Hydrogen and methane production through two stage anaerobic digestion of straw residues.

Anaerobic digestion of agricultural waste can contribute to the European renewable energy needs. The 71% of the 20,000 anaerobic digestion plants in operation already uses these agro-waste as feedstock; part of these plants can be converted into two stage processes to produce hydrogen and methane in the same plant. Biomethane enriched in hydrogen can replace natural gas in grids while contributing to the sector decarbonisation. Straw is the most abundant agricultural residue (156 Mt/y) and its conventional final fate is uncontrolled soil disposal, landfilling, incineration or, in the best cases, composting. The present research work focuses on the fermentation of spent mushroom bed, an agricultural lignocellulosic byproduct, composed mainly from wheat straw. The substrate has been characterized and semi-continuous tests were performed evaluating the effect of the hydraulic retention time on hydrogen and volatile fatty acids production. It was found that all the tests confirmed the feasibility of the process even on this lignocellulosic substrate, and also, it was identified HRT 4.0 d as the best option to optimize the productivity of volatile fatty acids (17.09 gCODVFAs/(KgVS*d)), and HRT 6.0 d for hydrogen (7.98 LH2/(KgVS*d)). The fermentation effluent was used in biomethanation potential tests to evaluate how this process affects a subsequent digestion phase, reporting an increase in the energetical feedstock exploitation up to 30%.

Upcycling of PHA-producing bacteria for biostimulants production and polyhydroxyalkanoates recovery.

The use of petroleum-based plastic has led to its accumulation in the environment, with negative impacts on the ecosystem and the biota. Polyhydroxyalkanoates (PHAs), biobased and biodegradable plastics produced by microbes, have many commercial applications, however their high production cost means they cannot yet compete with traditional plastics. At the same time, the problem of the growing human population implies that improved crop production is needed to avoid malnutrition. Biostimulants enhance plant growth and have the potential to improve agricultural yields; they can be obtained from biological feedstock, including microbes. Therefore, there is scope for coupling the production of PHAs with that of biostimulants, making the process more cost-efficient and minimizing by-products generation. In this work, low-value agro-zoological residues were processed to obtain PHA-storing bacteria via acidogenic fermentation; PHAs destined for the bioplastic market were extracted, and the protein-rich by-products were turned into protein hydrolysates using different treatment methods, assessing their biostimulant effects in growth trials with tomato and cucumber plants. The results indicate that the best hydrolysis treatment, realizing the highest amount of organic nitrogen (6.8 gN-org/L) while achieving the best PHA recovery (63.2 % gPHA/gTS), is obtained with strong acids. All the protein hydrolysates were effective in improving either roots or leaf development, with various results, depending on the species and the growth method. The acid hydrolysate was the most effective treatment to enhance the development of shoots (21 % increase compared to the control) and roots (16 % increase for the dry weight and 17 % for main root length) of hydroponically-grown cucumber plants, while pot-grown tomatoes, biostimulated via foliar spray, developed bigger shoots (up to 41 %) with the hydrolysate obtained from the alkaline treatment. These preliminary results indicate that simultaneous production of PHAs and biostimulants is feasible, and that commercialization could be achievable given the expected reduction in production costs.

Polyhydroxyalkanoates production from ethanol- and lactate-rich fermentate of confectionary industry effluents.

Polyhydroxyalkanoate (PHA) production has been the focus of considerable research to increase productivities and reduce production costs. In this study, a fermented confectionary industry wastewater was used as feedstock for mixed microbial culture PHA production. The feedstock was dominated by lactate and ethanol (60-90 % of all soluble fermentation products). The culture selection reactor was inoculated with municipal activated sludge and was operated at an organic loading rate (OLR) of 100 Cmmol·L-1·d-1, achieving a robust PHA-accumulating enrichment, which produced up to 52.6 ± 0.4 wt% of PHA in accumulation assays. An OLR increase in the culture selection stage to 150 Cmmol·L-1·d-1 led to a PHA content of 59.1 ± 0.6, a yield of 0.93 ± 0.01 Cmol-PHA·Cmol-S-1 and a productivity of 0.93 ± 0.01 g-PHA L-1·h-1. A correlation analysis of the impact of ethanol concentrations from 3.19 to 20.3 Cmmol·L-1 in the reactor showed that ethanol inhibited PHA production rate and yield and the consumption of other carbon sources available. Microbial community analysis revealed the increase of Amaricoccus genus during the bioreactor operation time, a known PHA accumulator. The produced polymer was poly(3-hydroxybutyrate) with an average molecular weight of 4.3 × 105 Da and a polydispersity index of 1.88.

