Incorporating saline microalgae biomass in anaerobic digester treating sewage sludge: Impact on performance and microbial populations.

The aim of this study was to acclimate anaerobic prokaryotes to saline microalgae biomass. Semi-continuous experiments were conducted using two 1.5 L mesophilic reactors for 10 weeks, (hydraulic retention time of 21 days). The first reactor was solely fed with sewage sludge (control), while the second received a mixture of sewage sludge and microalgal biomass (80/20 %w/w) cultivated at 70 g·L-1 salinity. The in-reactor salinity reached after the acclimation phase was 14 g·L-1. Biomethane production was comparable between the control and acclimated reactors (205 ± 29 NmLMethane·gVolatileSolids-1). Salinity tolerance assessment of methanogenic archaea revealed that salinity causing 50% inhibition of methane production increased from 10 to 27 g·L-1 after acclimation. Microbial diversity analyses revealed notable changes in methanogenic archaea populations during co-digestion of saline microalgae biomass, particularly methylotrophic (+27%) and acetotrophic (-26%) methanogens. This study has highlighted the possibility of treating efficiently saline microalgae in co-digestion with sewage sludge in future industrial biogas plants.

Integrating microalgae growth in biomethane plants: Process design, modelling, and cost evaluation.

The integration of microalgae cultivation in anaerobic digestion (AD) plants can take advantage of relevant nutrients (ammonium and ortho-phosphate) and CO2 loads. The proposed scheme of microalgae integration in existing biogas plants aims at producing approximately 250 t·y-1 of microalgal biomass, targeting the biostimulants market that is currently under rapid expansion. A full-scale biorefinery was designed to treat 50 kt·y-1 of raw liquid digestate from AD and 0.45 kt·y-1 of CO2 from biogas upgrading, and 0.40 kt·y-1 of sugar-rich solid by-products from a local confectionery industry. An innovative three-stage cultivation process was designed, modelled, and verified, including: i) microalgae inoculation in tubular PBRs to select the desired algal strains, ii) microalgae cultivation in raceway ponds under greenhouses, and iii) heterotrophic microalgae cultivation in fermenters. A detailed economic assessment of the proposed biorefinery allowed to compute a biomass production cost of 2.8 ± 0.3 €·kg DW-1, that is compatible with current downstream process costs to produce biostimulants, suggesting that the proposed nutrient recovery route is feasible from the technical and economic perspective. Based on the case study analysis, a discussion of process, bioproducts and policy barriers that currently hinder the development of microalgae-based biorefineries is presented.

Wastewater from textile digital printing as a substrate for microalgal growth and valorization.

This study aims at evaluating an innovative biotechnological process for the concomitant bioremediation and valorization of wastewater from textile digital printing technology based on a microalgae/bacteria consortium. Nutrient and colour removal were assessed in lab-scale batch and continuous experiments and the produced algae/bacteria biomass was characterized for pigment content and biomethane potential. Microbial community analysis provided insight of the complex community structure responsible for the bioremediation action. Specifically, a community dominated by Scenedesmus spp. and xenobiotic and dye degrading bacteria was naturally selected in continuous photobioreactors. Data confirm the ability of the microalgae/bacteria consortium to grow in textile wastewater while reducing the nutrient content and colour. Improvement strategies were eventually identified to foster biomass growth and process performances. The experimental findings pose the basis of the integration of a microalgal-based process into the textile sector in a circular economy perspective.

Microbial community and performance of a partial nitritation/anammox sequencing batch reactor treating textile wastewater.

Implementation of onsite bioremediation technologies is essential for textile industries due to rising concerns in terms of water resources and quality. Partial nitritation-anaerobic ammonium oxidation (PN/A) processes emerged as a valid, but unexplored, solution. In this study, the performance of a PN/A pilot-scale (9 m3) sequencing batch reactor treating digital textile printing wastewater (10-40 m3 d-1) was monitored by computing nitrogen (N) removal rate and efficiencies. Moreover, the structure of the bacterial community was assessed by next generation sequencing and quantitative polymerase chain reaction (qPCR) analyses of several genes, which are involved in the N cycle. Although anaerobic ammonium oxidation activity was inhibited and denitrification occurred, N removal rate increased from 16 to 61 mg N g VSS-1 d-1 reaching satisfactory removal efficiency (up to 70%). Ammonium (18-70 mg L-1) and nitrite (16-82 mg L-1) were detected in the effluent demonstrating an unbalance between the aerobic and anaerobic ammonia oxidation activity, while constant organic N was attributed to recalcitrant azo dyes. Ratio between nitrification and anammox genes remained stable reflecting a constant ammonia oxidation activity. A prevalence of ammonium oxidizing bacteria and denitrifiers suggested the presence of alternative pathways. PN/A resulted a promising cost-effective alternative for textile wastewater N treatment as shown by the technical-economic assessment. However, operational conditions and design need further tailoring to promote the activity of the anammox bacteria.

