Features of aerobic granular sludge formation treating fluctuating industrial saline wastewater at pilot scale.

A pilot-scale sequencing batch reactor, with a working volume of 3 m3, was installed in a fish cannery to develop aerobic granular sludge treating the produced effluents. Depending on the nitrogen (N) and organic matter (COD) concentration, the effluents were named in this study as medium-low-strength (Stage I) and high-strength (Stage II) wastewater. The composition of the wastewater was found to be a crucial factor to select granule-forming organisms. With medium-low-strength wastewater as feeding, the first granules were observed after 30 days, but the extremely high COD/N ratios of the wastewater provoked the overgrowth of filamentous bacteria after 4 months of operation (Stage I). When treating high-strength wastewater, stable aggregates with good settleability appeared, but well-shaped granules were not observed since the granulation process was not completed. The system was able to remove both COD (70-95%) and N (30-90%) treating both types of effluents. Biomass growth was the main N removal pathway. The reactor was found to be robust against factory production stops and, thus, a suitable alternative to treat wastewater from industries with discontinuous operation.

Assessment of a fast method to predict the biochemical methane potential based on biodegradable COD obtained by fractionation respirometric tests.

The biochemical methane potential test (BMP) is the most common analytical technique to predict the performance of anaerobic digesters. However, this assay is time-consuming (from 20 to over than 100 days) and consequently impractical when it is necessary to obtain a quick result. Several methods are available for faster BMP prediction but, unfortunately, there is still a lack of a clear alternative. Current aerobic tests underestimate the BMP of substrates since they only detect the easily biodegradable COD. In this context, the potential of COD fractionation respirometric assays, which allow the determination of the particulate slowly biodegradable fraction, was evaluated here as an alternative to early predict the BMP of substrates. Seven different origin waste streams were tested and the anaerobically biodegraded organic matter (CODmet) was compared with the different COD fractions. When considering adapted microorganisms, the appropriate operational conditions and the required biodegradation time, the differences between the CODmet, determined through BMP tests, and the biodegradable COD (CODb) obtained by respirometry, were not significant (CODmet (57.8026 ± 21.2875) and CODb (55.6491 ± 21.3417), t (5) = 0.189, p = 0.853). Therefore, results suggest that the BMP of a substrate might be early predicted from its CODb in only few hours. This methodology was validated by the performance of an inter-laboratory studyconsidering four additional substrates.

Potential of endogenous PHA as electron donor for denitrification.

The use of wastewater streams to obtain polyhydroxyalkanoates (PHA) as high added-value products is widely studied. However, nitrogen removal is not well integrated into this process. In this study, the optimal conditions to track the specific endogenous denitrifying activity (SEDA) driven by PHA as carbon source were selected as: sludge concentration of 0.5-2 g VSS/L, CODPHA/N ratio higher than 5.4 g/g and between 40 and 60 mg NO3--N/L. The seeding biomass used to perform the activity tests was collected from two sequencing batch reactors and was able to store up to 69% wt/wt of PHA. SEDA values of 0.26-0.39 g N2-N/(g VSSact d) were achieved, which proved the potential of PHA-accumulating mixed microbial cultures to be used in nitrogen removal processes. The results indicated that there is not a preference in the consumption of hydroxybutyrate over hydroxyvalerate and that PHA concentrations lower than 5% wt/wt do not allow the obtainment of the maximum SEDA value. Finally, N2O gas production was not detected in the SEDA experiments.

Does the feeding strategy enhance the aerobic granular sludge stability treating saline effluents?

