Optimizing a biorefinery design for the valorization of Rugulopteryx okamurae by extracting bioactive compounds and enhancing methane production.

The Mediterranean Sea has suffered recently from the unprecedent invasion of the alien macroalga Rugulopteryx okamurae due to global warming and climate change putting at risk the natural local ecosystem. Since 2015 this alga has colonized a great area on the western coast of the Mediterranean Sea and it has been also spotted in other areas such as the Azores Islands or the south of France. The arrival of alga tides into the coasts also provokes collateral environmental problems that need to be addressed. Seaweed-based biorefineries are considered a promising alternative within a circular economy model. This study aims to assess the potential of R. okamurae as raw material for the extraction of reducing sugars (RS) and total polyphenolic compounds (TPC) with antioxidant capacities, the subsequent production of methane from the extracted residue, and the final use of the anaerobic digestate as fertilizer. However, the presence of bioactive compounds greatly varies due to seasonality, location or even natural degradation. In order to provide some insights about these issues, two different batches were assessed: i.e. natural and ashore R. okamurae. As brown algae are characterised by a cell wall composed of crystalline cellulose and lignin, the biomasses studied were mechanically pretreated (dried at 100 °C and milled during 60 s) before the single and sequential extraction processes. Results showed that the extraction of the targeted compounds increased by 30-80 % when the biomasses were extracted sequentially. Similarly, the biochemical methane potential of the extracted solid residues increased as the RS and TPC content was reduced (120-150 NLCH4 kg-1VS), with no significant impact regarding the biomass origin (natural or ashore) or the extraction process order. An increase in the kinetic constant k (first-order model) of 150% and 75% was observed when the fresh biomass was extracted with water and ethanol, respectively, compared to the value obtained for the unextracted biomass. Finally, the physicochemical characteristics of the different anaerobic digestates generated were assessed for their potential use as biofertilizer. In this study, most of the digestate' liquid fractions (7 out of 10) comply with European regulations for organic fertilizers and could be used directly.

Effect of metals on mesophilic anaerobic digestion of strawberry extrudate in batch mode.

According to recent studies, the anaerobic digestion of strawberry extrudate is a promising option with potential in the berry industry biorefinery. However, the lack and/or unbalance of concentrations of metals in some agro-industrial residues could hamper methane production during the anaerobic digestion of these kinds of wastes. In this study, a fractional factorial design was applied to screen the supplementation requirements regarding six metals (Co, Ni, Fe, Cu, Mn, and Zn) for methane production from strawberry extrudate (SE). The logistic model was used to fit the experimental data of methane production-time. It allowed identifying two different stages in the anaerobic process and obtaining the kinetic parameters for each step. Maximum methane production obtained in the first (Bmax) kinetic stage, the methane production in the second stage (P), and the maximum methane production rates (Rmax) concluded a statistically significant effect for Ni and Zn. The second set of experiments was carried out with Ni and Zn through a central composite design to study the concentration effect in the anaerobic digestion process of the strawberry extrudate. The parameters P and Rmax demonstrated a positive interaction between Ni and Zn. Although, Bmax did not prove a statistically significant effect between Ni and Zn.

Evaluation and modelling of methane production from corn stover pretreated with various physicochemical techniques.

Lignocellulosic by-products from agricultural crops represent an important raw material for anaerobic digestion and clean renewable, which is a key component of the circular economy. Lignocellulose is recalcitrant to biodegradation and pretreatments are required to increase methane yield during anaerobic digestion. In this work, the efficacy of different physicochemical pretreatments was compared using corn stover biomass as substrate. Anaerobic digestion of untreated and pretreated corn stover was performed in batch mode at mesophilic temperature (38°C) and organic matter solubilization of pretreated substrates was also investigated. The highest organic matter solubilization occurred in autoclave pretreatment (soluble chemical oxygen demand = 5630 ± 42 mg O2 L-1). However, the highest methane yield was obtained using alkaline pretreatment (367 ± 35 mL CH4 g-1 VSadded). Alkaline pretreatment increased methane yield by 43.3% compared to untreated control (256 ± 15 mL CH4 g-1 VSadded). Two mathematical models (i.e. first-order kinetics and transfer function) were utilized to fit the experimental data with the aim of assessing anaerobic biodegradation and to obtain the kinetic constants in all cases studied. Both models adequately fit the experimental results. The kinetic constant, k, of the first-order model increased by 92.8% when stover was pretreated with sulphuric acid compared with control. The transfer function model revealed that the maximum methane production rate, Rm, was obtained for the sulphuric acid treatment, which was 63.5% higher compared to control.

