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Third generation biofuels, e.g. biofuels production from algal biomass, have gained attention due to increased interest on global renewable energy. However, crop-based biofuels compete with food production and should be avoided. Microalgal cultivation for biofuel production offers an alternative to crops and can become economically viable when combined with the use of used water resources. Besides nutrients and water, harvesting microalgal biomass represents one of the major costs related to biofuel production and thus efficient and cheap solutions are needed. In bacterial-algal systems, there is the potential to produce energy by co-digesting the two types of biomass. We present an innovative approach to recover microalgal biomass via a two-step flocculation using bacterial biomass after the destabilisation of microalgae with conventional cationic polymer. A short solids retention time (SRT) enhanced biological phosphorus removal (EBPR) system was combined with microalgal cultivation. Two different bacterial biomass removal strategies were assessed whereby bacterial biomass was collected from the solid-liquid separation after the anaerobic phase and after the aerobic phase. Microalgal recovery was tested by jar tests where three different chemical coagulants in coagulation-flocculation tests (AlCl3, PDADMAC and Greenfloc 120) were assessed. Furthermore, jar tests were conducted to assess the microalgal biomass recovery by a two-step flocculation method, involving chemical coagulants in the first step and bacterial biomass used in the second step to enhance the flocculation. Up to 97% of the microalgal biomass was recovered using 16 mg polymer/g algae and 0.1 g algae/g bacterial biomass. Moreover, the energy recovery by the short-SRT EBPR system combined with microalgal cultivation was assessed via biomethane potential tests. Up to 560 ± 24 mL CH4/gVS methane yield was obtained by co-digesting bacterial biomass collected after the anaerobic phase and microalgal biomass. The energy recovery in terms of methane production obtained in the short-SRT EBPR system is about 40% of the influent chemical energy.

Anaerobic ammonium oxidation (anammox) is a cost-effective process to treat high-strength nitrogenous wastewater. Even without organic carbon input, the effluent contains bioproducts from autotrophic and heterotrophic bacteria. In this work, excitation-emission matrix (EEM) fluorescence spectroscopy was used to characterize the effluent dissolved organic matter (EfOM) from an anammox reactor treating synthetic wastewater. Two dominant EEM components were identified as humic acid-like (component 1) and protein-like (component 2) substances with excitation/emission peaks at <240, 355, 420/464 nm and <240, 280, 330/346 nm, respectively. The presence of both compounds in the effluent was tracked during an activity recovery period (nitrogen load increased from 0.2 to 1.3 kg Nm(-3)d(-1)). The effluent concentration of both components increased during this period, indicating correlation between production and bacterial activity. The dynamics of these bioproducts during both substrate consumption and starvation phases was analyzed in batch experiments. Component 1 was only formed during substrate consumption in a rate proportional to ammonium removal and was considered an up-take associated product characteristic of anammox activity. The results show that the composition of the EfOM was qualitatively and quantitatively influenced by process performance. Monitoring the EfOM could, therefore, offer a useful approach to assess anammox process performance and must be further explored.

Batch autotrophic ammonia oxidation tracked through oxygen uptake measurements displays a preliminary acceleration phase. Failure to recognize the acceleration phase and fitting batch ammonia oxidation profiles with standard Monod‐type mathematical models can result in meaningless kinetic parameter estimates. The objectives of this study were to examine the factors controlling the acceleration phase and to derive and test empirical and metabolic models for its description. Because of possible sustained reducing power limitation during batch ammonia oxidation, the extent of the acceleration phase (1) increased with increasing initial ammonia concentration, (2) did not systematically vary with initial biomass concentrations, and (3) increased in response to starvation. Concurrent hydroxylamine oxidation significantly reduced the acceleration phase potentially by relieving reducing power limitation. A nonlinear empirical model described the acceleration phase more accurately than a linear empirical model. The metabolic model also captured experimental trends exceedingly well, but required determination of additional parameters and variables.

AbstractAddition of hydroxylamine (NH2OH) to autotrophic biomass in nitrifying bioreactors affected the activity, physical structure, and microbial ecology of nitrifying aggregates. When NH2OH is added to nitrifying cultures in 6‐h batch experiments, the initial NH3‐N uptake rates were physiologically accelerated by a factor of 1.4–13. NH2OH addition caused a 20–40% decrease in the median aggregate size, broadened the shape of the aggregate size distribution by up to 230%, and caused some of the microcolonies to appear slightly more dispersed. Longer term NH2OH addition in fed batch bioreactors decreased the median aggregate size, broadened the aggregate size distribution, and decreased NH3‐N removal from >90% to values ranging between 75% and 17%. This altered performance is explained by quantitative fluorescence in situ hybridization (FISH) results that show inhibition of nitrifying populations, and by qPCR results showing that the copy numbers of amoA and nxrA genes gradually decreased by up to an order‐of‐magnitude. Longer term NH2OH addition damaged the active biomass. This research clarifies the effect of NH2OH on nitrification and demonstrates the need to incorporate NH2OH‐related dynamics of the nitrifying biomass into mathematical models, accounting for both ecophysiological and structural responses. Biotechnol. Bioeng. 2009; 102: 714–724. © 2008 Wiley Periodicals, Inc.