The development and stability of aerobic granular sludge (AGS) was studied in two Sequencing Batch Reactors (SBRs) treating fish canning wastewater. R1 cycle comprised a fully aerobic reaction phase, while R2 cycle included a plug-flow anaerobic feeding/reaction followed by an aerobic reaction phase. The performance of the AGS reactors was compared treating the same effluents with variable salt concentrations (4.97-13.45 g NaCl/L) and organic loading rates (OLR, 1.80-6.65 kg CODs/(m3·d)). Granulation process was faster in R2 (day 34) than in R1 (day 90), however the granular biomass formed in the fully aerobic configuration was more stable to the variable feeding composition. Thus, in R1 solid retention times (SRT), up to 15.2 days, longer than in R2, up to 5.8 days, were achieved. These long SRTs values helped the retention of nitrifying organisms and provoked the increase of the nitrogen removal efficiency to 80% in R1 while it was approximately of 40% in R2. However, the presence of an anaerobic feeding/reaction phase increased the organic matter removal efficiency in R2 (80-90%) which was higher than in R1 with a fully aerobic phase (75-85%). Furthermore, in R2 glycogen-accumulating organisms (GAOs) dominated inside the granules instead of phosphorous-accumulating organisms (PAOs), suggesting that GAOs resist better the stressful conditions of a variable and high-saline influent. In terms of AGS properties an anaerobic feeding/reaction phase is not beneficial, however it enables the production of a better quality effluent.

Novel system configuration with activated sludge like-geometry to develop aerobic granular biomass under continuous flow.

A novel continuous flow system with "flat geometry" composed by two completely mixed aerobic tanks in series and a settler was used to promote the formation of aerobic granular sludge. Making similarities of this system with a typical sequencing batch reactor (SBR), for aerobic granules cultivation, the value of the tank 1/tank 2 vol ratio and the biomass recirculation rate would correspond with the feast/famine length ratio and the length of the operational cycle, respectively, while the settler upflow liquid velocity imposed would be related to the settling time. From the three experiments performed the best results were obtained when the tank 1/tank 2 vol ratio was of 0.28, the sludge recycling ratio of 0.25 and the settler upflow velocity of 2.5 m/h. At these conditions the aggregates had settling velocities between 29 and 113 m/h, sludge volume index at 10 min (SVI10) of 70 mL/g TSS and diameters between 1.0 and 5.0 mm.

Short- and long-term orange dye effects on ammonium oxidizing and anammox bacteria activities.

The effects of orange azo dye over ammonia oxidizing bacteria (AOB) and anammox bacteria activities were tested. Performed batch tests indicated that concentrations lower than 650 mgorange/L stimulated AOB activity, while anammox bacteria activity was inhibited at concentrations higher than 25 mgorange/L. Long-term performance of a continuous stirred tank reactor (CSTR) for the partial nitritation and a sequencing batch reactor (SBR) for the anammox process was tested in the presence of 50 mgorange/L. In the case of the partial nitritation process, both the biomass concentration and the specific AOB activity increased after 50 days of orange azo dye addition. Regarding the anammox process, specific activity decreased down to 58% after 12 days of operation with continuous feeding of 50 mgorange/L. However, the anammox activity was completely recovered only 54 days after stopping the dye addition in the feeding. Once the biomass was saturated the azo dye adsorption onto the biomass was insignificant in the CSTR for the partial nitritation process fed with 50 mgorange/L. However, in the SBR the absorption was determined as 6.4 mgorange/g volatile suspended solids. No biological decolorization was observed in both processes.

Biomass aggregation influences NaN3 short-term effects on anammox bacteria activity.

The main bottleneck to maintain the long-term stability of the partial nitritation-anammox processes, especially those operated at low temperatures and nitrogen concentrations, is the undesirable development of nitrite oxidizing bacteria (NOB). When this occurs, the punctual addition of compounds with the capacity to specifically inhibit NOB without affecting the process efficiency might be of interest. Sodium azide (NaN3) is an already known NOB inhibitor which at low concentrations does not significantly affect the ammonia oxidizing bacteria (AOB) activity. However, studies about its influence on anammox bacteria are unavailable. For this reason, the objective of the present study was to evaluate the effect of NaN3 on the anammox activity. Three different types of anammox biomass were used: granular biomass comprising AOB and anammox bacteria (G1), anammox enriched granules (G2) and previous anammox granules disaggregated (F1). No inhibitory effect of NaN3 was measured on G1 sludge. However, the anammox activity decreased in the case of G2 and F1. Granular biomass activity was less affected (IC50 90 mg/L, G2) than flocculent one (IC50 5 mg/L, F1). Summing up, not only does the granular structure protect the anammox bacteria from the NaN3 inhibitory effect, but also the AOB act as a barrier decreasing the inhibition.

