PH-Controlled fermentation of strawberry waste as phenol solubilisation method

1 Postprint of pH-Controlled fermentation of strawberry waste as phenol solubilisation method Journal of Cleaner Production Volume 266, 1 September 2020, 121924 DOI: https://doi.org/10.1016/j.jclepro.2020.121924 PH-CONTROLLED FERMENTATION OF STRAWBERRY WASTE AS PHENOL SOLUBILISATION METHOD Antonio Serrano1,2,*, Georgia Newton1, Bernabe Alonso-Fariñas3, Fernando G. Fermoso2, Denys K. Villa-Gomez1 1 School of Civil Engineering, The University of Queensland, Campus St. Lucia - AEB Ed 49, St Lucia, 4067, QLD, Australia 2 Instituto de Grasa, Spanish National Research Council (CSIC). Ctra. de Utrera, km. 1, Seville, Spain. 3 Departamento de Ingeniería Química y Ambiental, Escuela Técnica Superior de Ingeniería, Universidad de Sevilla, Camino de los Descubrimientos s/n, 41092 Seville, Spain. *Corresponding author: Tel.: +61 (07) 3365 3857; E-mail: a.serrano[email protected] Graphical Abstract Highlights  Soluble phenols were triplicated from initial concentration in reactors at pH 4  The process at pH 4 consumed 96% less energy than thermal extractions of phenols 2  Operation at pH 9 favoured the accumulation of acetic and propionic acids Abstract The present study aims to use pH as a control parameter for the accumulation of target compounds. Solubilisation of wasted strawberry through fermentation at different operational pH (pH 4, 7, and 9) was evaluated. Methane as a target compound was achieved at pH 7, reaching a value up to 353 ± 10 mL CH4/g VS (VS, volatile solids). Operation at pH 9 was hardly controlled due to the low pH of the substrate and the accumulation of VFA. pH 9 resulted in the highest accumulation of VFA, mainly acetic acid. The operation at pH 4 resulted in the inhibition of the biogas production, leading to the concentration of soluble phenols up to 3 times the initial concentration in the reactors. The concentrations of pelargonidin 3-glucoside and p-coumaric acid showed a significant increase at pH 4 (p=0.157 and p=0.058, respectively). Acidogenic fermentation could be a suitable method to solubilise bioactive compounds from wasted strawberries without the necessity of high-energy pretreatments. The low energy demand, i.e. 19 equivalent kJ/g gallic acid, as well as the high phenol solubilization capacity, indicates that the acidogenic fermentation at pH 4 it is a promising method for the recovery of phenols from wasted strawberry. Keywords: anthocyanins; energy balance; fermentation; methane; soluble phenols; volatile fatty acids 1. INTRODUCTION Strawberry is the most important berry crop worldwide, with an annual production of over 9 million tonnes in 2017. The production is concentrated in Mediterranean climate zones of China, USA, and Mexico, producing 40%, 17% and 7% of the global production respectively (FAOSTAT, 2018). An important percentage of this fruit is lost during 3 agricultural production, postharvest handling and distribution, which could reach up to 10- 30% of the total production (Warner et al., 2005). This huge volume of easily putrefactive waste entails a high polluting risk if not properly managed, such as uncontrolled global warming gas production, unpleasant smells, insect pests, or pollution of local watercourses from lixiviates (Siles et al., 2013). Management of these waste aligned with the circular economy concept includes resource recovery, reuse and recycling. Due to its high content of fermentable sugars, strawberry waste can be used to produce valuable products such as volatile fatty acids (VFA) and methane (Fermoso et al., 2018). Bioactive compounds are also contained in strawberry waste (Giampieri et al., 2015; Giampieri et al., 2012; Rodríguez-Gutiérrez et al., 2019). The recovery of bioactive compounds is economically interesting due to their extensive applications in pharmacy, cosmetics or food industry (Baby et al., 2018; Zhang et al., 2008). However, these compounds are usually integrated into the lignocellulosic fibres of strawberry, making it necessary to first solubilise the fibres to extract the bioactive compounds (Rubio-Senent et al., 2013; Trujillo-Reyes et al., 2019). To achieve this, different methods have been previously proposed, which include hydrothermal treatments, electric pulses and microwave or ultrasound-assisted extraction (Fermoso et al., 2018). Most of these methods have been successfully applied for the extraction of bioactive compounds from other biomass sources, such as the olive mill solid waste (Rubio-Senent et al., 2013), fig leaves (Qin & Chen, 2015) and pomegranate (Pan et al., 2012). However, most of these high-energy solubilisation methods might negatively affect some of the bioactive