Thauera sp. Sel9, a new bacterial strain for polyhydroxyalkanoates production from volatile fatty acids.

Thauera is one of the main genera involved in polyhydroxyalkanoate (PHA) production in microbial mixed cultures (MMCs) from volatile fatty acids (VFAs). However, no Thauera strains involved in PHA accumulation have been obtained in pure culture so far. This study is the first report of the isolation and characterization of a Thauera sp. strain, namely Sel9, obtained from a sequencing batch reactor (S-SBR) set up for the selection of PHA storing biomass. The 16S rRNA gene evidenced a high sequence similarity with T. butanivorans species. Genome sequencing identified all genes involved in PHA synthesis, regulation and degradation. The strain Sel9 was able to grow with an optimum of chemical oxygen demand-to-nitrogen (COD:N) ratio ranging from 4.7 to 18.9. Acetate, propionate, butyrate and valerate were used as sole carbon and energy sources: a lag phase of 72 h was observed in presence of propionate. Final production of PHAs, achieved with a COD:N ratio of 75.5, was 60.12 ± 2.60 %, 49.31 ± 0.7 %, 37.31 ± 0.43 % and 18.06 ± 3.81 % (w/w) by using butyrate, acetate, valerate and propionate as substrates, respectively. Also, the 3-hydroxybutyrate/3-hydroxyvalerate ratio reflected the type of carbon sources used: 12.30 ± 0.82 for butyrate, 3.56 ± 0.02 for acetate, 0.93 ± 0.03 for valerate and 0.76 ± 0.02 for propionate. The results allow a better elucidation of the role of Thauera in MMCs and strongly suggest a possible exploitation of Thauera sp. Sel9 for a cost-effective and environmentally friendly synthesis of PHAs using VFAs as substrate.

Integrated selection of PHA-storing biomass and nitrogen removal via nitrite from sludge reject water: a mathematical model.

One of the most recent innovations to promote a circular economy during wastewater treatment is the production of biopolymers. It has recently been demonstrated that it is possible to integrate the production of biopolymers in the form of polyhydroxyalkanoates (PHA) with nitrogen removal via nitrite during the treatment of sludge reject water. In the present study, simulation of a new process for bioresource recovery was conducted by an appropriate modification of the Activated Sludge Model 3. The process consists of the integrated nitrogen removal via nitrite from sludge reject water and the selection of PHA-storing biomass by inducing a feast and famine regime under aerobic and anoxic conditions. According to the results, it is anticipated that simulation data matched very satisfactorily with the experimental data and confirmed the main experimental observation, showing that during the famine period the PHA depletion was almost complete due to the availability of nitrite as the electron acceptor. Simulation results indicate that the selection of the volumetric organic loading rate and of the relative duration of the aerobic feast/anoxic famine duration is critical in order to allow for the effective denitritation of the internally stored PHA during the famine phase.

Expanding ASM models towards integrated processes for short-cut nitrogen removal and bioplastic recovery.

In next-generation water resource recovery facilities (WRRFs), it is becoming increasingly important to save energy costs and promote resource recovery of valuable products. One way of reducing the substantial aeration energy costs at WRRFs is to employ shortcut N removal, while polyhydroxyalkanoate (PHA) production and recovery as bioplastic is a promising means of recovering a valuable product from biosolids. Both objectives can be achieved simultaneously through the Short-Cut Enhanced Phosphorus and PHA Recovery (SCEPPHAR) process. However, current mathematical models have not previously been employed to describe the behavior of such a process, which limits engineering design and optimisation of process operation. This work focusses on extending the ASM3 model towards the description of short-cut nitrogen removal and simultaneous PHA recovery in a sidestream treatment process. The calibrated and validated model described very well the nitritation process coupled with the aerobic feast/anoxic famine process for the selection of PHA producing organisms at a pilot-scale facility operated in Carbonera, Italy, where the normalised root mean squared error (NRMSE) was consistently <20%. Furthermore, the model applied to the PHA selection stage could effectively describe the PHA accumulation stage without recalibration. A simulation study was performed using the modified ASM3 model to assess the relative benefits of the SCEPPHAR process strategy as compared to the fully aerobic selection process for mixed culture PHA production. While the level of PHA production was found to be 34% lower with SCEPPHAR, a 43% savings in volatile fatty acids (VFAs) demand, a 15% decrease in Total suspended solids (TSS) production and a 28% decrease in oxygen demand were also achievable, which could lead to savings in operational costs. This study facilitates the design and optimisation of WRRFs that integrate short-cut N removal with PHA production, saving aeration energy costs while achieving resource recovery.