Assessment of anammox, microalgae and white-rot fungi-based processes for the treatment of textile wastewater.

The treatability of seven wastewater samples generated by a textile digital printing industry was evaluated by employing 1) anammox-based processes for nitrogen removal 2) microalgae (Chlorella vulgaris) for nutrient uptake and biomass production 3) white-rot fungi (Pleurotus ostreatus and Phanerochaete chrysosporium) for decolorization and laccase activity. The biodegradative potential of each type of organism was determined in batch tests and correlated with the main characteristics of the textile wastewaters through statistical analyses. The maximum specific anammox activity ranged between 0.1 and 0.2 g N g VSS-1 d-1 depending on the sample of wastewater; the photosynthetic efficiency of the microalgae decreased up to 50% during the first 24 hours of contact with the textile wastewaters, but it improved from then on; Pleurotus ostreatus synthetized laccases and removed between 20-62% of the colour after 14 days, while the enzymatic activity of Phanerochaete chrysosporium was inhibited. Overall, the findings suggest that all microbes have great potential for the treatment and valorisation of textile wastewater after tailored adaptation phases. Yet, the depurative efficiency can be probably enhanced by combining the different processes in sequence.

Balancing Microalgae and Nitrifiers for Wastewater Treatment: Can Inorganic Carbon Limitation Cause an Environmental Threat?

The first objective of this study is to assess the predictive capability of the ALBA (ALgae-BActeria) model for a pilot-scale (3.8 m2) high-rate algae-bacteria pond treating agricultural digestate. The model, previously calibrated and validated on a one-year data set from a demonstrative-scale raceway (56 m2), successfully predicted data from a six-month monitoring campaign with a different wastewater (urban wastewater) under different climatic conditions. Without changing any parameter value from the previous calibration, the model accurately predicted both online monitored variables (dissolved oxygen, pH, temperature) and off-line measurements (nitrogen compounds, algal biomass, total and volatile suspended solids, chemical oxygen demand). Supported by the universal character of the model, different scenarios under variable weather conditions were tested, to investigate the effect of key operating parameters (hydraulic retention time, pH regulation, kLa) on algae biomass productivity and nutrient removal efficiency. Surprisingly, despite pH regulation, a strong limitation for inorganic carbon was found to hinder the process efficiency and to generate conditions that are favorable for N2O emission. The standard operating parameters have a limited effect on this limitation, and alkalinity turns out to be the main driver of inorganic carbon availability. This investigation offers new insights in algae-bacteria processes and paves the way for the identification of optimal operational strategies.

ALBA: A comprehensive growth model to optimize algae-bacteria wastewater treatment in raceway ponds.

This paper proposes a new model describing the algae-bacteria ecosystem evolution in an outdoor raceway for wastewater treatment. The ALBA model is based on mass balances of COD, C, N and P, but also H and O. It describes growth and interactions among algae, heterotrophic and nitrifying bacteria, while local climate drives light and temperature. Relevant chemical/physical processes are also included. The minimum-law was used as ground principle to describe the multi-limitation kinetics. The model was set-up and calibrated with an original data set recorded on a 56 m2 raceway located in the South of France, continuously treating synthetic wastewater. The main process variables were daily measured along 443 days of operations and dissolved O2 and pH were on-line recorded. A sub-dataset was used for calibration and the model was successfully validated, along the different seasons over a period of 414 days. The model proved to be effective in reproducing both the short term nycthemeral dynamics and the long-term seasonal ones. The analysis of different scenarios reveals the fate of nitrogen and the key role played by oxygen and CO2 in the interactions between the different players of the ecosystem. On average, the process turns out to be CO2 neutral, as compared to a standard activated sludge where approximately half of the influent carbon will end up in the atmosphere. The ALBA model revealed that a suboptimal regulation of the paddle wheel can bring to several detrimental impacts. At high velocity, the strong aeration will reduce the available oxygen provided by photo-oxygenation, while very low aeration can rapidly lead to oxygen inhibition of the photosynthetic process. On the other hand, during night, the paddle wheel is fundamental to ensure enough oxygen in the system to support algal-bacteria respiration. The model can be used to support advanced control strategies, including smart regulation of the paddle wheel velocity to more efficiently balance the mixing, aeration and degassing effects.

Integration of a side-stream microalgae process into a municipal wastewater treatment plant: A life cycle analysis.

