Strategies to suppress hydrogen-consuming microorganisms affect macro and micro scale structure and microbiology of granular sludge.

Treatment of anaerobic granules with heat and two chemical treatments, contacting with 2-bromoethanesulfonate (BES) and with BES + Chloroform, were applied to suppress hydrogen-consuming microorganisms. Three mesophilic expanded granular sludge bed (EGSB) reactors-R(Heat), R(BES), and R(BES + Chlo)--were inoculated with the treated sludges and fed with synthetic sugar-based wastewater (5 g(COD) L(-1), HRT 20-12 h). Morphological integrity of granules and bacterial communities were assessed by quantitative image analysis and 16S rRNA gene based techniques, respectively. Hydrogen production in R(Heat) was under 300 mL H(2) L(-1) day(-1), with a transient peak of 1,000 mL H(2) L(-1) day(-1) after decreasing HRT. In R(BES + Chlo) hydrogen production rate did not exceed 300 mL H(2) L(-1) day(-1) and there was granule fragmentation, release of free filaments from aggregates, and decrease of granule density. In R(BES), there was an initial period with unstable hydrogen production, but a pulse of BES triggered its production rate to 700 ± 200 mL H(2) L(-1) day(-1). This strategy did not affect granules structure significantly. Bacteria branching within Clostridiaceae and Ruminococcaceae were present in this sludge. This work demonstrates that, methods applied to suppress H(2)-consuming microorganisms can cause changes in the macro- and microstructure of granular sludge, which can be incompatible with the operation of high-rate reactors.

Zeolite addition to improve biohydrogen production from dark fermentation of C5/C6-sugars and Sargassum sp. biomass.

Thermophilic biohydrogen production by dark fermentation from a mixture (1:1) of C5 (arabinose) and C6 (glucose) sugars, present in lignocellulosic hydrolysates, and from Sargassum sp. biomass, is studied in this work in batch assays and also in a continuous reactor experiment. Pursuing the interest of studying interactions between inorganic materials (adsorbents, conductive and others) and anaerobic bacteria, the biological processes were amended with variable amounts of a zeolite type-13X in the range of zeolite/inoculum (in VS) ratios (Z/I) of 0.065-0.26 g g-1. In the batch assays, the presence of the zeolite was beneficial to increase the hydrogen titer by 15-21% with C5 and C6-sugars as compared to the control, and an increase of 27% was observed in the batch fermentation of Sargassum sp. Hydrogen yields also increased by 10-26% with sugars in the presence of the zeolite. The rate of hydrogen production increased linearly with the Z/I ratios in the experiments with C5 and C6-sugars. In the batch assay with Sargassum sp., there was an optimum value of Z/I of 0.13 g g-1 where the H2 production rate observed was the highest, although all values were in a narrow range between 3.21 and 4.19 mmol L-1 day-1. The positive effect of the zeolite was also observed in a continuous high-rate reactor fed with C5 and C6-sugars. The increase of the organic loading rate (OLR) from 8.8 to 17.6 kg m-3 day-1 of COD led to lower hydrogen production rates but, upon zeolite addition (0.26 g g-1 VS inoculum), the hydrogen production increased significantly from 143 to 413 mL L-1 day-1. Interestingly, the presence of zeolite in the continuous operation had a remarkable impact in the microbial community and in the profile of fermentation products. The effect of zeolite could be related to several properties, including the porous structure and the associated surface area available for bacterial adhesion, potential release of trace elements, ion-exchanger capacity or ability to adsorb different compounds (i.e. protons). The observations opens novel perspectives and will stimulate further research not only in biohydrogen production, but broadly in the field of interactions between bacteria and inorganic materials.

Advanced monitoring of high-rate anaerobic reactors through quantitative image analysis of granular sludge and multivariate statistical analysis.

Four organic loading disturbances were performed in lab-scale EGSB reactors fed with ethanol. In load disturbance 1 (LD1) and 2 (LD2), the organic loading rate (OLR) was increased between 5 and 18.5 kg COD m(-3) day(-1), through the influent ethanol concentration increase, and the hydraulic retention time decrease from 7.8 to 2.5 h, respectively. Load disturbances 3 (LD3) and 4 (LD4) were applied by increasing the OLR to 50 kg COD m(-3) day(-1) during 3 days and 16 days, respectively. The granular sludge morphology was quantified by image analysis and was related to the reactor performance, including effluent volatile suspended solids, indicator of washout events. In general, it was observed the selective washout of filamentous forms associated to granules erosion/fragmentation and to a decrease in the specific acetoclastic activity. These phenomena induced the transitory deterioration of reactor performance in LD2, LD3, and LD4, but not in LD1. Extending the exposure time in LD4 promoted acetogenesis inhibition after 144 h. The application of Principal Components Analysis determined a latent variable that encompasses a weighted sum of performance, physiological and morphological information. This new variable was highly sensitive to reactor efficiency deterioration, enclosing variations between 27% and 268% in the first hours of disturbances. The high loadings raised by image analysis parameters, especially filaments length per aggregates area (LfA), revealed that morphological changes of granular sludge, should be considered to monitor and control load disturbances in high rate anaerobic (granular) sludge bed digesters.

