Biogas digestate as a sustainable phytosterol source for biotechnological cascade valorization

Every year, several million tonnes of anaerobic digestate are produced worldwide as a by-product of the biogas industry, most of which is applied as agricultural fertilizer. However, in the context of a circular bioeconomy, more sustainable uses of residual digestate biomass would be desirable. This study investigates the fate of the sterol lipids β-sitosterol and cholesterol from the feedstocks to the final digestates of three agricultural and one biowaste biogas plants to assess if sterols are degraded during anaerobic digestion or if they remain in the digestate, which could provide a novel opportunity for digestate cascade valorization. Gas chromatographic analyses showed that feedstock sterols were not degraded during anaerobic digestion, resulting in their accumulation in the digestates to up to 0.15% of the dry weight. The highest concentrations of around 1440 mg β-sitosterol and 185 mg cholesterol per kg dry weight were found in liquid digestate fractions, suggesting partial sterol solubilization. Methanogenic batch cultures spiked with β-sitosterol, cholesterol, testosterone and β-oestradiol confirmed that steroids persist during anaerobic digestion. Mycobacterium neoaurum was able to transform digestate sterols quantitatively into androstadienedione, a platform chemical for steroid hormones, without prior sterol extraction or purification. These results suggest that digestate from agricultural and municipal biowaste is an untapped resource for natural sterols for biotechnological applications, providing a new strategy for digestate cascade valorization beyond land application.     

Fungal fermentation on anaerobic digestate for lipid-based biofuel production

Background

Anaerobic digestate is the effluent from anaerobic digestion of organic wastes. It contains a significant amount of nutrients and lignocellulosic materials, even though anaerobic digestion consumed a large portion of organic matters in the wastes. Utilizing the nutrients and lignocellulosic materials in the digestate is critical to significantly improve efficiency of anaerobic digestion technology and generate value-added chemical and fuel products from the organic wastes. Therefore, this study focused on developing an integrated process that uses biogas energy to power fungal fermentation and converts remaining carbon sources, nutrients, and water in the digestate into biofuel precursor-lipid.

Results

The process contains two unit operations of anaerobic digestion and digestate utilization. The digestate utilization includes alkali treatment of the mixture feed of solid and liquid digestates, enzymatic hydrolysis for mono-sugar release, overliming detoxification, and fungal fermentation for lipid accumulation. The experimental results conclude that 5 h and 30 °C were the preferred conditions for the overliming detoxification regarding lipid accumulation of the following fungal cultivation. The repeated-batch fungal fermentation enhanced lipid accumulation, which led to a final lipid concentration of 3.16 g/L on the digestate with 10% dry matter. The mass and energy balance analysis further indicates that the digestate had enough water for the process uses and the biogas energy was able to balance the needs of individual unit operations.

Conclusions

A fresh-water-free and energy-positive process of lipid production from anaerobic digestate was achieved by integrating anaerobic digestion and fungal fermentation. The integration addresses the issues that both biofuel industry and waste management encounter—high water and energy demand of biofuel precursor production and few digestate utilization approaches of organic waste treatment.

     

Improvement of sludge digestate biodegradability by thermophilic bioaugmentation

The sludge digestate stabilized by mesophilic anaerobic digestion was further degraded through thermophilic anaerobic digestion using 0–10 % (v/v) of thermophilic, proteolytic Coprothermobacter proteolyticus, and/or methanogenic granular sludge. The results demonstrated that the temperature shift to thermophilic condition promoted abiotic solubilization of proteins and reactivated the fermentative bacteria and methanogens indigenous in the sludge digestate, resulting in a final methane yield of 6.25 mmol-CH₄/g-volatile suspended solid (VSS) digestate. The addition of C. proteolyticus accelerated the hydrolysis and fermentation of proteins and polysaccharides in the digestate during the early stage of thermophilic anaerobic digestion and stimulated methane production by syntrophic cooperation with methanogenic granular sludge. In the treatment with granular sludge and inoculated with 10 % (v/v) of C. proteolyticus, a final methane yield of 7 mmol-CH₄/g-VSS digestate was obtained, and 48.4 % proteins and 27.0 % polysaccharides were degraded. The dissolved proteins were contributed by abiotic factor, C. proteolyticus, and indigenous digestate bacteria, respectively, by around 16, 28, and 56 %.     