This study investigates the environmental improvements associated to the integration of a microalgae unit as a side-stream process within an existing municipal wastewater treatment facility in northern Italy. Microalgae are fed on the centrate from sludge dewatering, rich in nutrients, and on the CO2 in the flue-gas of the combined heat and power unit. The produced biomass is recirculated upflow the water line where it settles and undergoes anaerobic digestion generating extra biogas. A life cycle assessment was performed collecting primary data from an algal pilot-scale plant installed at the facility. Fifteen environmental indicators were evaluated. Compared to the baseline wastewater treatment, the new algal configuration allows an improvement for 7 out of 15 indicators mainly thanks to the electricity savings in the facility. Some recommendations are provided to improve the performance of the algal system in the scaling up.

Comparison of the Performance and Microbial Community Structure of Two Outdoor Pilot-Scale Photobioreactors Treating Digestate.

This study aimed at examining and comparing the nutrient removal efficiency, biomass productivity and microbial community structure of two outdoor pilot-scale photobioreactors, namely a bubble column and a raceway pond, treating the liquid fraction of an agricultural digestate. Bacterial and eukaryotic communities were characterized using a metabarcoding approach and quantitative PCR. The abundance, composition, diversity, and dynamics of the main microbes were then correlated to the environmental conditions and operational parameters of the reactors. Both photobioreactors were dominated either by Chlorella sp. or Scenedesmus sp. in function of temperature, irradiance and the nitrogen compounds derived by nitrification. Other species, such as Chlamydomonas and Planktochlorella, were sporadically present, demonstrating that they have more specific niche requirement. Pseudomonas sp. always dominated the bacterial community in both reactors, except in summertime, when a bloom of Calothrix occurred in the raceway pond. In autumn, the worsening of the climate conditions decreased the microalgal growth, promoting predation by Vorticella sp. The study highlights the factors influencing the structure and dynamics of the microbial consortia and which ecological mechanisms are driving the microbial shifts and the consequent reactor performance. On these bases, control strategies could be defined to optimize the management of the microalgal-based technologies.

Bioremediation of aquaculture wastewater with the microalgae Tetraselmis suecica: Semi-continuous experiments, simulation and photo-respirometric tests.

Tetraselmis suecica was cultivated in a semi-continuously operated tubular photobioreactor fed on aquaculture wastewater (AW) testing two hydraulic retention times (HRT): 10 and 7 days (RUN_1 and RUN_2, respectively). The integrated mechanistic model BIO_ALGAE was validated with experimental data in order to simulate the biomass production and nutrient uptake of T. suecica. Moreover, AW was used as substitute synthetic cultivation medium to test the production of lipids, proteins, and carbohydrates in the microalgal biomass. Preliminary photo-respirometric tests were carried out on the AW suspension containing microalgae and bacteria. Dissolved Inorganic Nitrogen (DIN) and Dissolved Inorganic Phosphorus (DIP) were analyzed for the two RUNs, and no significant difference was highlighted (p > 0.05). On the contrary, the productivity of the Total suspended solids (TSS) was significantly higher (p < 0.05) for RUN_1 (900 mg TSS/L) than for RUN_2 (550 mg TSS/L). The analysis of the biochemical composition of biomass has demonstrated a higher content of proteins than of lipids and carbohydrates for the two RUNs. BIO_ALGAE model was validated by comparing simulated results to experimental data. The model was able to reproduce the pattern of these experimental data quite well, for both nutrient uptake and biomass production. The simulated curve follows the same pattern as the experimental data for both RUNs. The wavelike trend indicates the good accuracy of the simulated curves to reproduce the microalgae growth and nutrient uptake that occurring during daytime and at night. With this study, BIO_ALGAE Model was demonstrated to be useful to simulate bioremediation and microalgae production in aquaculture wastewater in a semi-continuous system with different environmental factors. The photo-respirometric outputs were compared with the process rates affecting dissolved oxygen dynamics computed by the mathematical model.

Free ammonia inhibition in microalgae and cyanobacteria grown in wastewaters: Photo-respirometric evaluation and modelling.

