Rapid acclimation of methanogenic granular sludge into denitrifying sulfide removal granules

Rapid formation of denitrifying sulfide removal granules is of practical interest to start up an expanded granular sludge bed reactor for wastewater treatment. This study demonstrates that methanogenic granules can be easily acclimated into DSR granules in one day, removing all 1.30 kg m⁻³ d⁻¹ sulfide and converting >90% of 0.56 kg-N m⁻³d⁻¹ nitrate into di-nitrogen gas. Under high loadings, reactor performance, however, declined. Under high loading rates, sulfide first inhibited the heterotrophic denitrifier (Caldithrix sp.), thereby accumulating nitrite in the system; the autotrophic denitrifier (Pseudomonas sp. C23) was then inhibited by accumulated nitrite, leading to breakdown of the entire DSR process.     

Functional consortium for denitrifying sulfide removal process

Denitrifying sulfide removal (DSR) process simultaneously converts sulfide, nitrate, and chemical oxygen demand from industrial wastewaters to elemental sulfur, nitrogen gas, and carbon dioxide, respectively. This investigation utilizes a dilution-to-extinction approach at 10⁻² to 10⁻⁶ dilutions to elucidate the correlation between the composition of the microbial community and the DSR performance. In the original suspension and in 10⁻² dilution, the strains Stenotrophomonas sp., Thauera sp., and Azoarcus sp. are the heterotrophic denitrifiers and the strains Paracoccus sp. and Pseudomonas sp. are the sulfide-oxidizing denitrifers. The 10⁻⁴ dilution is identified as the functional consortium for the present DSR system, which comprises two functional strains, Stenotrophomonas sp. strain Paracoccus sp. At 10⁻⁶ dilution, all DSR performance was lost. The functions of the constituent cells in the DSR granules were discussed based on data obtained using the dilution-to-extinction approach.     

Reduction of produced elementary sulfur in denitrifying sulfide removal process

Denitrifying sulfide removal (DSR) processes simultaneously convert sulfide, nitrate, and chemical oxygen demand from industrial wastewater into elemental sulfur, dinitrogen gas, and carbon dioxide, respectively. The failure of a DSR process is signaled by high concentrations of sulfide in reactor effluent. Conventionally, DSR reactor failure is blamed for overcompetition for heterotroph to autotroph communities. This study indicates that the elementary sulfur produced by oxidizing sulfide that is a recoverable resource from sulfide-laden wastewaters can be reduced back to sulfide by sulfur-reducing Methanobacterium sp. The Methanobacterium sp. was stimulated with excess organic carbon (acetate) when nitrite was completely consumed by heterotrophic denitrifiers. Adjusting hydraulic retention time of a DSR reactor when nitrite is completely consumed provides an additional control variable for maximizing DSR performance.     

Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor

High-rate biological conversion of sulfide and nitrate in synthetic wastewater to, respectively, elemental sulfur (S⁰) and nitrogen-containing gas (such as N₂) was achieved in an expanded granular sludge bed (EGSB) reactor. A novel strategy was adopted to first cultivate mature granules using anaerobic sludge as seed sludge in sulfate-laden medium. The cultivated granules were then incubated in sulfide-laden medium to acclimate autotrophic denitrifiers. The incubated granules converted sulfide, nitrate, and acetate simultaneously in the same EGSB reactor to S⁰, N-containing gases and CO₂ at loading rates of 3.0 kg S m⁻³ d⁻¹, 1.45 kg N m⁻³ d⁻¹, and 2.77 kg Ac m⁻¹ d⁻¹, respectively, and was not inhibited by sulfide concentrations up to 800 mg l⁻¹. Effects of the C/N ratio on granule performance were identified. The granules cultivated in the sulfide-laden medium have Pseudomonas spp. and Azoarcus sp. presenting the heterotrophs and autotrophs that co-work in the high-rate EGSB-SDD (simultaneous desulfurization and denitrification) reactor.     