Bacterial community dynamics in long-term operation of a pilot plant using aerobic granular sludge to treat pig slurry.

Aerobic granular sludge represents an interesting approach for simultaneous organic matter and nitrogen removal in wastewater treatment plants. However, the information about microbial communities in aerobic granular systems dealing with industrial wastewater like pig slurry is limited. Herein, bacterial diversity and dynamics were assessed in a pilot scale plant using aerobic granular sludge for organic matter and nitrogen elimination from swine slurry during more than 300 days. Results indicated that bacterial composition evolved throughout the operational period from flocculent activated sludge, used as inoculum, to mature aerobic granules. Bacterial diversity increased at the beginning of the granulation process and then declined due to the application of transient organic matter and nitrogen loads. The operational conditions of the pilot plant and the degree of granulation determined the microbial community of the aerobic granules. Brachymonas, Zoogloea and Thauera were attributed with structural function as they are able to produce extracellular polymeric substances to maintain the granular structure. Nitrogen removal was justified by partial nitrification (Nitrosomonas) and denitrification (Thauera and Zoogloea), while Comamonas was identified as the main organic matter oxidizing bacteria. Overall, clear links between bacterial dynamics and composition with process performance were found and will help to predict their biological functions in wastewater ecosystems improving the future control of the process. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1212-1221, 2016.

Transient concentrations of NaCl affect the PHA accumulation in mixed microbial culture.

The present study explores the feasibility of the accumulation of polyhydroxyalkanoates (PHAs) under the presence of transient concentrations of added sodium chloride, by means of a mixed microbial culture (MMC). This culture was enriched on a mixture of volatile fatty acids (VFAs) containing 0.8g Na(+)/L as NaOH. This MMC presented a maximum PHA accumulation capacity of 53wt% with 27Cmol% HV. Accumulation experiments performed with added NaCl at concentrations of 7, 13 and 20g/L shown that this salt provoked a decrease of the biomass PHA production rate, with an IC50 value close to 7gNaCl/L. The accumulated PHA was lower than the corresponding value of the assay without the addition of salt. Furthermore, the composition of the biopolymer, in terms of HB:HV ratio, changed from 2.71 to 6.37Cmol/Cmol, which means a HV decrease between 27 and 14Cmol%. Summarizing, the PHA accumulation by a MMC non-adapted to saline conditions affected the polymer composition and lead to lower production yields and rates than in absence of added NaCl.

Influence of dissolved oxygen concentration on the start-up of the anammox-based process: ELAN®.

The anammox-based process ELAN® was started-up in two different sequencing batch reactor (SBR) pilot plant reactors treating municipal anaerobic digester supernatant. The main difference in the operation of both reactors was the dissolved oxygen (DO) concentration in the bulk liquid. SBR-1 was started at a DO value of 0.4 mg O2/L whereas SBR-2 was started at DO values of 3.0 mg O2/L. Despite both reactors working at a nitrogen removal rate of around 0.6 g N/(L d), in SBR-1, granules represented only a small fraction of the total biomass and reached a diameter of 1.1 mm after 7 months of operation, while in SBR-2 the biomass was mainly composed of granules with an average diameter of 3.2 mm after the same operational period. Oxygen microelectrode profiling revealed that granules from SBR-2 where only fully penetrated by oxygen with DO concentrations of 8 mg O2/L while granules from SBR-1 were already oxygen penetrated at DO concentrations of 1 mg O2/L. In this way granules from SBR-2 performed better due to the thick layer of ammonia oxidizing bacteria, which accounted for up to 20% of all the microbial populations, which protected the anammox bacteria from non-suitable liquid media conditions.

