gradually weaker, starting in the bottom layer. In the range of oleate loads evaluated, 6 kg of COD m(-3) day(-1) was found as the maximum value that could be applied to the system. A further increase to 8 kg of oleate-COD m(-3) day(-1) induced a maximal shift on the microbial structure of the sludges. At this time point, methanogenic acetoclastic activity was not detected and only very low methanogenic activity on H(2)/CO(2) was exhibited by the sludges.