Mechanical shear contributes to granule formation resulting in quick start-up and stability of a hybrid anammox reactor

It appears that if suspended biomass washout can be reduced effectively, granule formation will be fastened in fluidized bed. Quicker reactor start-up can be anticipated especially for those system keeping slow growth bacteria such as anammox. A hybrid reactor combined fixed-bed with nonwoven fabrics as biomass carrier and fluidized bed with slow speed mechanical stirring was therefore developed, and its nitrogen removal performances was evaluated experimentally. Only in 38 days, the total nitrogen removal rate (NRR) reached to 1.9 kg(N) m⁻³ day⁻¹ and then doubled within 17 days, with total nitrogen removal efficiency kept above 70%. After 180 days reactor operating, the NRR reached a maximum value of 6.6 kg(N) m⁻³ day⁻¹ and the specific anammox activity was gradually constant in 0.32 kg(N) kg(VSS)⁻¹ day⁻¹. Biomass attached on nonwoven fabrics could additionally improve reactor nitrogen removal by 8%. The dominant size of granular sludge reached to 0.78 mm with stirring speed adjusted from 30 to 80 rpm and the hydraulic retention time (HRT) from 8 to 1.5 h during the whole operating time. Scanning electron microscope observation showed especially compact structure of granular sludge. A 70% of anammox bacteria percentage was identified by fluorescence in situ hybridization analysis.     

Potential cause of aerobic granular sludge breakdown at high organic loading rates

Aerobic sludge granules are compact, strong microbial aggregates that have excellent settling ability and capability to efficiently treat high-strength and toxic wastewaters. Aerobic granules disintegrate under high organic loading rates (OLR). This study cultivated aerobic granules using acetate as the sole carbon and energy source in three identical sequencing batch reactors operated under OLR of 9–21.3 kg chemical oxygen demand (COD) m⁻³ day⁻¹. The cultivated granules removed 94–96% of fed COD at OLR up to 9–19.5 kg COD m⁻³ day⁻¹, and disintegrated at OLR of 21.3 kg COD m⁻³ day⁻¹. Most tested isolates did not grow in the medium at >3,000 mg COD l⁻¹; additionally, these strains lost capability for auto-aggregation and protein or polysaccharide productivity. This critical COD regime correlates strongly with the OLR range in which granules started disintegrating. Reduced protein quantity secreted by isolates was associated with the noted poor granule integrity under high OLR. This work identified a potential cause of biological nature for aerobic granules breakdown.     

An upflow microaerobic sludge blanket reactor operating at high organic loading and low dissolved oxygen levels

The activated sludge process (ASP) has high operational costs due to the need for aeration at dissolved O₂ (DO) levels of ≥2 mg l⁻¹ and high capital costs to construct large reactors due to a low organic loading [typically 1 kg chemical oxygen demand (COD) m⁻³ day⁻¹]. A novel method for improving the energy use and treatment efficiency of the ASP via limited oxygenation (0.4 mg DO l⁻¹) and high organic loading (6.2 kg COD m⁻³ day⁻¹) is proposed based on a laboratory-scale ASP for ammonia-rich industrial wastewaters. The sludge blanket phenomenon and granulation occurred simultaneously in the upflow microaerobic reactor.     

Microbial community structure in anaerobic co-digestion of grass silage and cow manure in a laboratory continuously stirred tank reactor

The impacts of feeding ratio and loading rate on the microbial community during co-digestion of grass silage with cow manure in an anaerobic laboratory continuously stirred tank reactor were investigated by 16S rRNA gene-based fingerprints. The microbial community remained stable when the reactor was fed with cow manure alone and with up to 20% of grass silage in feedstock at an organic loading rate (OLR) of 2 kg VS m⁻³ day⁻¹. Large changes in the bacterial community were observed when the loading ratio of grass was increased to 40%, while there was little change in the archaeal community. During the increase in OLR from 2 to 4 kg VS m⁻³ day⁻¹ the bacterial community structure showed few differences, whereas Archaea was undetectable. Sequencing of the major DGGE bands indicated that the phylum Bacteriodetes predominated in the bacterial community. Two unclassified bacteria with high abundance survived throughout the operation of the reactor.     

Enhancing denitrifying sulfide removal with functional strains under micro-aerobic condition

The biological denitrifying sulfide reaction (DSR) frequently proceeds in anaerobic environments since excess oxygen inhibits the activity of the denitrifiers. This study isolated a consortium H7, comprising two strains, Penibacillus sp. and Aneurinibacillus aneurinilyticus. It indicated that the DSR performance with the H7 in a mixotrophic medium is significantly enhanced at high sulfide concentrations. The H7 was inhibited by adding >200 mg l⁻¹ of S²⁻ under anaerobic conditions. However, when 280 mg ⁻¹ of S²⁻ was added, H7 could still degrade some of the sulfide, nitrate and acetate under micro-aerobic conditions. Micro-aerobic conditions stimulated the activity of sulfide oxidase and increased the removal rate of highly concentrated sulfide, reducing the inhibition of sulfide on denitrifiers and improving DSR performance.     