The inhibitory effects of free ammonia (FA) on microalgae/cyanobacteria in wastewater-treating photobioreactors (PBR) can strongly reduce their treatment efficiency, increasing the operational costs and undermining the stability of the system. Although FA-promoting conditions (high pH, temperature and ammoniacal nitrogen concentration) are commonly met in outdoor PBRs, photosynthesis inhibition from FA has been scarcely explored and is rarely considered in microalgae-bacteria growth models. Two pilot systems and a series of lab-scale monocultures were tested using a photo-respirometry approach, to evaluate the effects of FA (8.5-136 mg NH3 L-1) on photosynthesis. Two mathematical inhibition models were compared, with the aim of selecting best-fitting equations to describe photo-respirometric experiments. A set of calibrated inhibition parameters was obtained for microalgae and cyanobacteria, growing in monocultures or in mixed algae-bacteria consortia. Cyanobacteria were more sensitive to FA than green microalgae and mixed phototrophs-bacteria consortia showed a higher resistance compared to monocultures. Estimated inhibition parameters were used to describe common operational/environmental conditions in algae-bacteria systems, demonstrating the potential drop in photosynthetic activity under those relevant operational conditions.

Outdoor pilot-scale raceway as a microalgae-bacteria sidestream treatment in a WWTP.

This study aims at demonstrating the feasibility of using microalgae-bacteria consortia for the treatment of the sidestream flow of the supernatant from blackwater dewatering (centrate) in an urban wastewater treatment plant in Northern Italy. A 1200 L raceway reactor was used for the outdoor cultivation of a diverse community of Chlorella spp., Scenedesmus spp. and Chlamydomonas spp. in continuous operation mode with 10 days hydraulic retention time. During the trial, an average daily areal productivity of 5.5 ± 7.4 g TSS m-2 day-1 was achieved while average nutrient removal efficiencies were 86% ± 7% and 71% ± 10% for NH4-N and PO4-P, respectively. The microalgal nitrogen assimilation accounted for 10% of the nitrogen in the centrate while 34% was oxidized to nitrite and nitrate. The oxygen produced by microalgae fully covert the oxygen demand for nitrification. This suggests that the proposed process would reduce the aeration demand for nitrification in the water line of the plant, while producing algal biomass to be further valorized for energy or material recovery.

Two-stage granular sludge partial nitritation/anammox process for the treatment of digestate from the anaerobic digestion of the organic fraction of municipal solid waste.

The increasing amount of source separated organic fraction of municipal solid wastes (OFMSW) treated by anaerobic digestion for energy recovery requires the implementation of cost-efficient processes for the treatment of the produced digestate, especially in terms of nitrogen removal. The autotrophic nitrogen removal process, based on the coupling of two biological processes, partial nitritation (PN) and anammox (A), appears as a suitable solution due to important savings in operational costs compared to conventional treatment processes. However, its application could be hampered by the high salinity and inhibitory potential of this kind of digestate. In this contribution, two lab-scale granular sludge reactors performing the PN and anammox processes, respectively, were used to treat (opportunely diluted) real OFMSW digestate originating from full-scale biogas plants with the aim of assessing their treatment feasibility in a two-stage PN/A configuration. The PN process was implemented in an air-lift granular sludge reactor and was able to treat a nitrogen loading rate of about 1 g N L-1 d-1 at 30 ±â€¯0.5 °C; moreover, its effluent was suitable for the subsequent anammox treatment, with an appropriate effluent NO2-/NH4+ ratio and marginal inhibiting effects. In the anammox granular sludge reactor, the anammox activity was affected by high salinity levels, nonetheless a stable reactor performance at a nitrogen removing rate of 0.83 ±â€¯0.20 and 0.31 ±â€¯0.04 g N L-1 d-1 at 35 ±â€¯0.5 °C, were achieved when treating 50% and 30% diluted real wastewaters at a conductivity in the reactor of 9.1 and 11.2 mS cm-1, respectively.

Use of solid digestate for lignocellulolytic enzymes production through submerged fungal fermentation.

Studies were performed on the use of the solid fraction of digestate (D) for the production of lignocellulolytic enzymes (endo- and exo-glucanase, xylanase, ß-glucosidase and laccase) by fungi, in comparison with wheat straw (benchmark) (W). To date, this is the first report on the use of such an inexpensive substrate in a liquid environment. Submerged instead of solid state fermentation was applied to overcome pH inhibition and increase surface accessibility. A total of 21 fungal strains were tested: the most performing ones were Irpex lacteus DSM1183 for both ß-glucosidase (52 IU/g with D, + 400% compared to W) and endo-glucanase (236 IU/g with D, + 470% compared to W), Schizophyllum commune CBS30132 for xylanase (715 IU/g with W, + 145% compared to D) and Pleurotus ostreatus ATCC96997 for laccase (124 IU/g with D, +230% compared to D). Cultures from S. commune and P. ostreatus were analyzed at the beginning and at the end of the growth test to determine soluble COD, total (TS) and volatile (VS) solids. COD was always lower at the end of the test suggesting a faster uptake than hydrolysis. P. ostreatus evidenced a higher VS reduction (-11% rather than -32%), suggesting a more effective growth of this strain on D. Results may open up new avenues for the utilization of solid digestate, an inexpensive agricultural by-product, for the production of value-added products as well as to increase biodegradation of lignocellulosic materials.