The methane yield of digestate: effect of organic loading rate, hydraulic retention time, and plant feeding

In biogas plants, huge volumes of digestate are produced daily and stored in uncovered tanks, which leak methane into the atmosphere and cause negative environmental impacts. To better understand the effect that different operating parameters of anaerobic digestion plants have on digestate residual methane yield, four digestate samples collected from plants with very different operations were analysed in batch reactors. Their methane yields were very heterogeneous and varied between 2.88 and 37.63 NL/kgVS. The methane yield was shown to be highly influenced by the A.D. plant Organic Loading Rate and by feedstock quality; hydraulic retention time had only limited effects.     

Legacy effects override soil properties for CO2 and N2O but not CH4 emissions following digestate application to soil

The application of organic materials to soil can recycle nutrients and increase organic matter in agricultural lands. Digestate can be used as a nutrient source for crop production but it has also been shown to stimulate greenhouse gas (GHG) emissions from amended soils. While edaphic factors, such as soil texture and pH, have been shown to be strong determinants of soil GHG fluxes, the impact of the legacy of previous management practices is less well understood. Here we aim to investigate the impact of such legacy effects and to contrast them against soil properties to identify the key determinants of soil GHG fluxes following digestate application. Soil from an already established field experiment was used to set up a pot experiment, to evaluate N₂O, CH₄ and CO₂ fluxes from cattle‐slurry‐digestate amended soils. The soil had been treated with farmyard manure, green manure or synthetic N‐fertilizer, 18 months before the pot experiment was set up. Following homogenization and a preincubation stage, digestate was added at a concentration of 250 kg total N/ha eq. Soil GHG fluxes were then sampled over a 64 day period. The digestate stimulated emissions of the three GHGs compared to controls. The legacy of previous soil management was found to be a key determinant of CO₂ and N₂O flux while edaphic variables did not have a significant effect across the range of variables included in this experiment. Conversely, edaphic variables, in particular texture, were the main determinant of CH₄ flux from soil following digestate application. Results demonstrate that edaphic factors and current soil management regime alone are not effective predictors of soil GHG flux response following digestate application. Knowledge of the site management in terms of organic amendments is required to make robust predictions of the likely soil GHG flux response following digestate application to soil.     

Residual biogas potential from the storage tanks of non-separated digestate and digested liquid fraction

Biogas plants daily produce enormous volumes of digestate that can be handled in its raw form or after mechanical separation. In Italy, effluents are usually stored within aboveground, uncovered tanks, which make them potential emitters of biogas into the atmosphere. The purpose of this study was to estimate the amount of biogas emitted to the atmosphere during the storage phase of non-separated digestate and digested liquid fraction. The trials were performed at two northwest Italy 1 MWel. biogas plants. A floating system for the residual biogas recovery, and a set of three wind tunnels for NH3 emission measurement were used. The experiment demonstrated significant loss to the atmosphere for each of the gases; specifically, on average, 19.5 and 7.90 N m3 biogas MWhel.(-1) were emitted daily from the storage tanks of non-separated digestate and digested liquid fraction, respectively.     

Polyhydroxyalkanoate production using waste vegetable oil and filtered digestate liquor of chicken manure

Abstract

The production of polyhydroxyalkanoates (PHA) using digestate of chicken manure combined with waste sunflower oil as no-cost feedstocks in a multi-stage process was investigated. Using Cupriavidus necator H16 in combined culture media, a maximum PHA accumulation of 4.6?±?0.2?g/L at 75.1?±?1.4% of cell dry matter and a residual cell matter yield of 1.5?±?0.1?g/L were obtained after 96?hr of cultivation (30?°C, 160?rpm, pH 7.0) in flask-based experiments. Manure was acidogenically fermented in a continuous stirring tank reactor in fed-batch mode. The bioreactor was operated at varying organic loading rates (OLR) and hydraulic retention times (HRT) ranging from 1–4?g volatile solids (VS)/L/d and 4–8?days, respectively. Optimal operation was observed at an OLR of 4?g VS/L/d and an HRT of 4?days. Analysis showed the presence of significant amounts of ammonia, potassium, magnesium, calcium, and trace elements, i.e. Fe, Cu, Ni, Mn, Co, Zn, Cr in the digestate. The micro-filtered digestate was utilized as a complex culture media base while waste oil served as an additional carbon source and supplemented for effective PHA production. The total volatile fatty acid content of digestate greatly affected the growth performance of the PHA-producing microorganism Cupriavidus necator H16.     