Biotreatment of phenol-contaminated wastewater in a spiral packed-bed bioreactor

A spiral packed-bed bioreactor inoculated with microorganisms obtained from activated sludge was used to conduct a feasibility study for phenol removal. The reactor was operated continuously at various phenol loadings ranging from 53 to 201.4 g m⁻³ h⁻¹, and at different hydraulic retention times (HRT) in the range of 20–180 min to estimate the performance of the device. The results indicated that phenol removal efficiency ranging from 82.9 to 100% can be reached when the reactor is operated at an HRT of 1 h and a phenol loading of less than 111.9 g m⁻³ h⁻¹. At an influent phenol concentration of 201.4 g m⁻³, the removal efficiency increased from 18.6 to 76.9% with an increase in the HRT (20–120 min). For treatment of phenol in the reactor, the maximum biodegradation rate (V _m) was 1.82 mg l⁻¹ min⁻¹; the half-saturation constant (K _s), 34.95 mg l⁻¹.     

Biological nitrification–denitrification with alternating oxic and anoxic operations using aerobic granules

Efficient nitrification and denitrification of wastewater containing 1,700 mgl⁻¹ of ammonium-nitrogen was achieved using aerobic granular sludge cultivated at medium-to-high organic loading rates. The cultivated granules were tested in a sequencing batch reactor (SBR) fed with 6.4 or 10.2 kg NH₄⁺-N m⁻³ day⁻¹, a loading significantly higher than that reported in literature. With alternating 2 h oxic and 2 h anoxic operation (OA) modes, removal rate was 45.5 mg NH₄⁺-N g⁻¹ volatile suspended solids⁻¹ h⁻¹ at 6.4 kg NH₄⁺-N m⁻³ day⁻¹ loading and 41.3 ± 2.0 at 10.2 kg NH₄⁺-N m⁻³ day⁻¹ loading. Following the 60 days SBR test, granules were intact. The fluorescence in situ hybridization and confocal laser scanning microscopy results indicate that the SBR-OA granules have a distribution with nitrifers outside and heterotrophs outside that can effectively expose functional strains to surrounding substrates at high concentrations with minimal mass transfer limit. This microbial alignment combined with the smooth granule surface achieved nitrification–denitrification of wastewaters containing high-strength ammonium using aerobic granules. Conversely, the SBR continuous aeration mode yielded a distribution with nitrifers outside and heterotrophs inside with an unsatisfactory denitrification rate and floating granules as gas likely accumulated deep in the granules.     

Treatment of food processing wastewater in a full-scale jet biogas internal loop anaerobic fluidized bed reactor

A full-scale jet biogas internal loop anaerobic fluidized bed (JBILAFB) reactor, which requires low energy input and allows enhanced mass transfer, was constructed for the treatment of food processing wastewater. This reactor has an active volume of 798 m³ and can treat 33.3 m³ wastewater per hour. After pre-treating the raw wastewater by settling, oil separating and coagulation-air floating processes, the reactor was operated with a relatively shorter start-up time (55 days). Samples for the influent and effluent of the JBILAFB reactor were taken and analyzed daily for the whole process including both the start-up and stable running periods. When the volumetric COD loading fluctuated in the range of 1.6–5.6 kg COD m⁻³ day⁻¹, the COD removal efficiency, the volatile fatty acid(VFA)/alkalinity ratio, the maximum biogas production and the content of CH₄ in total biogas of the reactor were found to be 80.1 ± 5%, 0.2–0.5, 348.5 m³ day⁻¹ and 94.5 ± 2.5%, respectively. Furthermore, the scanning electron microscope (SEM) results showed that anaerobic granular sludge and microorganism particles with biofilm coexisted in the reactor, and that the bacteria mainly in bacilli and cocci were observed as predominant species. All the data demonstrated that the enhanced mass transfer for gas, liquid and solid phases was achieved, and that the formation of microorganism granules and the removal of inhibitors increased the stability of the system.     