Sulfur-based mixotrophic denitrification with the stoichiometric S0/N ratio and methanol supplementation: effect of the C/N ratio on the process.

The impact of the organic carbon to nitrate ratio (C/N ratio) on mixotrophic denitrification rate has been scarcely studied. Thus, this work aims to investigate the effect of the C/N ratio on the mixotrophic denitrification when methanol is used as a source of organic matter and elemental sulfur as an electron donor for autotrophic denitrification. For this, two initial concentrations of NO3--N (50 and 25 mg/L) at a stoichiometric ratio of S0/N, and four initial C/N ratios (0, 0.6, 1.2, and 1.9 mg CH3OH/mg NO3- -N) were used at 25 (±2) °C. The results showed that when using a C/N ratio of 0.6, the highest total nitrogen removal was obtained and the accumulation of nitrites was reduced, compared to an autotrophic system. The most significant contribution to nitrate consumption was through autotrophic denitrification (AuDeN) for a C/N ratio of 0.6 and 1.2, while for C/N = 1.9 the most significant contribution of nitrate consumption was through heterotrophic denitrification (HD). Finally, organic supplementation (methanol) served to increase the specific nitrate removal rate at high and low initial concentrations of substrate. Therefore, the best C/N ratio was 0.6 since it allowed for increasing the removal efficiency and the denitrification rate.

Comparative effect of biochar and activated carbon addition on the mesophilic anaerobic digestion of piggery waste in batch mode.

A comparative study of the batch mesophilic anaerobic digestion of piggery waste was carried out with the addition of 5% biochar and 5% activated carbon. The results obtained showed that the bioreactors amended with biochar increased cumulative methane production, the kinetic constant for methane production and the COD removal efficiency compared to the control reactors and reactors with activated carbon addition. The maximum methane production and the kinetic constant were 6.9% higher in the reactors with biochar addition compared to the controls; while the COD removal efficiency was 3% higher in the case of biochar addition. In the case of activated carbon, only a slight improvement in anaerobic digestion performance was observed compared to the control.

Modeling of the effect of zeolite concentration on the biological nitrification process in the presence of sulfide and organic matter.

The evaluation of the nitrification kinetics in the simultaneous presence of sulfide and organic matter using zeolite as improver was the main goal of this work. According to the sensitivity and collinearity analyses, five parameters were the most sensitive in the model, whose calibrated values were: µ max, AOB = 0.02642 ± 0.002 h-1; µ max, NOB = 0.3307 ± 0.416 h-1; K S,NOB = 1.65·10-6 ± 2.85·10-6 mgHNO2-N/L; k S2 = 0.8213 ± 0.076 and n = 0.6537 ± 0.030. A good fit between the experimental data and the model's results including the effect of zeolite on the kinetic parameters was obtained, with Theil inequality coefficient values between 0.109 and 0.007 for all the variables studied, with all of these values lower than 0.3. Thus, the model proposed is robust and can simulate the nitrification process in the presence of sulfide and organic matter when zeolite was used as improver.

Fly ash as stimulant for anaerobic digestion: effect over hydrolytic stage and methane generation rate.

Thermoelectric fly ash was used as a micronutrient source for microorganisms in the anaerobic digestion process of thermally pretreated (1 hour, 120 °C) secondary sludge. The obtained results not only suggest that fly ash improves methane generation in the conversion of volatile fatty acids into methane, but also show a new observation, that the fly ash contributes in the particulate organic solubilization. The maximum methane production rate increased from 6.52 mL/L/d to 22.59 mL/L/d when fly ash was added at a dosage of 150 mg/L in biochemical methane potential tests compared with tests with no added ash. Additionally, the kinetic constants of the hydrolysis of particulate organic matter were obtained in both cases (with and without added ash) in batch reactors using a first-order kinetic model; in the case of no addition, the first-order kinetic parameter was 0.019 ± 0.002 d-1, while with ashes this value increased to 0.045 ± 0.000 d-1. Therefore, the addition of fly ash improves methane generation and hydrolytic kinetics in different orders of magnitude.