Filamentous bacteria existence in aerobic granular reactors.

Filamentous bacteria are associated to biomass settling problems in wastewater treatment plants. In systems based on aerobic granular biomass they have been proposed to contribute to the initial biomass aggregation process. However, their development on mature aerobic granular systems has not been sufficiently studied. In the present research work, filamentous bacteria were studied for the first time after long-term operation (up to 300 days) of aerobic granular systems. Chloroflexi and Sphaerotilus natans have been observed in a reactor fed with synthetic wastewater. These filamentous bacteria could only come from the inoculated sludge. Thiothrix and Chloroflexi bacteria were observed in aerobic granular biomass treating wastewater from a fish canning industry. Meganema perideroedes was detected in a reactor treating wastewater from a plant processing marine products. As a conclusion, the source of filamentous bacteria in these mature aerobic granular systems fed with industrial effluents was the incoming wastewater.

Implications of full-scale implementation of an anammox-based process as post-treatment of a municipal anaerobic sludge digester operated with co-digestion.

The feasibility of treating the supernatant of a municipal sludge digester supplemented with co-substrates by means of an anammox-based process (ELAN(®)) was tested in Guillarei (NW of Spain). Ammonia concentration measured in the supernatant of the sludge digester varied in the range 800-1,500 g N/m(3) due to the fact that the sludge produced in the plant was co-digested with wastes coming from surrounding food industries. Treating this supernatant in the ELAN(®) reactor, nitrogen removal rates up to 1.1 kg N/(m(3) d) were reached in experiments run in a pilot plant reactor operated in batch mode. No nitrite oxidation was registered after several months of operation despite the average dissolved oxygen (DO) concentrations being 1.5 g O2/m(3) and the temperature reaching values as low as 18 °C. By keeping the DO set point at 1-2 g O2/m(3) and tuning the hydraulic retention time, the stability of the process was guaranteed and the presence of co-substrates in the anaerobic digester did not affect negatively the operation of the autotrophic nitrogen removal process. Due to the success of the pilot plant experiment, an upscale of the process to full scale is proposed. Mass balances applied to Guillarei wastewater treatment plant revealed that in the main stream line the average denitrification rate calculated with the data of year 2011 was 226 kg N/d. Since the nitrogen removal efficiency is limited by the amount of readily biodegradable organic matter available to carry out denitrification in the water line, the implementation of an anammox-based process to treat the supernatant seems the best option to improve the effluent quality in terms of nitrogen content. The nitrogen removal rate in the sludge line would be 30 times higher than the one in the water line. The implementation of the process would improve the energetic balance and the nitrogen removal performance of the plant.

Enhanced ammonia removal at room temperature by pH controlled partial nitrification and subsequent anaerobic ammonium oxidation.

The Anammox-based processes are suitable for the treatment of wastewaters characterized by a low carbon to nitrogen (C/N) ratio. The application of the Anammox process requires the availability of an effluent with a NO2- -N/NH4+ -N ratio composition around 1 g g-1, which involves the necessity of a previous step where the partial nitrification is performed. In this step, the inhibition of the nitrite-oxidizing bacteria (NOB) is crucial. In the present work, a combined partial nitrification-ANaerobic AMmonia OXidation (Anammox) two-units system operated at room temperature (20 degreeC) has been tested for the nitrogen removal of pre-treated pig slurry. To achieve the successful partial nitrification and inhibit the NOB activity, different ammonium/inorganic carbon (NH4+/IC) ratios were assayed from 1.19 to 0.82g NH4+-Ng-1 HCO3-C. This procedure provoked a decrease of the pH value to 6.0 to regulate the inhibitory effect over ammonia-oxidizing bacteria caused by free ammonia. Simultaneously, the NOB experienced the inhibitory effect of free nitrous acid which avoided the presence of nitrate in the effluent. The NH4+/IC ratio which allowed the obtaining of the desired effluent composition (50% of both ammonium and nitrite) was 0.82 +/- 0.02 g NH4+-N g-1 HCO3- -C. The Anammox reactor was fed with the effluent of the partial nitrification unit containing a NO2 -N/NH4+ -N ratio of 1 g g-1' where a nitrogen loading rate of 0.1 g N L-1 d-1 was efficiently removed.