Life-history, thallus ontogeny, and the effects of temperature, irradiance and salinity on growth of the edible green seaweed Gayralia spp. (Chlorophyta) from Southern Brazil

Gayralia K.L. Vinogr. is a monostromatic green alga of commercial importance in the southern Brazil, and its cultivation is being considered. This paper reports some basic aspects of the biology of this poorly known genus. Two populations of Gayralia spp., from outer and inner sectors of Paranaguá Bay, showed an asexual life history with a distinct pattern of thallus ontogeny. In one population (Gayralia sp. 1), zooids developed an expanded monostromatic blade directly, while in the other (Gayralia sp. 2) zooids produced an intermediate saccate stage, before giving rise to a monostromatic blade. Thalli of the two species differ in size and in cell diameter. The effects of temperature (16–30°C), irradiance (50–100 μmol photons m⁻² s⁻¹), and salinity (5–40 psu) on the growth of both populations were assessed. Plantlets of Gayralia sp. 1 from in vitro cultures showed a broader tolerance to all salinity and irradiance levels tested, with the highest growth rate (GR; mean 17% day⁻¹) at 21.5°C and 100 μmol photons m⁻² s⁻¹. Plantlets of Gayralia sp. 1 collected during the winter in the field showed higher GR, ranging from 5% day⁻¹ to 7.5% day⁻¹ in salinities from 20 to 40 psu, and 2.0% day⁻¹ and 4.3% day⁻¹ for plantlets collected during the summer. Gayralia sp. 2 from the field showed highest GR at salinity of 15 psu. These results suggest distinct physiological responses of the two species, in accordance with their distribution: Gayralia sp. 2 is limited to the inner areas of the estuary, while Gayralia sp. 1 grows in outer areas, where salinity values are higher than 20 psu. These data indicate that Gayralia sp. 1 has a higher potential for aquaculture than Gayralia sp. 2 due to its larger thalli, higher GR, and wider tolerance to environmental variations.     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.     

Utilization of <Emphasis Type="Italic">Anabaena</Emphasis> sp. in CO<Subscript>2</Subscript> removal processes

This paper focuses on modelling the growth rate and exopolysaccharides production of Anabaena sp. ATCC 33047, to be used in carbon dioxide removal and biofuels production. For this, the influence of dilution rate, irradiance and aeration rate on the biomass and exopolysaccharides productivity, as well as on the CO₂ fixation rate, have been studied. The productivity of the cultures was maximum at the highest irradiance and dilution rate assayed, resulting to 0.5 g_bio l⁻¹ day⁻¹ and 0.2 g_eps l⁻¹ day⁻¹, and the CO₂ fixation rate measured was 1.0 gCO₂ l⁻¹ day⁻¹. The results showed that although Anabaena sp. was partially photo-inhibited at irradiances higher than 1,300 μE m⁻² s⁻¹, its growth rate increases hyperbolically with the average irradiance inside the culture, and so does the specific exopolysaccharides production rate. The latter, on the other hand, decreases under high external irradiances, indicating that the exopolysaccharides metabolism hindered by photo-damage. Mathematical models that consider these phenomena have been proposed. Regarding aeration, the yield of the cultures decreased at rates over 0.5 v/v/min or when shear rates were higher than 60 s⁻¹, demonstrating the existence of thus existence of stress damage by aeration. The behaviour of the cultures has been verified outdoors in a pilot-scale airlift tubular photobioreactor. From this study it is concluded that Anabaena sp. is highly recommended to transform CO₂ into valuable products as has been proved capable of metabolizing carbon dioxide at rates of 1.2 gCO₂ l⁻¹ day⁻¹ outdoors. The adequacy of the proposed equations is demonstrated, resulting to a useful tool in the design and operation of photobioreactors using this strain.     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Estimation of periphytic microalgae gross primary production with DCMU-fluorescence method in Yenisei River (Siberia, Russia)

Periphyton (epilithon) gross primary production (GPP) was estimated using the DCMU-fluorescence method in the Yenisei River. In the unshaded littoral zone, chlorophyll a concentration (Chl a) and GPP value varied from 0.83 to 973.74 mg m⁻²and 2–304,425 O₂ m⁻² day⁻¹ (0.64–95 133 mg C m⁻² day⁻¹), respectively. Positive significant correlation (r = 0.8) between daily GPP and periphyton Chl a was found. Average ratio GPP:Chl a for periphyton was 36.36 mg C mg Chl a m⁻² day⁻¹. The obtained GPP values for the Yenisei River have a high significant correlation with values predicted by a conventional empirical model for stream periphyton. We concluded that the DCMU-fluorescence method can be successfully used for measuring of gross primary production of stream phytoperiphyton at least as another useful tool for such studies.     