compounds present in the strawberry, such as the anthocyanins, which are highly thermosensitive (Jaster et al., 2018; Terefe et al., 2013). As an alternative to the current solubilisation methods, different authors have proposed that acidogenic fermentation could be an alternative for releasing important 4 bioactive compounds from biodegradable biomass sources (Girotto et al., 2017). Acidogenic fermentation is an integral step in anaerobic digestion where the fermentation metabolites such as VFA, ethanol or lactic acid are consumed by acetogenic and methanogenic microorganisms (Gu et al., 2018; Wu et al., 2015). If controlled conditions are met, full degradation of the organic matter to methane can be inhibited allowing the accumulation of these compounds. These conditions can be controlled by working at pH values outside that which is optimal for methanogenesis (Wheatley, 1990), but where hydrolytic and acidogenic activity still can occur (Zhang et al., 2009). For example, Cabrera et al. (2019) demonstrated that it is possible to accumulate phenolic compounds such as vanillin from olive mill solid waste through acidogenic fermentation at pH 5. However, at this pH, the methanogenic activity is not totally inhibited, so the degradation of phenolic compounds may still occur depending on the type of substrate. The present study aims to evaluate the solubilisation of the wasted strawberry through acidogenic fermentation at different operational pH, i.e. pH 4, 7, and 9. The processes were evaluated in terms of the inhibition of methane production, the accumulation of VFA and the accumulation of bioactive compounds such as phenolic acids and anthocyanins. 2. MATERIALS AND METHODS 2.1 Anaerobic inoculum and wasted strawberry The anaerobic inoculum was obtained from the wastewater treatment plant of the XXXX brewery company (www.xxxx.com.au), located in Brisbane, Australia (-27.467634, 153.006357). Wasted strawberry was provided by Luvaberry® and SunnyRidge® berries farms. Both farms are located in the Caboolture region, Queensland, Australia. The wasted strawberry was blended prior to the experiments to improve its homogeneity and then frozen at -10 °C to avoid undesirable fermentation processes. The characterization of the anaerobic inoculum and the wasted strawberry are shown in Table 1. 5 2.2 Experimental set-up and design Batch experiments were carried out at each operational pH (4, 7, and 9) in mesophilic conditions (35 °C) and manually shacked once per day. These pH conditions were chosen to evaluate the possibility of inhibiting methanogenesis without affecting hydrolytic and fermentative activity. For all the cases, the inoculum to substrate ratio was 2 on a grams of volatile solids (g VS) basis. Anaerobic conditions were achieved by flushing each bottle with N2 to displace the air from the headspace before sealing the bottle with a rubber septa and aluminium cap. The methane yield from the inoculum was distinguished by performing a blank experiment on the inoculum alone. All experiments, including the blank experiment, were performed in triplicate. For experiments at pH 7, biogas production was evaluated by using 24 serum bottles (140-mL). Alkalinity was initially added as 1 g of CaCO3. The characterization of the liquid phase throughout the experimental time was carried out by removing 2 serum bottles each day (destructive assays). The biogas production was quantified in all the remaining bottles through the pressure increase by using a manometer (Cole-Parmer Instrument Co Model 15.00 psig). For experiments at pH 4 and 9, biogas production and composition were monitored by using serum bottles (140-mL) in triplicate. In parallel to the serum bottles, effluent characterization was carried out at the same experimental conditions through 1-L glass reactors. The use of 1-L glass reactors was carried out to facilitate the measurement and correction, if necessary, of the operational pH during the experiments at pH 4 and 9. The operational pH was daily monitored and corrected, if necessary, at each reactor (including the serum bottles) by adding 0.1 M HCl or 0.1 M NaOH solutions as necessary. Samples from the 1-L digesters were daily taken to monitor the solubilisation of the wasted strawberry and 6 the accumulation of VFA. Alkalinity was only added as 1 g of CaCO3 to the batch operating at pH 9. 