Influence of the shear stress and salinity on Anammox biofilms formation: modelling results.

Anammox biomass has a long duplication time and low yield, thus the process must be operated in reactors with good sludge retention, such as biofilm systems. Therefore, it would be important to research the ability of Anammox biomass to form biofilms under different conditions. The effects of shear stress and salinity (NaCl and CaCl2) on Anammox biofilm formation were studied. Anammox bacteria showed good attachment capacity, with an initial adhesion phase lasting for 5-7 days at the different flow rates tested (Reynolds numbers 54, 63, 188 and 400). A four-parameter model was developed and the experimental data fitted well into the model. The presence of 5 g/L of each of the two salts favoured the formation of Anammox biofilm. The effects of CaCl2 were stronger than those caused by NaCl. 15 g/L of NaCl was detrimental for the biofilm, probably due to an inhibitory effect.

Effects of the cycle distribution on the performance of SBRs with aerobic granular biomass.

The aerobic granular systems are mainly sequencing batch reactors where the biomass is submitted to feast-famine regimes to promote its aggregation in the form of granules. In these systems, different cycle distributions can be applied for the simultaneous removal of organic matter, nitrogen and phosphorus. In this work two strategies were followed in order to evaluate the effects of the cycle distribution. In the first experiment, the length of the operational cycle was decreased in order to maximize the treatment capacity and consequently the famine/feast ratio was also decreased. In the second experiment, an initial anoxic phase was implemented to improve nitrogen removal efficiency. The results obtained showed that to reduce the famine/feast ratio from 10 to 5 was possible by increasing the treated organic and nitrogen loading rates in the system to 33%, without affecting the removal efficiencies of organic matter (97%) and nitrogen (64%) and producing a slight detriment of the granules characteristics. On the other hand, the implementation of an anoxic phase of 30 min previous to the aerobic one with a pulse-fed mode increased the nitrogen removal of pig manure from 20 to 60%, while the cycle configuration comprising a continuous feeding simultaneous with an anoxic phase of 60 min did not enhance the nitrogen removal and even worsen the ammonia oxidation.

Autotrophic denitrification with sulphide in a sequencing batch reactor.

In this study a sequencing batch reactor was used to simultaneously remove both sulphide and nitrate via an autotrophic denitrification process. The sulphide loading rates were gradually increased from 200 mg S(2-) L(-1) d(-1)-450 mg S(2-) L(-1)d(-1)while the nitrogen loading rates were kept at 450 mg NO(3)(-)-N L(-1)d(-1). The obtained results demonstrated that it was possible to carry out autotrophic denitrification in a Sequencing Batch Reactor with removal efficiencies of sulphide and nitrogen of 100% and 67%, respectively. The efficiency of the process was influenced by the pH value in the reactor. The operation at pH values higher than 9.0 decreased the efficiency of sulphide oxidation into sulphate to 11.3%. The main bacteria populations present in the sludge belonged to Thiobacillus genus.

Short- and long-term effects of ammonium and nitrite on the Anammox process.