Characterization of Polyhydroxyalkanoates Produced at Pilot Scale From Different Organic Wastes.

Polyhydroxyalkanoates (PHAs) production at pilot scale has been recently investigated and carried out exploiting different process configurations and organic wastes. More in detail, three pilot platforms, in Treviso (North-East of Italy), Carbonera (North-East of Italy) and Lisbon, produced PHAs by open mixed microbial cultures (MMCs) and different organic waste streams: organic fraction of municipal solid waste and sewage sludge (OFMSW-WAS), cellulosic primary sludge (CPS), and fruit waste (FW), respectively. In this context, two stabilization methods have been applied, and compared, for preserving the amount of PHA inside the cells: thermal drying and wet acidification of the biomass at the end of PHA accumulation process. Afterward, polymer has been extracted following an optimized method based on aqueous-phase inorganic reagents. Several PHA samples were then characterized to determine PHA purity, chemical composition, molecular weight, and thermal properties. The polymer contained two types of monomers, namely 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) at a relative percentage of 92.6-79.8 and 7.4-20.2 w/w, respectively, for Treviso and Lisbon plants. On the other hand, an opposite range was found for 3HB and 3HV monomers of PHA from Carbonera, which is 44.0-13.0 and 56.0-87.0 w/w, respectively. PHA extracted from wet-acidified biomass had generally higher viscosity average molecular weights (M v ) (on average 424.8 ± 20.6 and 224.9 ± 21.9 KDa, respectively, for Treviso and Lisbon) while PHA recovered from thermally stabilized dried biomass had a three-fold lower M v .

Effects of the Sludge Retention Time and Carbon Source on Polyhydroxyalkanoate-Storing Biomass Selection under Aerobic-Feast and Anoxic-Famine Conditions.

Polyhydroxyalkanoates (PHAs) are versatile biodegradable polymers produced by bacteria and are suitable for many downstream applications. They can be produced inexpensively from mixed microbial cultures under feast and famine conditions in the presence of biobased volatile fatty acids (VFAs). Here, we investigated the effect of changing the sludge retention time (SRT) and the addition of fermented cellulosic primary sludge (CPS) as a carbon source on the selection of PHA-storing biomass when applying the feast and famine strategy under aerobic and anoxic conditions, respectively. Increasing the SRT from 5 to 7-10 days enhanced PHA yields under feast conditions from 0.18 gCODPHA/gCODVFA (period 1) to 0.40 gCODPHA/gCODVFA (period 2). The use of fermented CPS as a carbon source (period 3) increased PHA yields to 0.62 gCODPHA/gCODVFA despite the presence of biodegradable non-VFA fractions. Microbial characterization by denaturing gradient gel electrophoresis and fluorescence in situ hybridization revealed high microbial speciation during the three experimental periods. In period 3, the dominant genera were Thauera, Paracoccus, and Azoarcus, which accounted for ∼95% of the total microbial biomass.

Volatile fatty acids production from biowaste at mechanical-biological treatment plants: Focusing on fermentation temperature.

The impact of temperature (20, 35, 45, 55, 70 °C) on volatile fatty acid (VFA) production from biowaste collected at a mechanical-biological treatment plant was analysed. Additionally, relevant streams of the treatment plant were characterised to assess seasonality effects and conceive the integration of a fermentation unit. Batch fermentation tests at 35 °C showed the highest VFA yields (0.49-0.59 gCODVFA/gVS). The VFA yield at 35 °C was 2%, 6%, 10% and 14% higher than at 55, 45, 20 and 70 °C, respectively. The VFA profile was not affected by the fermentation temperature nor seasonality and was dominated by acetic, propionic and butyric acid (75-86% CODVFA). The concentration of non-VFA soluble COD and ammoniacal nitrogen in the fermentation liquor increased with temperature. The fermentation unit in the treatment plant was conceived after the pulper and hydrocyclones and before the anaerobic digester, while the fermenter temperature depends on the VFA application.