Investigating the effects of anaerobic and aerobic post-treatment on quality and stability of organic fraction of municipal solid waste as soil amendment

The use of OFMSW for biogas and compost production is considered as a sustainable strategy in saving valuable landfill space while producing valuable product for soil application. This study examines the effects of anaerobic and aerobic post-treatment of OFMSW on the stability of anaerobic digestate and compost and soil quality using seed germination tests. Anaerobic digestion of OFMSW was carried out for fifteen days after which the residual anaerobic digestate was subjected to aerobic post-treatment for seventy days. Seed germination tests showed that fresh feedstock and digestates collected during anaerobic digestion and during the early stages of aerobic post-treatment were phytotoxic. However, phytotoxic effects were not observed in soils amended with the fully stabilised anaerobic digestate compost, ADC. It was also found that seed germination increases with dilution and incubation time, suggesting that lower soil application rates and longer lag periods between soil application of ADC and planting can reduce the amount of biodegradable organics in the ADC, thus enhancing the benefits of ADC as soil amendment.     

Anaerobic digestates are useful nutrient sources for microalgae cultivation: functional coupling of energy and biomass production

We investigated the extent to which nitrogenous and phosphorus nutrients from liquid anaerobic digestates could be recycled for photosynthetic growth of a microalga, Scenedesmus sp. AMDD. Digestates recovered from the anaerobic digestion of cow manure and swine manure and a co-digestion of swine manure and algal biomass were diluted in distilled water and used for algal growth with and without supplemental CO₂ addition. Nutrient assimilation and final biomass yield were retarded in all but the swine manure/algae co-digestate cultures supplemented with high CO₂. Swine manure digestate cultures supplemented with the typical complement of micronutrients normally added with a commonly used growth medium or with Fe/EDTA failed to grow any better than unamended controls. When the culture medium was prepared by blending swine manure digestate with 25 or 50 % algal biomass digestate, diluting it with lake water or by supplementing with magnesium, nutrient assimilation and final algal biomass yields were maximized, indicating that magnesium was critically limiting for algal growth in swine manure digestates. Magnesium amendment thus appears to be essential if nutrients from swine manure digestates are recycled for algal growth. No such requirement is necessary for recycling nutrients from digestates generated wholly or in part from algal biomass.     

Cultivation of Scenedesmus dimorphus using anaerobic digestate as a nutrient medium

Bioprocess and Biosystems Engineering - In this study, the microalga Scenedesmus dimorphus was cultivated phototrophically using unsterilized anaerobic digestate as a nutrient medium. A bench-scale...     

Physicochemical and microbiological indicators of surface water body contamination with different sources of digestate from biogas plants

Transition from fossil energy sources to biogas production has resulted in a strong increase of leakage accidents from fermenters, but knowledge on the effects of fermentation product runoff into freshwater systems is currently restricted to direct toxicity due to oxygen depletion. This study provides first information about the influence of digestate runoff on the physicochemical habitat properties and the bacterial community composition of the hyporheic interstitial which is important in determining ecosystem functioning. We exposed natural stream beds to different concentrations of two different digestates from fermenters (corn and manure feedstock), hypothesizing that the digestate addition causes acute changes of the physicochemical parameters and has distinct effects on microbial community composition of the hyporheic interstitial depending on concentration and type of digestate. In line with the hypotheses, pH value, conductivity, redox potential and ammonium differed significantly from controls and among treatments after digestate addition, but only for a maximum of two days. pH values (controls: 7.8; corn: 7.9; manure: 7.9) and conductivity (controls: 813 μS/cm; corn: 969 μS/cm; manure: 1097 μS/cm) increased, the redox potential (controls: 153 mV; corn: 145 mV; manure: 144 mV) decreased the first two days. A high peak of ammonium-N was detected in the corn and manure treatments (controls: 5 mg/l, corn: 80 mg/l; manure: 60 mg/l) at day 1. In contrast, changes in bacterial community composition were detectable for longer periods of time (>5 days). Seventeen unique T-RF fingerprints of bacterial community response to each of the different digestate treatments (11 unique T-RFs in manure and 6 unique T-RFs in corn treatments) were found, suggesting that this approach provides a suitable ecological indicator for source tracking, e.g. in case of a biogas power plant leakage accident.     

Viability of Plant–Pathogenic Fungi Reduced by Anaerobic Digestion

Feedstock of anaerobic digestion infected with phytopathogens could enhance the risk of spreading those pathogens to uninfested field through digestate. The viability of Fusarium proliferatum, Fusarium verticillioides, Sclerotinia sclerotiorum, and Rhizoctonia solani was investigated in anaerobic digestion experiments using infected plant material of sorghum (Sorghum bicolor), sugar beet (Beta vulgaris subsp. vulgaris var. altissima), and potato (Solanum tuberosum L.). Results from lab-scale reactors were confirmed in full-scale biogas plants. Anaerobic digestion under mesophilic conditions (35–42 °C) reduced most of the phytopathogens of feedstocks investigated. Thus, S. sclerotiorum and R. solani lost their viability within 6 h. In the case of sorghum, however, Fusarium spp. infected feedstock required a maximum of 138 h for sanitation. Thus, the risk of spreading plant pathogens with the digestate can only be decreased when the feedstock would undergo an additional treatment before anaerobic digestion or of the resulting digestate.     