Autotrophic anaerobic ammonium oxidation (Anammox) is a biological process in which Planctomycete-type bacteria combine ammonium and nitrite to generate nitrogen gas. Both substrates can exert inhibitory effects on the process, causing the decrease of the specific activity of the biomass and the loss of the stable operation of reactors. The aim of the present work is to evaluate these effects in short- and long-term experiments. The short-term effects were carried out with two different types of Anammox biomass, biofilm on inorganic carriers and flocculent sludge. The effects of ammonium on both kinds of biomass were similar. A decrease of the Specific Anammox Activity (SAA) of 50% was observed at concentrations about 38 mg NH(3)-N·L(-1), while 100 mg NH(3)-N·L(-1) caused an inhibition of 80%. With regards to nitrite, the SAA was not affected at concentrations up to 6.6 µg HNO(2)-N·L(-1) but it suffered a decrease over 50% in the presence of 11 µg HNO(2)-N·L(-1) in the case of the biofilm. The flocculent biomass was much less resistant and its SAA sharply decreased up to 30% of its initial value in the presence of 4.4 µg HNO(2)-N·L(-1). The study of the long-term effects was carried out in lab-scale Sequencing Batch Reactors (SBR) inoculated with the biofilm biomass. Concentrations up to 20 mg NH(3)-N·L(-1) showed no effects on either reactor efficiency or biomass activity. However, when free ammonia concentrations reached values between 35 and 40 mg NH(3)-N·L(-1), the operation turned unstable and the efficiency was totally lost. Nitrous acid concentrations around 1.5 µg HNO(2)-N·L(-1) caused a loss of the efficiency of the treatment and a destabilization of the system. However, a total restoration of the SAA was observed after the stoichiometric feeding was applied to the SBR.

Use of biopolymers as solid substrates for denitrification.

The conventional process to remove nitrate from water, the biological denitrification, uses the addition of dissolved organic carbon that has the potential risk to further deteriorate water quality. Thus, this work aimed to evaluate the specific denitrification activity of a mixed microbial culture and a pure culture of Pseudomonas stutzeri with solid substrates such as polycaprolactone (PCL), polylactic acid (PLA), and starch. The highest nitrate reduction activity was obtained with a microbial mixed culture using starch, 104 mg N(2)-N/(g VSS.d), and PCL, 97 mg N(2)-N/(g VSS.d), followed by PLA, 53 mg N(2)-N/(g VSS.d). A considerable advantage of using biopolymers in water denitrification is the reduced risk of contaminating the water with soluble biodegradable organic carbon.

Aerobic granular SBR systems applied to the treatment of industrial effluents.

Four lab scale sequencing batch reactors (SBRs) were operated to remove organic matter and nitrogen from four different industrial wastewaters. The biomass grew in the reactors in the form of aerobic granules characterized by good settling properties. The high biomass concentrations achieved inside the reactors allowed reducing the solids concentration in the effluent down to 0.2 g VSS L(-1). The organic loading rates (OLR) applied to reactors ranged between 0.7 and 5.0 g CODL(-1)d(-1) with removal efficiencies of 60-95%. The nitrogen loading rates (NLR) applied varied between 0.15 and 0.65 g NH(4)(+)-NL(-1)d(-1) with variable removal efficiencies in the four systems (between 15% and 76%).

Aerobic granulation in a mechanical stirred SBR: treatment of low organic loads.

Aerobic granular sludge was produced in a sequencing batch reactor (SBR) characterized by a height to diameter ratio of 2.5 and the use of mechanical stirring. Compact and regular aerobic granules of up to 1.75 mm of average diameter were formed in the reactor with an organic loading rate of 1.75 kg COD/(m3 d). Settling properties of the obtained aggregates were: sludge volumetric index of 30-40 mL/g VSS and settling velocity higher than 8 m/h. The effects of different carbon to nitrogen ratios (TOC/N) in the feeding on the organic matter oxidation and nitrification process were studied. The concentration of organic matter in the feeding was stepwise reduced (from 190.0 to 37.5 mg TOC/L) and ammonium increased (from 25 to 50 mg NH4+ -N/L). TOC/N ratios of 7.50, 3.00, 1.50 and 0.75 g/g in the feeding were tested. The TOC removal percentage was around 80-95% during the whole operational period and the N removal percentages obtained in the reactor were up to 40%, however, physical properties of the granules were not maintained.