Anaerobic digestion of wastewater rich in sulfate and sulfide: effects of metallic waste addition and micro-aeration on process performance and methane production.

This work explores the effect of two metallic wastes (mining wastes, MW; fly ashes, FA) and micro-aeration (MA) on the anaerobic digestion of wastewater which is rich in sulfate and sulfide. Two initial COD concentrations (5,000 and 10,000 mg/L) were studied under both conditions in batch systems at 35 °C, with a fixed COD/SO42- ratio = 10, with 100 mg/L of S2-. It was observed that the use of MW and FA in the assays with an initial COD concentration of 10,000 mg/L resulted in a simultaneous increase in COD removal, sulfate removal, sulfide removal and methane generation, while MA only improved the COD and sulfide removals in comparison with the control system. On the contrary, the use of MW, FA or MA in systems with initial COD concentrations equal to or lower than 5,000 mg/L did not show any improvement with respect to the control system in terms of COD removal, sulfate removal or methane generation, with only sulfide removal being positively affected by MW and FA.

Sequential adaptation of Nannochloropsis gaditana to table olive processing water.

The main objective of this study was to evaluate the suitability of Nannochloropsis gaditana to grow by sequential adaptation to TOPW (Table olive processing water) at increased substrate concentrations (10-80%). Sequential adaptation allows growing Nannochloropsis gaditana up to 80% TOPW, although the maximum microalgae biomass productions were achieved for percentages of 20-40%, i.e. 0.308 ± 0.005 g VSS (Volatile Suspended Solids)/L. In all growth experiments, proteins were the majority compound in the grown microalgae biomass (0.44 ± 0.05 g/g VSS), whereas phenols were retained up to a mean concentration of 12.1 ± 1.9 mg total phenols/g VSS. The highest microalgae biomass production rate at rate of 80% TOPW took place in the first two days when most nutrients were also removed. Average removal efficiencies at this percentage of TOPW were 69.1%, 50.9%, 54.3% and 71.8% for total organic carbon, total soluble nitrogen, phosphate and total phenols, respectively. Sequential adaptation can ensure the obtaining of a sustainable microalgae culture as a treatment method for TOPW.

Batch anaerobic co-digestion of waste activated sludge and microalgae (Chlorella sorokiniana) at mesophilic temperature.

The microalgae Chlorella sorokiniana are used as co-substrate for waste activated sludge (WAS) anaerobic digestion. The specific objective of this research was to evaluate the feasibility of improving methane production from anaerobic digestion of WAS in co-digestion with this microalga, based on an optimized mixture percentage. Thus, the anaerobic co-digestion of both substrates aims to overcome the drawbacks of the anaerobic digestion of single WAS, simultaneously improving its management. Different co-digestion mixtures (0% WAS-100% microalgae; 25% WAS-75% microalgae; 50% WAS-50% microalgae; 75% WAS-25% microalgae; 100% WAS-0% microalgae) were studied. The highest methane yield (442 mL CH4/g VS) was obtained for the mixture with 75% WAS and 25% microalgae. This value was 22% and 39% higher than that obtained in the anaerobic digestion of the sole substrates WAS and microalgae, respectively, as well as 16% and 25% higher than those obtained for the co-digestion mixtures with 25% WAS and 75% microalgae and 50% WAS and 50% microalgae, respectively. The kinetic constant of the process increased 42%, 42% and 12%, respectively, for the mixtures with 25%, 50% and 75% of WAS compared to the substrate without WAS. Anaerobic digestion of WAS, together with C. sorokiniana, has been clearly improved by ensuring its viability, suitability and efficiency.

Performance evaluation of micro-aerobic hydrolysis of mixed sludge: Optimum aeration and effect on its biochemical methane potential.