Proteomic investigation of natural killer cell microsomes using gas-phase fractionation by mass spectrometry

We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-charge (m/z) dimension (GPF(m/z)) for increasing the effective number of protein identifications in cases where sample quantity limits the use of multi-dimensional chromatographic fractionation. A peptide digestate from proteins isolated from the membrane fraction of natural killer (NK) cells was analyzed by microcapillary reversed-phase liquid chromatography coupled online to an ion-trap (IT) mass spectrometer. Performing GPF(m/z) using eight narrow precursor ion scan m/z ranges enabled the identification of 340 NK cell proteins from 12 microg of digestate, representing more than a fivefold increase in the number of proteins identified as compared to the same experiment employing a standard precursor ion survey scan m/z range (i.e., m/z 400-2000). The results show that GPF(m/z) represents an effective technique for increasing protein identifications in global proteomic investigations especially when sample quantity is limited.     

‘Bioenergy from cattle manure? Implications of anaerobic digestion and subsequent pyrolysis for carbon and nitrogen dynamics in soil’

Cattle manure can be processed to produce bioenergy, resulting in by‐products with different physicochemical characteristics. To evaluate whether application of such bioenergy by‐products to soils would be beneficial compared with their unprocessed counterpart, we quantified differences in greenhouse gas emissions and carbon (C) and nitrogen (N) dynamics in soil. Three by‐products (¹⁵N‐labeled cattle manure, from which anaerobic digestate was obtained, which was subsequently pyrolysed) were applied to a loess and a sandy soil in a laboratory incubation study. The highest losses of soil C from biological activity (CO₂ respiration) were observed in manure treatments (39% and 32% for loess and sandy soil), followed by digestate (31% and and 18%), and biochar (15% and and 7%). Emissions of nitrous oxide (N₂O) ranged from 0.6% of applied N from biochar to 4.0% from manure. Isotope labeling indicated that manure N was most readily mineralized, contributing 50% to soil inorganic N. The anaerobic digestate was the only by‐product increasing the mineral N pool, while reducing emissions of N₂O compared with manure. In biochar treatments, less than 18.3% of soil mineral N derived from the biochar, while it did not constrain mineralization of native soil N. By‐products of anaerobic digestion and pyrolysis revealed soil fertility in addition to environmental benefits. However, the reported advantages lessen when the declining yields of C and N over the bioenergy chain are considered.     

Mixotrophic cultivation of <Emphasis Type="Italic">Chlorococcum</Emphasis> sp. under non-controlled conditions using a digestate from pig manure within a biorefinery

A number of business opportunities may arise from microalgae and wastewater treatment becoming an integrated system, as biofuels and high-added value products could be obtained simultaneously. This study, performed under controlled and non-controlled conditions, aimed at cultivating Chlorococcum sp. using a digestate from pig manure as culture medium and assessing its growth and biochemical composition for further applications. Under controlled conditions, cultures of Chlorococcum sp. were established testing various digestate dilutions (v/v). It was found that all tested dilutions (up to 8% v/v) promoted a higher biomass density, compared to the control culture in modified Bold’s Basal Medium (modified BBM). Under non-controlled conditions, it was found that the biomass productivity using the digestate diluted 5.6% v/v (23.4 mg L^?1 day^?1) was statistically similar to the one obtained using modified BBM (26.4 mg L^?1 day^?1). The volatile fatty acids contained in the digestate might have allowed mixotrophic growth for Chlorococcum sp. The intracellular lipid content in Chlorococcum sp. remained constant throughout the experiments in both, treatment and control cultures, while carbohydrates increased from 20 to 45% of the cell dry weight in the treatment and from 20 to 42% in the control one. It was concluded that conditions of nitrogen starvation and fluctuating irradiance and temperature benefit carbohydrate accumulation in this strain, since intracellular carbohydrate content increased nearly two-fold during this period. Additionally, the obtained biomass has the potential to be used as feedstock for bioethanol production. This system can meet the concept of a microalgae-based biorefinery, in which biofuels and high-added value products are produced from microalgae and wastewater.     