Sieving of municipal wastewater and recovery of bio-based volatile fatty acids at pilot scale.

This study combined at pilot scale the recovery of cellulosic primary sludge from the sieving of municipal wastewater followed by the production of bio-based VFAs through acidogenic fermentation. The sieving of municipal wastewater was accomplished by a rotating belt filter which allowed the removal of around 50% of suspended solids when operated at solids loading rates higher than 30-35 kgTSS/m2 h. The solids recovered by sieving contained around 40% of cellulose, which is a suitable raw material for the production of bio-based VFAs. Initially, fermentation batch tests of cellulosic primary sludge were carried out adjusting the initial pH of the sludge at values of 8, 9, 10 and 11, in order to evaluate the best production yields of bio-based VFAs and their composition. The highest VFAs yield achieved was 521 mgCODVFA/gVS occurring when pH was adjusted at 9, while propionic acid reached 51% of the total VFAs. Then, the optimal conditions were applied at long term in a sequencing batch fermentation reactor where the highest potential productivity of bio-based VFAs (2.57 kg COD/m3 d) was obtained by adjusting the pH feeding at 9 and operating with an hydraulic retention time of 6 days under mesophilic conditions. The cost-benefit analyses for the implementation of cellulosic primary sludge recovery was carried out consideringthe anaerobic digestion as reference scenario. The economical assessment showed that the production of bio-based VFAs from cellulosic primary sludge as carbon source and/or as chemical precursors give higher net benefits instead of the only biogas production.

Pilot scale cellulose recovery from sewage sludge and reuse in building and construction material.

The recovery of cellulose in toilet paper from municipal wastewater is one of the most innovative actions in the circular economy context. In fact, fibres could address possible new uses in the building sector as reinforcing components in binder-based materials. In this paper, rotating belt filters were tested to enhance the recovery of sludge rich in cellulose fibres for possible valorisation in construction applications. Recovered cellulosic material reached value up to 26.6 gm-3 with maximum solids removal of 74%. Content of cellulose, hemicellulose and lignin was found averagely equal to 87% of the total composition. Predictive equation of cellulosic material was further obtained. The addition of recovered cellulose fibres in mortars bring benefits in terms of lightness, microstructure and moisture buffering value (0.17 g/m2%UR). Concerning mechanical properties, flexural strength was improved with the addition of 20% of recovered cellulose fibres. In addition, a simplified economic assessment was reported for two possible pre-mixed blends with 5% and 20% of recovered fibres content.

Volatile fatty acids production from food wastes for biorefinery platforms: A review.

Volatile fatty acids (VFAs) are a class of largely used compounds in the chemical industry, serving as starting molecules for bioenergy production and for the synthesis of a variety of products, such as biopolymers, reduced chemicals and derivatives. Because of the huge amounts of food waste generated from household and processing industry, 47 and 17 million tons per year respectively only in the EU-28 Countries, food wastes can be the right candidate for volatile fatty acids production. This review investigates all the major topics involved in the optimization of VFAs production from food wastes. Regarding the best operative conditions for the anaerobic fermenter controlled pH in the neutral range (6.0-7.0), short HRT (lower than 10 days), thermophilic temperatures and an organic loading rate of about 10 kgVS/m3d, allowed for an increase in the VFAs concentration between 10 and 25%. It was also found that additions of mineral acids, from 0.5 to 3.0%, and thermal pretreatment in the range 140-170 °C increase the organic matter solubilisation. Applications of VFAs considered in this study were biofuels and bioplastics production as well as nutrients removal in biological wastewater treatment processes.

Environmental and sustainability evaluation of livestock waste management practices in Cyprus.