This study evaluated the performance of a micro-aerobic hydrolysis of mixed sludge and its influence as a pretreatment of this waste for its subsequent anaerobic digestion. Three experimental series were carried out to evaluate the optimum micro-aeration levels in the range from 0.1 to 0.5 air volume/min.reactor volume (vvm) and operation times within the range of 24-60 h. The maximum methane yield [35 mL CH4/g volatile suspended solids (VSS) added] was obtained for an aeration level of 0.35 vvm. This methane yield value increased 114% with respect to that obtained with the non-aerated sludge. In the micro-aeration process carried out at an aeration level of 0.35 vvm, increases in soluble proteins and total sugars concentrations of 185% and 192% with respect to their initial values were found, respectively, after 48 h of aeration. At the above micro-aerobic conditions, soluble chemical oxygen demand (CODS) augmented 150%, whereas VSS content decreased until 40% of their initial respective values. Higher COD increases and VSS decreases were found at 60 h of micro-aeration, but the above parameters did not vary significantly with respect to the values found at 48 h.

Autotrophic and heterotrophic denitrification for simultaneous removal of nitrogen, sulfur and organic matter.

The aim of this investigation was to assess the startup and operation of a laboratory-scale hybrid UASB-Anaerobic Filter Reactor (UASFB) of 1 L volume, kept at 30°C, in order to carry out a simultaneous autotrophic and heterotrophic denitrification process. First, the heterotrophic and autotrophic populations were separately enriched, with specific cultures and subsequently the UASFB was inoculated with 2 g L(-1) of volatile suspended solids (VSS), with a ratio of 1.5:1 (autotrophs: heterotrophs). The influent or synthetic wastewater used was composed of: Na2S2O3·5H2O, CH3COOK, NaNO3, NaHCO3, K2HPO4, NH4Cl and saline solution. The concentrations varied depending on the organic loading rate (OLR), nitrogen loading rate (NLR) and sulfur loading rate (SLR) applied. In the UASFB reactor, two experimental conditions were tested and assessed: (i) COD/N ratio of 3.6 and SLR of 0.75 kg S m(-3) d(-1); and (ii) COD/N ratio of 5.8 and SLR of 0.25 kg S m(-3) d(-1). The results obtained demonstrated that an inoculum coming from an anaerobic reactor was able to carry out the process, obtaining a maximum nitrate removal of 85.3% in the first stage of operation and 99.5% in the second stage. The recovery of sulfur in form of sulfate in the effluent did not present a tendency to stabilize during the measured time, with a maximum thiosulfate removal of 32.5%, when the SLR was lowered to 0.25 kg S m(-3) d(-1). The maximum organic matter elimination, measured as COD, was 75.8%, which indicates the relatively good performance and behavior of the heterotrophic microorganisms.

Screening of biomethane production potential from dominant microalgae.

The use of microalgae for biomethane production has been considerably increasing during the recent years. In this study, four dominant species belonging to the genera Scenedesmus, Chlorella, Dunaliella and Nostoc were selected. The influence of different genera with several morphological, structural and physicochemical characteristics on methane production was assessed in biochemical methane potential (BMP) tests. The ultimate methane yield values were 332 ± 24, 211 ± 2, 63 ± 17 and 28 ± 10 mL CH4/g VSadded for Scenedesmus obliquus, Chlorella sorokiniana, Dunaliella salina and Nostoc sp., respectively. The highest methane production was achieved by microalga species that had no complex cell wall or wall basically composed by proteins and simple sugars such as in S. obliquus, whereas lower methane yields were found for D. salina and Nostoc sp., due to the salinity effects and cell wall composition in terms of complex polysaccharide and glycolipid layers, respectively. Kinetic constant values obtained in the BMP tests ranged between 1.00 ± 0.08 and 0.097 ± 0.005 days(-1) for D. salina and S. obliquus, respectively.

Effect of dissolved oxygen and temperature on macromolecular composition and PHB storage of activated sludge.

The macromolecular composition of activated sludge (lipids, intracellular proteins and intracellular polysaccharides) was studied together with its capacity to store macromolecules such as polyhydroxybutyrate (PHB) in a conventional activated sludge system fed with synthetic sewage water at an organic load rate of 1.0 kg COD/(m(3)·d), varying the dissolved oxygen (DO) and temperature. Six DO concentrations (0.8, 1.0, 1.5, 2.0, 2.5 and 8 mg/L) were studied at 20°C with a sludge retention time (SRT) of 6 days. In addition, four temperatures (10ºC, 15ºC, 20ºC and 30ºC) were assessed at constant DO (2 mg/L) with 2 days SRT in a second experimental run. The highest lipid content in the activated sludge was 95.6 mg/g VSS, obtained at 30°C, 2 mg/L of DO and a SRT of 2 days. The highest content of intracellular proteins in the activated sludge was 87.8 mg/g VSS, obtained at 20°C, 8 mg/L of DO and a SRT of 6 days. The highest content of intracellular polysaccharides in the activated sludge was 76.6 mg/g VSS, which was achieved at 20°C, a SRT of 6 days and a wide range of DO. The activated sludge PHB storage was very low for all the conditions studied.