2.3 Analytical techniques The characterization of the anaerobic inoculum, wasted strawberry and the effluents from the anaerobic digesters were carried out in triplicate according to the analytical techniques described as followed. Total solids (TS, g/kg), VS (g/kg) content and pH were performed using standards methods (APHA, 2005). Soluble chemical oxygen demand (sCOD, mgO2/L) was determined using COD cell test (ranges 25-1500 mg/l and 500-10000 mg/L, Merck) and measured in a spectrophotometer (Spectroquant® Prove 300) at 620 nm. Soluble phenols concentration was determined by the Folin-Ciocalteau method (García et al., 2016). Individual VFA (C2-C7) were analysed using gas chromatography (Agilent Technologies 7890A GC System, CA, USA). All the soluble determinations were carried out after filtration using 0.22 µm pore size filters. Ellagic acid was analysed in negative ESI mode using a modified method published by Yan et al. (2014). Individual bioactive compounds including anthocyanins, i.e. cyanidin 3-glucoside, pelargonidin 3-glucoside, and cyanidin glucuronide; and other phenolics, i.e. p-coumaric acid, quercetin glucuronide/quercetin hexoside, kaempferol glucuronide/kaempferol hexoside, isorhamnetin, and ferulic acid; were analysed using a modified method published by Gasperotti et al. (2015) by liquid chromatography-mass spectrometry (LC-MS/MS, Shimadzu 8060). The anthocyanins were analysed in positive electrospray ionization mode, whereas the phenolics were analysed in negative electrospray ionization mode. The biogas composition was determined at the end of each test with a gas chromatograph (Perkin Elmer, Clarus® 590 GC), equipped with serially connected columns (7’ HayeSep N 60/80, 1/8” Sf and 9’ Molecular Sieve 13x 45/60, 1/8” Sf) and a thermal conductivity detector. 7 2.4 Data analysis The energy efficiency of the proposed method (fermentation at pH 4) was evaluated by comparing its specific energy consumption with those obtained applying a high-temperature thermal treatment, which is the alternative extraction method reported in the literature for phenols extraction from strawberry waste (Fermoso et al., 2018; Rodríguez-Gutiérrez et al., 2019). The specific energy consumption was defined as the consumed energy per g gallic acid separated in the liquid phase (see Figure 1). The energy consumed was expressed as equivalent kJ by multiplying the consumed electricity and thermal energy by a factor of 2.6 and 1.1 respectively (Ryu & Shin, 2013). All assumptions for the mass and energy balances are listed below: - Data from the serum bottle and 1L experiments section was used in the mass and energy balances for both extraction strategies (Table 1). - The enhanced extraction of phenolic compounds observed in the experiments was applied in the efficiency analysis. - The electricity consumption included the energy requirement for pumping, 1800 kJ/m3, and stirring, 300 kJ/(m3·day) (Ferrer et al., 2009). The thermal energy necessary to increase the temperature of the substrate to the operational temperature of 35 °C was calculated assuming a heat capacity of 4.18 kJ/kg/K for the waste (Ferrer et al., 2009). To compensate thermal losses in the fermenter, an extra 10% was added to the theoreticalcalculated value of required thermal energy (He et al., 2019). 20 °C was fixed as the average environmental temperature. - In the thermal extraction method, the strawberry waste is thermally treated for 1 hour by the injection of steam at 150 °C and 5 kg/cm2 (Rodríguez-Gutiérrez et al., 2019). The mass ratio steam:waste was set on 1 (Trujillo-Reyes et al., 2019). All the steam added during the thermal pre-treatment is considered to remain in the strawberry stream. 8 Thermal energy recovery of 80% was imposed, reducing the thermal energy consumption (Franchetti, 2013). According to the data reported by Rodríguez-Gutiérrez et al. (2019), the amount of soluble phenols after the thermal treatment was duplicated respect to that in the untreated strawberry waste. - Separation stage. A centrifuge was used for separation. The mass ratio liquid-phase:solidphase was set to 80:20 after centrifugation. The electricity consumption for centrifugation was 2 kWh per ton of treated-waste stream (Kookos et al., 2019). 2.5 Data processing and statistical analysis The data processing and statistical analysis (means and standard deviations) were carried out using SigmaPlot version 11.0 and Microsoft Excel 2010VR software. The tstudent test (p=0.05) was used as statistics analysis to evaluate the significant differences in the concentrations of individual anthocyanins and phenolic compounds throughout the experimental time in the experiments at pH 4. 