Anaerobic digestibility of marine microalgae <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> in a lab-scale anaerobic membrane bioreactor

The biomass of industrially grown Phaeodactylum tricornutum was subjected in a novel way to bio-methanation at 33°C, i.e., in an anaerobic membrane bioreactor (AnMBR) at a hydraulic retention time of 2.5 days, at solid retention times of 20 to 10 days and at loading rates in the range of 2.6–5.9 g biomass-COD L⁻¹ day⁻¹ with membrane fluxes ranging from 1 to 0.8 L m⁻² h⁻¹. The total COD recovered as biogas was in the order of 52%. The input suspension was converted to a clear effluent rich in total ammonium nitrogen (546 mg TAN L⁻¹) and phosphate (141 mg PO₄-P L⁻¹) usable as liquid fertilizer. The microbial community richness, dynamics, and organization in the reactor were interpreted using the microbial resource management approach. The AnMBR communities were found to be moderate in species richness and low in dynamics and community organization relative to UASB and conventional CSTR sludges. Quantitative polymerase chain reaction analysis revealed that Methanosaeta sp. was the dominant acetoclastic methanogen species followed by Methanosarcina sp. This work demonstrated that the use of AnMBR for the digestion of algal biomass is possible. The fact that some 50% of the organic matter is not liquefied means that the algal particulates in the digestate constitute a considerable fraction which should be valorized properly, for instance as slow release organic fertilizer. Overall, 1 kg of algae dry matter (DM) could be valorized in the form of biogas (€2.07), N and P in the effluent (€0.02) and N and P in the digestate (€0.04), thus totaling about €2.13 per kilogram algae DM.     

Functional Characterization of the Microbial Community in Geothermally Heated Marine Sediments

The microbial population of geothermally heated sediments in a shallow bay of Vulcano Island (Italy) was characterized with respect to metabolic activities and the putatively catalyzing hyperthermophiles. Site-specific anoxic culturing media, most of which were amended with combinations of electron donors (glucose or carboxylic acids) and acceptors (sulfate), were used for selective enrichment of metabolically defined subpopulations. The mostly archaeal chemoautotrophs produced formate at rates of 3.25 and 0.46 fmol cell⁻¹ day⁻¹ with and without sulfate, respectively. The glucose fermenting heterotrophs produced acetate (18 fmol cell⁻¹ day⁻¹) and lactate (2.6 fmol cell⁻¹ day⁻¹) and were identified as predominantly Thermus sp. and coccoid archaea. These archaeal cells also metabolized lactate (5.6 fmol cell⁻¹ day⁻¹), but neither formate nor acetate. The heterotrophic culture enriched on formate/acetate/propionate/sulfate utilized mainly formate (27 fmol cell⁻¹ day⁻¹) and lactate (89–195 fmol cell⁻¹ day⁻¹), and consumed sulfate (38–68 fmol cell⁻¹ day⁻¹). These formate or lactate consuming sulfate reducers were dominated by Archaeoglobales (7% in situ) and unidentified Archaea. The in situ benthic community comprised 15% Crenarchaeota, a significant group only in the autotrophic cultures, and 3% Thermus sp., the putatively predominant group involved in fermentative metabolism. The role of Thermoccales (4% in situ) remained undisclosed in our experiments. This first comprehensive data set established plausible links between several groups of hyperthermophiles in shallow marine hydrothermal systems, their metabolic function within the benthic microbial community, and biogeochemical turnover rates.     

Cell growth and nutritive value of the tropical benthic diatom, <Emphasis Type="Italic">Amphora</Emphasis> sp., at varying levels of nutrients and light intensity,and different culture locations

Two series of experiments were conducted to determine suitable growth factors for the mass propagation of the local algal isolate Amphora sp. A higher growth rate of 0.2 doubling (μ) day⁻¹ was attained at a lower photosynthetic photon flux density (PPFD; 11.4 μmol photon m⁻²s⁻¹) compared to cultures exposed to higher levels of PPFD (16.1 μmol photon m⁻²s⁻¹, −0.1 μ day ⁻¹; 31.3 μmol photon m⁻²s⁻¹, 0.0 μ day⁻¹). Cultures located inside the laboratory had a significantly higher cell density (133 × 10⁴ cells cm⁻²) and growth rate (0.3 μ day⁻¹) compared to those located outdoors (100 × 10⁴ cells cm⁻², 0.2 μ day⁻¹). A comparison of nutrient medium across two locations showed that lipid content was significantly higher in cultures enriched with F/2MTM (macronutrients + trace metals) and F/2MV (macronutrients + vitamins). Saturated fatty acids were also present in high concentrations in cultures enriched with F/2M (macronutrients only). Significantly higher amounts of saturated fatty acids were observed in cultures located outdoors (33.1%) compared to those located indoors (26.6%). The protein, carbohydrates and n-6 fatty acid content of Amphora sp. were influenced by the location and enrichment of the cultures. This study has identified growth conditions for mass culture of Amphora sp. and determined biochemical composition under those culture conditions. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.