A comprehensive and critical review of the unstandardized Folin-Ciocalteu assay to determine the total content of polyphenols: The conundrum of the experimental factors and method validation.

The Folin-Ciocalteu method can be considered to be the most widely used in laboratories around the world, to quantify the total polyphenols content. Many different variations found in this assay have been reported in the scientific literature. In this review, the full experimental conditions influencing the Folin-Ciocalteu assay have been comparatively assessed and discussed. Furthermore, few studies relating to the method validation have been evaluated according to the results of selectivity, linearity, precision, trueness, limit of determination, limit of quantification and robustness. In general, the results derived from the reviewed literature are widely variable according to both, the experimental factors selected and the performance parameters reported, making difficult the comparison of the overall results published.

Performance and kinetic evaluation of the semi-continuous anaerobic digestion of sunflower oil cake pretreated with ultrasound.

A study of the semi-continuous anaerobic digestion of sunflower oil cake previously sonicated (at a specific energy of 24,000 kJ/kg TS, constant sonication frequency of 20 kHz and ultrasonic power of 120 W) was carried out in laboratory-scale completely stirred tank reactors at mesophilic temperature (35°C). Two anaerobic inocula were used: a mixture of flocculant biomass (I) from a full-scale anaerobic reactor treating waste activated sludge and a granular inoculum (II) from an industrial UASB reactor treating brewery wastewater. Soluble COD (CODs) removal efficiencies ranged between 67.7% and 70.1% and between 61.3% and 67.7% at hydraulic retention times (HRTs) of between 24-10 days for inoculum I and 24-8 days for inoculum II. However, for HRTs lower than 8 days and 6.7 days, equivalent to organic loading rates (OLRs) higher than 2.62 and 3.15 g COD/(L·d), respectively, a sudden decrease in the CODs removal efficiency was observed in both cases. In any case, inoculum II allowed for a more stable and efficient operation for a wider range of both OLRs and HRTs, permitting an appropriate and reliable operation for OLRs as high as 3.15 g COD/(L·d) and HRTs as low as 6.7 days. The methane production rates achieved with inoculum II were always higher than those reached with inoculum I. The overall methane yield obtained with inoculum II was 13% higher than that achieved with inoculum I. In addition, this value was 1.9 times higher than the methane yield obtained with untreated (non-sonicated) SuOC. A second-order kinetic model was found to be adequate to fit the experimental results obtained for the two inocula used. The kinetic constant obtained with inoculum I was 3.5 times higher than that achieved with inoculum II.

Influence of the type and source of inoculum on the start-up of anammox sequencing batch reactors (SBRs).

Anammox (anaerobic ammonium oxidation) is an attractive option for the treatment of wastewaters with a low carbon/nitrogen ratio. This is due to its low operating costs when compared to the classical nitrification-denitrification processes. However, one of the main disadvantages of the Anammox process is slow biomass growth, meaning a relatively slow reactor start-up. This becomes even more complicated when Anammox microorganisms are not present in the inoculum. Four inocula were studied for the start-up of Anammox sequencing batch reactors (SBRs) 2 L in volume agitated at 100 rpm, one of them using zeolite as a microbial support. Two inocula were taken from UASB reactors and two from aerobic reactors (activated sludge and SBR). The Anammox SBRs studied were operated at 36 ± 0.5°C. The results showed that the only inoculum that enabled the enrichment of the Anammox biomass came from an activated sludge plant treating wastewaters from a poultry slaughterhouse. This plant was designed for organic matter degradation and nitrogen removal (nitrification). This could explain the presence of Anammox microorganisms. This SBR operated without zeolite and achieved nitrite and ammonium removals of 96.3% and 68.4% respectively, at a nitrogen loading rate (NLR) of 0.1 kg N/m(3)/d in both cases. The lower ammonium removal was due to the fact that a sub-stoichiometric amount of nitrite (1 molar ratio) was fed. The specific Anammox activity (SAA) achieved was 0.18 g N/g VSS/d.