3. RESULTS AND DISCUSSION 3.1 Methane production As expected, the fixation of the operational pH to 4 resulted in the almost total inhibition of methane production (Figure 2), while the anaerobic digestion of wasted strawberry at pH 7 resulted in the highest final methane yield, reaching a value up to 353 ± 10 mL CH4/g VS. This value is within the range described by Trujillo-Reyes et al. (2019), which reported methane yields from 324 to 391 mL CH4/g VS for the anaerobic digestion of strawberry waste at mesophilic conditions. The methane yield at pH 9 reached a final value of 215 ± 10 mL CH4/g VS. However, the time required to achieve this yield was significantly lower than at pH 7 (Figure 2). The high methane production rate can be a consequence of the enhancement of the hydrolysis 9 activity due to the alkaline operational pH since this stage is usually the rate-limiting for the anaerobic digestion of organic solids (Ortega et al., 2008). Alkaline pH has been reported to enhance the hydrolysis rate by enhancing the activity of some enzymes involved in the disintegration and solubilization of proteins and carbohydrates (Fang et al., 2020; Jie et al., 2014). Although the methane yield coefficient was around 40% lower than that obtained at pH 7, the methanogenesis was only partially inhibited. Methanogenesis at pH 9 is rarely reported (Zhang et al., 2009), but there are some cases in literature reporting methane production at pH 9 (Cabrera et al., 2019). Methanogenesis in the present study at pH 9 might be explained by the variation in the operational pH (Figure 3), which allowed the partial conversion of the soluble compounds into methane before the pH corrections. Contrary to this, maintaining the operational pH at 4 throughout the experimental time (Figure 3) did not require adding any extra reagents (data not shown) due to the acidic character of the wasted strawberry (Table 1). The biogas composition at the end of the experiment varied depending on the operational pH (Table 2). The biogas at pH 7 was composed of 71.87 ± 0.24% of CH4 and 28.13 ± 0.19% of CO2 (Table 2). The high methane production and methane content in the biogas at pH 7 was in line with the pH of the reactors, which fell within the optimal values for the methanogenic activity, i.e. 6.6-7.8 (Yuan et al., 2011) (Figure 3). At pH 4 the low amount of biogas that was produced had a low percentage of methane as well as hydrogen, showing that methanogenesis and particularly hydrogenotrophic methanogenesis was inhibited in the proposed controlled process (Sivagurunathan et al., 2016). 3.2 Accumulation of soluble matter and VFA The concentration of sCOD did not significantly vary in the reactors operated at pH 4 (Figure 4a). After an initial decrease from 8575 ± 50 mg O2/L to 6680 ± 50 mg O2/L, the sCOD gradually increased up to values around 9000 mg O2/L. The concentration of VFA 16 Giampieri, F., Tulipani, S., Alvarez-Suarez, J.M., Quiles, J.L., Mezzetti, B., Battino, M., 2012. The strawberry: Composition, nutritional quality, and impact on human health. Nutr. 28(1), 9-19. 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Zhang, Y., Seeram, N.P., Lee, R., Feng, L., Heber, D., 2008. Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties. J. Agri. Food Chem. 56(3), 670-675. 20 Figure captions Figure 1. Alternative processes for phenols extraction from strawberry waste: A) Fermentation at pH = 4, and B) Thermal treatment 1 hour at 150 °C. Figure 2. Accumulated methane yield coefficient (mL CH4/g VS) at pH 7, 4 and 9. Figure 3. Measured pH over time on the reactors operated at pH 7, pH 4 and pH 9. Figure 4. Concentration of soluble chemical oxygen demand (sCOD) and volatile fatty acids (VFA) over time on the reactors operated at (a) pH 4, (b) pH 7, and (c) pH 9. Figure 5. Characterization of volatile fatty acids (VFA) produced over the experimental time at (a) pH 4, (b) pH 7, and (c) pH 9. Figure 6. Concentration of soluble phenols over time on the reactors operated at pH 4, pH 7 and pH 9. 21 Figure 1. 22 Figure 2. Time (d) 0 2 4 6 8 10 Methane yield coefficient (mL CH4/g VS) 0 100 200 300 400 pH 4 pH 7 pH 9 23 Figure 3. Time (d) 02468 pH 3 4 5 6 7 8 9 10 pH 4 pH 7 pH 9 24 Figure 4. A) Time (d) 0 2 4 6 8 sCOD (mg O2/L), VFA (mg O2/L) 0 2000 4000 6000 8000 10000 sCOD VFA B) Time (d) 0 2 4 6 8 sCOD (mg O2/L), VFA (mg O2/L) 0 2000 4000 6000 8000 10000 sCOD VFA C) Time (d) 0 1 2 3 4 5 6 7 sCOD (mg O2/L), VFA (mg O2/L) 0 2000 4000 6000 8000 10000 sCOD VFA 25 Figure 5. A) Time (d) 2 4 6 8 10 12 14 VFA (mg/L) 0 1000 2000 3000 4000 5000 Acetic acid Propionic acid iso-Butyric acid Butyric acid iso-Valeric acid Valeric acid 4-Methyl valeric acid Hexanoic acid Heptanoic acid B) Time (d) 0 1 2 3 4 5 6 7 VFA (mg/L) 0 1000 2000 3000 4000 Acetic acid Propionic acid iso-Butyric acid Butyric acid iso-Valeric acid Valeric acid 4-Methyl valeric acid Hexanoic acid Heptanoic acid C) Time (d) 0 1 2 3 4 5 6 VFA (mg/L) 0 1000 2000 3000 4000 5000 Acetic acid Propionic acid iso-Butyric acid Butyric acid iso-Valeric acid Valeric acid 4-Methyl valeric acid Hexanoic acid Heptanoic acid