The aim of this study was to compare the environmental performance and sustainability of different management options for livestock waste in Cyprus. The two most common practices in the country, i.e. the use of anaerobic lagoons and conventional biogas plants, were compared with the innovative scheme developed in the LiveWaste project (LIFE12 ENV/CY/000544), which aims not only to produce bioenergy, but also to treat the digestate for nutrient recovery and water reuse. The Life Cycle Assessment (LCA) methodology was combined with the Analytic Hierarchy Process (AHP) to compare the performance of these alternatives. Four relevant indicators were selected for each dimension of sustainability (environmental, social and economic). The results of the evaluations showed that anaerobic lagoons are not an appropriate option for the sustainable management of livestock waste due to environmental (e.g. climate change, acidification and eutrophication) and social impacts (e.g. noise exposure, visual impact and risk perception for human health). The most important strengths and weaknesses of anaerobic treatment with and without digestate treatment were identified. Compared to conventional anaerobic digestion where digestate is directly applied as an organic fertiliser, the technology proposed in the project entails higher technological complexity due to nitrogen removal and phosphorus recovery. The rise in chemical and electricity requirements increased the impacts on some indicators, such as climate change and operational cost (emissions of greenhouse gases and operation costs were around 50% higher), while reduced impacts in others due to proper nutrient management, as acidification and eutrophication impacts (which were 10 and almost two times lower, respectively). For the specific Cypriot conditions, where the overapplication of nutrients leads to pollution of water bodies, the innovative treatment scheme with higher technological development presents an interesting approach. Nevertheless, the treatment of the digestate should be analysed taking into account the specific characteristics of each scenario.

Biorefinery of cellulosic primary sludge towards targeted Short Chain Fatty Acids, phosphorus and methane recovery.

Cellulose from used toilet paper is a major untapped resource embedded in municipal wastewater which recovery and valorization to valuable products can be optimized. Cellulosic primary sludge (CPS) can be separated by upstream dynamic sieving and anaerobically digested to recover methane as much as 4.02 m3/capita·year. On the other hand, optimal acidogenic fermenting conditions of CPS allows the production of targeted short-chain fatty acids (SCFAs) as much as 2.92 kg COD/capita·year. Here propionate content can be more than 30% and can optimize the enhanced biological phosphorus removal (EBPR) processes or the higher valuable co-polymer of polyhydroxyalkanoates (PHAs). In this work, first a full set of batch assays were used at three different temperatures (37, 55 and 70 °C) and three different initial pH (8, 9 and 10) to identify the best conditions for optimizing both the total SCFAs and propionate content from CPS fermentation. Then, the optimal conditions were applied in long term to a Sequencing Batch Fermentation Reactor where the highest propionate production (100-120 mg COD/g TVSfed·d) was obtained at 37 °C and adjusting the feeding pH at 8. This was attributed to the higher hydrolysis efficiency of the cellulosic materials (up to 44%), which increased the selective growth of Propionibacterium acidopropionici in the fermentation broth up to 34%. At the same time, around 88% of the phosphorus released during the acidogenic fermentation was recovered as much as 0.15 kg of struvite per capita·year. Finally, the potential market value was preliminary estimated for the recovered materials that can triple over the conventional scenario of biogas recovery in existing municipal wastewater treatment plants.

Is SCENA a good approach for side-stream integrated treatment from an environmental and economic point of view?

The environmental and economic benefits and burdens of including the first Short Cut Enhanced Nutrient Abatement (SCENA) into a real municipal wastewater treatment plant were evaluated using life cycle assessment (LCA) and life cycle cost (LCC). The implications of accomplishing nitrogen (N) removal and phosphorus (P) recovery via nitrite in the side stream were assessed taking into account the actual effluent quality improvement, the changes in the electricity and chemical consumption, N2O, CO2 and CH4 emissions and the effects of land application of biosolids, among others. In addition, a case-specific estimation of the P availability when sludge is applied to land, therefore replacing conventional fertilizer, was performed. Furthermore, to account for the variability in input parameters, and to address the related uncertainties, Monte Carlo simulation was applied. The analysis revealed that SCENA in the side stream is an economic and environmentally friendly solution compared to the traditional plant layout with no side-stream treatment, thanks to the reduction of energy and chemical use for the removal of N and P, respectively. The uncertainty analysis proved the validity of the LCA results for global warming potential and impact categories related to the consumption of fossil-based electricity and chemicals, while robust conclusions could not be drawn on freshwater eutrophication and toxicity-related impact categories. Furthermore, three optimization scenarios were also evaluated proving that the performance of the WWTP can be further improved by, for instance, substituting gravitational for mechanical thickening of the sludge or changing the operational strategy to the chemically enhanced primary treatment, although this second alternative will increase the operational cost by 5%. Finally, the outcomes show that shifting P removal from chemical precipitation in the main line to biologically enhanced uptake in the side stream is key to reducing chemicals use, thus the operational cost, and increasing the environmental benefit of synthetic fertilizers replacement.