Impact of natural degradation of the invasive alga Rugulopteryx okamurae on anaerobic digestion: Heavy metal pollution and kinetic performance.

This study shows, for the first time, how the natural biodegradation of the Phaeophyceae Rugulopteryx okamurae (R.o.) affects its methane yield, by biochemical methane potential assays, and the methane production kinetics. Additionally, a mechanical (zeolite-assisted milling) and a thermal (120 °C, 45 min) pretreatments were assessed. The highest methane yield was obtained from the mechanically pretreated fresh ashore biomass (219 (15) NLCH4 kgVS-1), which presents the use of zeolite during milling as an economical alternative for heavy metal toxicity reduction. Moreover, no significant differences were observed between the other tests (with the exception of the lowest value obtained for the mechanically pretreated fresh R.o.). Low methane yields were linked to the heavy metal content. However, an increase of 28.5 % and 20.0 % in the k value was found for the untreated fresh R.o. biomass and fresh ashore biomass, respectively, when subjected to thermal pretreatment. Finally, an enhancement of 80.5 % in the maximum methane production rate was obtained for the fresh ashore biomass milled with zeolite compared to the untreated fresh ashore biomass.

Valorization of strawberry extrudate waste: Recovery of phenolic compounds by direct-hydrothermal treatment and subsequent methane production by mesophilic semi-continuous anaerobic digestion.

Strawberry extrudate (SE) is an underused by-product from strawberry industry. Recovery of the phenolic compounds present in SE would represent a very interesting valorisation option. Two main challenges need to be solved, firstly, the solubilisation and recovery of the phenolic compounds contained in SE, and, after that, the stabilisation of the resulted de-phenolized SE. The present research evaluates the potential of a biorefinery process combining a hydrothermal pre-treatment, followed by a phenolic extraction process and, finally, the anaerobic digestion of the remaining SE for producing energy that will contribute to compensate the energy requirements of the whole system. Following the hydrothermal pre-treatment at 170 °C for 60 min, an extraction of 0.6 ± 0.1 g of gallic acid per kilogram of SE was achieved using an adsorbent resin, representing a recovery rate of 64 %. Long-term semi-continuous anaerobic digestion of de-phenolized SE was evaluated at different organic loading rates to evaluate the stability of the process. The anaerobic digestion of pre-treated SE achieved a stable methane production value of 243 ± 34 mL CH4·g volatile solids-1·d-1 at an organic loading rate (ORL) of 1.25 g volatile solids·L-1·d-1. During the operation at this ORL, the control parameters including pH, alkalinity, soluble chemical organic demand (sCOD), and volatile fatty acid (VFA) remained stable and consistently constant. Specifically, the VFA in the reactor during this stable period achieved a value of 102 ± 128 mg O2/L. Also, an economic balance showed that the minimal price of the generated phenolic extract for having benefited from the proposed biorefinery system was 0.812 €·(g of gallic acid equivalents)-1, a price within the range of phenolic compounds used in the food industry.

Turning an invasive alien species into a valuable biomass: Anaerobic digestion of Rugulopteryx okamurae after thermal and new developed low-cost mechanical pretreatments.

The invasive alien seaweed Rugulopteryx okamurae (R.o.) has spread quickly through the Mediterranean Sea causing an unprecedented ecological impact. A solution integrated into a circular economy model is needed in order to curb the negative effects of its presence. Anaerobic digestion (AD) is proposed as a feasible process able to transform biomass into renewable energy. Nevertheless, in order to improve the methane yield and surpass the drawbacks associated with AD processes, this research proposes a thermal pretreatment and a new developed method where the macroalgae is mechanically pretreated with zeolite. Chemical and microstructure characterization of the algal biomass after pretreatments involved scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR). The highest methane yields of 240 (28) and 250 (20) NLCH4 kg-1 VSadded were obtained with the new mechanical pretreatment and the thermal pretreatment at 120 °C for 45 min without zeolite, achieving a 35 % improvement against the non-pretreated algae. A direct relationship between the crystallinity index of the samples and methane production was observed. The experimental data of methane production versus time were found to be in accordance with both first-order kinetic and Transference Function mathematical models.