Anaerobic digestion of source-segregated domestic food waste: Performance assessment by mass and energy balance

An anaerobic digester receiving food waste collected mainly from domestic kitchens was monitored over a period of 426 days. During this time information was gathered on the waste input material, the biogas production, and the digestate characteristics. A mass balance accounted for over 90% of the material entering the plant leaving as gaseous or digestate products. A comprehensive energy balance for the same period showed that for each tonne of input material the potential recoverable energy was 405 kWh. Biogas production in the digester was stable at 642 m³ tonne⁻¹ VS added with a methane content of around 62%. The nitrogen in the food waste input was on average 8.9 kg tonne⁻¹. This led to a high ammonia concentration in the digester which may have been responsible for the accumulation of volatile fatty acids that was also observed.     

Optimization of Spongiochloris sp. biomass production in the abattoir digestate

In the present study, the abattoir digestate was used as a culture medium for Spongiochloris sp. growth with added mineral components under optimized conditions in batch culture. Firstly, an Hadamard matrix was used to investigate the impact of certain influencing factors on the Spongiochloris sp. growth. Then, a fractional factorial design 2^7-4 was successfully employed to optimize the concentration of different added components to abattoir digestate for increased Spongiochloris sp. biomass production. The major influencing factors were NaHCO₃ and FeSO₄ at a level of 2000 mg/L and 5 mg/L, respectively. A high biomass production of 5.29 × 10⁶ cell/mL and an important content of chlorophyll a of 65.32 mg/L were obtained after 42 days of culture of Spongiochloris sp. on the defined abattoir medium at static conditions.     

Treatment of pig manure liquid digestate in horizontal flow constructed wetlands: Effect of aeration

With the rapid development of scaled anaerobic digestion of pig manure, the generation of liquid anaerobic digestate exceeds the farmland loading capacity, causing serious environmental pollution. Three laboratory‐scale horizontal subsurface flow constructed wetlands (CWs; planted + aeration, planted, and unplanted) were set up to investigate the feasibility of liquid digestate treatment in wetlands. Treatment capacity in different wetlands was evaluated under different influent concentrations (chemical oxygen demand [COD], 5 days biochemical oxygen demand [BOD₅], and nitrogen forms). The effect of aeration and effluent recirculation on organic matter and total nitrogen removal was investigated. Results showed that integrating intermittent aeration in CWs significantly improved the oxygen condition (p < 0.01) in the wetland bed and promoted BOD₅ removal to 90% in aerated CWs as compared with <15% in the unaerated CWs. Meanwhile, COD removal between these three wetlands did not show any difference and varied from 52 to 72% under influent concentration of 200–820 mg/L because of the high content of hard‐degradable organic matter in the liquid digestate. Intermittent aeration resulted in high ammonium removal (>98%) although the influent loading varied from 65 to 350 mg/L. However, intermittent aeration caused nitrate accumulation of 300 mg/L and limited total nitrogen (TN) removal of 33%. To intensify the TN removal, we verified effluent recirculation to increase the removal efficiency of TN to 78%. These results not only show the potential application of CWs for treatment of high‐strength liquid anaerobic digested slurry, but also indicate the significance of intermittent aeration on the enhanced removal of organic matter and ammonium.     

Energy cover crops for biogas production increase soil organic carbon stocks: A modeling approach

Energy cover crops for biogas production through anaerobic digestion (AD) are inserted between two primary crops. They replace either bare soil or nonharvested cover crops, and their management is usually intensified to produce more biomass. They allow the production of renewable energy as well as digestate, used as an organic fertilizer, without directly competing with food production. Because of the increased biomass production and export and of the return of a digested biomass to the soil, the impact of energy cover crops on soil organic carbon (SOC) is questioned. The objective of this paper was to study the difference in SOC stocks induced by the introduction of energy cover crops for AD coupled with the application of the resulting amount of digestate. We used the AD model Sys-Metha combined with the soil C model AMG to simulate SOC stocks for 13 case studies in France, with scenarios comparing different intercrop management practices, with or without cover crops, harvested or not. Our results indicated that the higher biomass production of energy cover crops (from 6.7 to 11.1 t DM ha⁻¹) in comparison with nonharvested cover crops (2 t DM ha⁻¹) or bare soil led to higher humified C input (belowground input and digestate), despite the high C fraction exported in AD. This resulted in an increase in SOC stocks in comparison with nonharvested cover crops or bare soil (from 0.01 to 0.12 t C ha⁻¹ year⁻¹ over 30 years). The uncertainties in the model parameters did not modify these results. However, in the case of equal biomass production between energy cover crops and nonharvested cover crops, SOC stocks would be lower with energy cover crops.     

Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants

Aims

To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis‐infected dairy herds.

Methods and Results

Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one‐stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two‐stage plant.

Conclusions

Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two‐stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit.

Significance and Impact of the Study

Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.