Microalgae Cultivation on Anaerobic Digestate of Municipal Wastewater, Sewage Sludge and Agro-Waste.

Microalgae are fast-growing photosynthetic organisms which have the potential to be exploited as an alternative source of liquid fuels to meet growing global energy demand. The cultivation of microalgae, however, still needs to be improved in order to reduce the cost of the biomass produced. Among the major costs encountered for algal cultivation are the costs for nutrients such as CO2, nitrogen and phosphorous. In this work, therefore, different microalgal strains were cultivated using as nutrient sources three different anaerobic digestates deriving from municipal wastewater, sewage sludge or agro-waste treatment plants. In particular, anaerobic digestates deriving from agro-waste or sewage sludge treatment induced a more than 300% increase in lipid production per volume in Chlorella vulgaris cultures grown in a closed photobioreactor, and a strong increase in carotenoid accumulation in different microalgae species. Conversely, a digestate originating from a pilot scale anaerobic upflow sludge blanket (UASB) was used to increase biomass production when added to an artificial nutrient-supplemented medium. The results herein demonstrate the possibility of improving biomass accumulation or lipid production using different anaerobic digestates.

Development of a Novel Process Integrating the Treatment of Sludge Reject Water and the Production of Polyhydroxyalkanoates (PHAs).

Polyhydroxyalkanoates (PHAs) from activated sludge and renewable organic material can become an alternative product to traditional plastics since they are biodegradable and are produced from renewable sources. In this work, the selection of PHA storing bacteria was integrated with the side stream treatment of nitrogen removal via nitrite from sewage sludge reject water. A novel process was developed and applied where the alternation of aerobic-feast and anoxic-famine conditions accomplished the selection of PHA storing biomass and nitrogen removal via nitrite. Two configurations were examined: in configuration 1 the ammonium conversion to nitrite occurred in the same reactor in which the PHA selection process occurred, while in configuration 2 two separate reactors were used. The results showed that the selection of PHA storing biomass was successful in both configurations, while the nitrogen removal efficiency was much higher (almost 90%) in configuration 2. The PHA selection degree was evaluated by the volatile fatty acid (VFA) uptake rate (-qVFAs) and the PHA production rate (qPHA), which were 239 ± 74 and 89 ± 7 mg of COD per gram of active biomass (Xa) per hour, respectively. The characterization of the biopolymer recovered after the accumulation step, showed that it was composed of 3-hydroxybutyrate (3HB) (60%) and 3-hydroxyvalerate (3HV) (40%). The properties associated with the produced PHA suggest that they are suitable for thermoplastic processing.

Use of external carbon sources derived from biowaste for short-cut nutrient removal from anaerobic effluents.

This work evaluated the use of different external carbon sources to promote the via-nitrite nutrient removal from anaerobic effluents. The carbon sources consisted of fermentation liquid produced from the organic fraction of municipal solid waste (OFMSW FL), drainage liquid produced from OFMSW, fermentation liquid produced from vegetable and fruit waste (VFW FL) and acetic acid. Denitritation and phosphorus uptake via nitrite were evaluated in two sequencing batch reactors, one treating the anaerobic supernatant produced from the co-digestion of OFMSW and activated sludge (highly nitrogenous anaerobic effluent - HNAE), and the other one treating the weakly nitrogenous anaerobic effluent (WNAE) from an upflow anaerobic sludge blanket reactor. The use of OFMSW FL to treat HNAE resulted in high nitrite (27 mgN/(gVSS·h) (VSS - volatile suspended solids) and phosphate uptake (15 mgP/gVSS·h). In the WNAE, nutrient kinetics were much slower. The use of acetic acid and VFW FL performed poorly, while the use of OFMSW FL, which was rich in butyric acid and propionic acid, resulted in significant nutrient removal (7 mgN/gVSS·h and 6 mgP/gVSS·h). The economic evaluation showed that the use of OFMSW FL is a less expensive option than the acetic acid use.