The NDHA model comprehensively describes nitrous oxide (N2O) producing pathways by both autotrophic ammonium oxidizing and heterotrophic bacteria. The model was calibrated via a set of targeted extant respirometric assays using enriched nitrifying biomass from a lab-scale reactor. Biomass response to ammonium, hydroxylamine, nitrite and N2O additions under aerobic and anaerobic conditions were tracked with continuous measurement of dissolved oxygen (DO) and N2O. The sequential addition of substrate pulses allowed the isolation of oxygen-consuming processes. The parameters to be estimated were determined by the information content of the datasets using identifiability analysis. Dynamic DO profiles were used to calibrate five parameters corresponding to endogenous, nitrite oxidation and ammonium oxidation processes. The subsequent N2O calibration was not significantly affected by the uncertainty propagated from the DO calibration because of the high accuracy of the estimates. Five parameters describing the individual contribution of three biological N2O pathways were estimated accurately (variance/mean < 10% for all estimated parameters). The NDHA model response was evaluated with statistical metrics (F-test, autocorrelation function). The 95% confidence intervals of DO and N2O predictions based on the uncertainty obtained during calibration are studied for the first time. The measured data fall within the 95% confidence interval of the predictions, indicating a good model description. Overall, accurate parameter estimation and identifiability analysis of ammonium removal significantly decreases the uncertainty propagated to N2O production, which is expected to benefit N2O model discrimination studies and reliable full scale applications.

A comprehensive and global sensitivity analysis was conducted under a range of operating conditions. The relative importance of mass transfer resistance versus kinetic parameters was studied and found to depend on the operating regime as follows: Operating under the optimal loading ratio of 1.90(gO(2)/m(3)/d)/(gN/m(3)/d), the system was influenced by mass transfer (10% impact on nitrogen removal) and performance was limited by AOB activity (75% impact on nitrogen removal), while operating above, AnAOB activity was limiting (68% impact on nitrogen removal). The negative effect of oxygen mass transfer had an impact of 15% on nitrogen removal. Summarizing such quantitative analyses led to formulation of an optimal operation window, which serves a valuable tool for diagnosis of performance problems and identification of optimal solutions in nitritation/anammox applications.

One-stage autotrophic nitrogen (N) removal, requiring the simultaneous activity of aerobic and anaerobic ammonium oxidizing bacteria (AOB and AnAOB), can be obtained in spatially redox-stratified biofilms. However, previous experience with Membrane-Aerated Biofilm Reactors (MABRs) has revealed a difficulty in reducing the abundance and activity of nitrite oxidizing bacteria (NOB), which drastically lowers process efficiency. Here we show how sequential aeration is an effective strategy to attain autotrophic N removal in MABRs: Two separate MABRs, which displayed limited or no N removal under continuous aeration, could remove more than 5.5 g N/m(2)/day (at loads up to 8 g N/m(2)/day) by controlled variation of sequential aeration regimes. Daily averaged ratios of the surficial loads of O(2) (oxygen) to NH(4)(+) (ammonium) (L(O(2))/L(NH(4))) were close to 1.73 at this optimum. Real-time quantitative PCR based on 16S rRNA gene confirmed that sequential aeration, even at elevated average O(2) loads, stimulated the abundance of AnAOB and AOB and prevented the increase in NOB. Nitrous oxide (N(2)O) emissions were 100-fold lower compared to other anaerobic ammonium oxidation (Anammox)-nitritation systems. Hence, by applying periodic aeration to MABRs, one-stage autotrophic N removal biofilm reactors can be easily obtained, displaying very competitive removal rates, and negligible N(2)O emissions.

Extracellular polymeric substances (EPS) have a presumed determinant role in the structure, architecture, strength, filterability, and settling behaviour of microbial solids in biological wastewater treatment processes. Consequently, numerous EPS extraction protocols have recently been published that aim to optimize the trade off between high EPS recovery and low cell lysis. Despite extensive efforts, the obtained results are often contradictory, even when analysing similar biomass samples and using similar experimental conditions, which greatly complicates the selection of an extraction protocol. This study presents a rigorous and critical assessment of existing physical and chemical EPS extraction methods applied to mixed-culture biomass samples (nitrifying, nitritation-anammox, and activated sludge biomass). A novel fluorescence-based method was developed and calibrated to quantify the lysis potential of different EPS extraction protocols. We concluded that commonly used methods to assess cell lysis (DNA concentrations or G6PDH activities in EPS extracts) do not correlate with cell viability. Furthermore, we discovered that the presence of certain chemicals in EPS extracts results in severe underestimation of protein and carbohydrate concentrations by using standard analytical methods. Keeping both maximum EPS extraction yields and minimal biomass lysis as criteria, it was identified a sonication-based extraction method as the best to determine and compare tightly-bound EPS fractions in different biomass samples. Protein was consistently the main EPS component in all analysed samples. However, EPS from nitrifying enrichments was richer in DNA, the activated sludge EPS had a higher content in humic acids and carbohydrates, and the nitritation-anammox EPS, while similar in composition to the nitrifier EPS, had a lower fraction of hydrophobic biopolymers. In general, the easily-extractable EPS fraction was more abundant in carbohydrates and humic substances, while DNA could only be found in tightly bound EPS fractions. In conclusion, the methodology presented herein supports the rational selection of analytical tools and EPS extraction protocols in further EPS characterization studies.