Garden and food waste co-fermentation for biohydrogen and biomethane production in a two-step hyperthermophilic-mesophilic process.

Co-fermentation of garden waste (GW) and food waste (FW) was assessed in a two-stage process coupling hyperthermophilic dark-fermentation and mesophilic anaerobic digestion (AD). In the first stage, biohydrogen production from individual substrates was tested at different volatile solids (VS) concentrations, using a pure culture of Caldicellulosiruptor saccharolyticus as inoculum. FW concentrations (in VS) above 2.9 g L-1 caused a lag phase of 5 days on biohydrogen production. No lag phase was observed for GW concentrations up to 25.6 g L-1. In the co-fermentation experiments, the highest hydrogen yield (46 ±â€¯1 L kg-1) was achieved for GW:FW 90:10% (w/w). In the second stage, a biomethane yield of 682 ±â€¯14 L kg-1 was obtained using the end-products of GW:FW 90:10% co-fermentation. The energy generation predictable from co-fermentation and AD of GW:FW 90:10% is 0.5 MJ kg-1 and 24.4 MJ kg-1, respectively, which represents an interesting alternative for valorisation of wastes produced locally in communities.

Quantitative image analysis as a diagnostic tool for identifying structural changes during a revival process of anaerobic granular sludge.

Due to unspecified operational problems, the specific acetoclastic activity (SAA) of the anaerobic granular sludge present in an industrial UASB reactor was considerably damaged (from 250 to less than 10mL CH(4)@STP/gVSS.d), significantly reducing the biogas production of that industrial unit. The hydrogenotrophic methanogenic activity exhibited a value of 600mL CH4@STP/gVSS.d, the settling velocity was 31.4+/-9.8m/h, the average equivalent diameter was 0.92+/-0.43mm, and about 70% of the VSS were structured in aggregates larger than 1mm. In order to study the recovery of the SAA, this sludge was collected and inoculated in a lab-scale expanded granular sludge blanket (EGSB) reactor. Ethanol was fed as the sole carbon source during a trial period of 106 days. Process monitoring included COD removal efficiency, methane production, and periodic determination of the specific methanogenic activity in the presence of acetate, propionate, butyrate, ethanol and H(2)/CO(2). Quantitative image analysis allowed for information to be obtained on granular fragmentation/erosion and filaments release. During the first operational period, biogas production was mainly due to the hydrogenotrophic activity. However, after 40 days, the SAA steadily increased achieving a maximum value of 183+/-13mL CH4@STP/gVSS.d. The onset of SAA recovery, granules breakdown and filaments release to the bulk occurred simultaneously. Further increase in SAA was accompanied by granular growth. In the last 25 days of operation, the size distribution was stable with more than 80% of projected area of aggregates corresponding to granules larger than 1mm (equivalent diameter). Confocal images from FISH hybridized sections of the granules showed that after SAA recovery, the granules developed an organized structure where an acidogenic/acetogenic external layer was apparent. Granular fragmentation and increase of filaments in the bulk, simultaneously with the increase in the acetoclastic activity are described for the first time and might represent a structural response of granular sludge to promote the optimal substrate uptake at minimal diffusion limitations.

Quantitative image analysis as a diagnostic tool for monitoring structural changes of anaerobic granular sludge during detergent shock loads.

Two shock loads of a commercial detergent (I-150 mg chemical oxygen demand (COD)/L, fed for 56 h; II-300 mg COD/L fed for 222 h) were applied in a lab-scale Expanded Granular Sludge Blanket (EGSB) reactor, fed with 1,500 mg COD/L of ethanol. The impact of the surfactant was assessed in terms of granular sludge morphology, specific methanogenic activity (SMA) in the presence of individual substrates, and reactor performance. COD removal efficiency remained unaffected in the shock I, but 80 h after starting exposure to the shock II, the COD removal efficiency decreased drastically from 75 to 17%. In the first 8 h of operation of shock I, the SMA was stimulated and decreased afterwards, being recovered 5 days after the end of exposure time. Concerning to shock II, the SMA was immediately and persistently reduced during the exposure time, although, the inhibition of SMA in presence of H(2)/CO(2) showed a trend to increase after the exposure time. Acetoclastic bacteria were observed as the most sensitive to the toxic effects of surfactant whereas the hydrogenotrophic bacteria were less affected. The inhibitory effects were dependent on surfactant concentration and exposure time. The ratio filaments length per total aggregates area (LfA) was an early-warning indicator of biomass washout, since it increased 3 and 5 days before effluent volatile suspended solids (VSS) rise, respectively, in shocks I and II.