Towards a standardization of biomethane potential tests.

Production of biogas from different organic materials is a most interesting source of renewable energy. The biomethane potential (BMP) of these materials has to be determined to get insight in design parameters for anaerobic digesters. Although several norms and guidelines for BMP tests exist, inter-laboratory tests regularly show high variability of BMPs for the same substrate. A workshop was held in June 2015, in Leysin, Switzerland, with over 40 attendees from 30 laboratories around the world, to agree on common solutions to the conundrum of inconsistent BMP test results. This paper presents the consensus of the intense roundtable discussions and cross-comparison of methodologies used in respective laboratories. Compulsory elements for the validation of BMP results were defined. They include the minimal number of replicates, the request to carry out blank and positive control assays, a criterion for the test duration, details on BMP calculation, and last but not least criteria for rejection of the BMP tests. Finally, recommendations on items that strongly influence the outcome of BMP tests such as inoculum characteristics, substrate preparation, test setup, and data analysis are presented to increase the probability of obtaining validated and reproducible results.

A full-scale plug-flow reactor for biological sludge ozonation.

The reduction of biological excess sludge production using ozone is a well-known technology and is applied in several full-scale plants around the world. Nevertheless, optimisation of the process is not yet adequately documented in the literature. Operational parameters are usually chosen by assuming a direct proportionality between ozone dose and excess sludge reduction. This paper investigates the role of ozone concentration on process efficiency and demonstrates the (non-linear) inverse relationship between ozone dose and specific particulate chemical oxygen demand solubilisation in plug-flow contact reactors. The influence of total suspended solids concentration is also studied and described. No short-term lethal effects on heterotrophic biomass have been observed.

Comparative performance of enzymatic and combined alkaline-enzymatic pretreatments on methane production from ensiled sorghum forage.

This study investigated the effect of enzymatic and combined alkaline-enzymatic pretreatments on chemical composition and methane production from ensiled sorghum forage. Four commercial enzymatic preparations were tested and the two yielding the highest sugars release were added to evaluate any hydrolytic effect on both untreated and alkaline pretreated samples. In the combined alkaline-enzymatic pretreatment trials, the highest sugar release was found with Primafast and BGL preparations (added at a final concentration 0.12 and 0.20 mL/g TS, respectively), with a total monomeric content of 12 and 6.5 g/L. Fibre composition analysis confirmed that the combined alkaline-enzymatic pretreatment led to cellulose (up to 32 %) and hemicelluloses (up to 56 %) solubilisation, compared to the enzymatic pretreatment alone. BMP tests were performed on both untreated and pretreated samples, and time courses of methane production were fitted. Both enzymatic and combined alkaline-enzymatic pretreatment led to a methane production increase (304 and 362 mL CH4/g VS), compared to that of untreated sorghum (265 mL CH4/g VS), as  +15 and  +37 %, respectively. Moreover, higher specific methane production rates, compared to that of untreated sorghum (20.31 mL CH4/g VS/d), were obtained by applying the enzymatic and combined alkaline-enzymatic pretreatment (33.94 and 31.65 mL CH4/g VS/d), respectively.

Maize mono-digestion efficiency: results from laboratory tests.

A laboratory experimental campaign was carried out in order to assess the optimal configuration for the anaerobic digestion of a mixture of sweet corn and ensiled maize. Batch hydrolysis tests were conducted at 35 and 55 °C and at four different particle sizes (2, 5, 20 and 50 mm) obtained by manual chopping and sieving. Chemical pre-treatment by 24 h incubation at various acid and alkaline pH was also considered for its potential to increase the maize methane yield. Results suggest that the hydrolytic phase proceeds significantly faster under thermophilic conditions. Significant differences in the solubilization rate were also observed when comparing coarse (20-50 mm) with fine (2-5 mm) particles, while 2 and 5 mm particles were solubilized at similar rates. No advantages from the chemical pre-treatment, in terms of solubilization efficiency and biomethanization potential were observed. According to these preliminary results, a two-stage semi-continuous laboratory plant consisting of a thermophilic hydrolytic reactor followed by a mesophilic methanogenic reactor was operated for 110 days. Steady state loading parameters were: influent concentration (maize mixture diluted in tap water) of 46 g VS/L, hydraulic retention time of 31 d, organic loading rate of 1.5 g VS/L/d. Alkalinity was dosed to the methanogenic reactor to avoid pH drops. Collected data allowed the average biodegradation efficiency to be estimated at around 60-65%.