Influence of inoculum to substrate ratio on the anaerobic digestion of a cassava starch polymer

Industrial Crops & Products 141 (2019) 111709

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Influence of inoculum to substrate ratio on the anaerobic digestion of a cassava starch polymer

T

Paulo André Cremoneza, , Silvio Cesar Sampaioa, Joel Gustavo Telekenb, Thompson Weiser Meierb, Jonathan Dieterb, Jhony Telekenc ⁎

a

University of West Paraná, Paraná, Brazil. Rua Universitária, 2069, CEP: 85.819-130, Bairro Faculdade, Cascavel – PR, Brazil Federal University of Paraná (UFPR-Campus Palotina), R. Pioneiro, 2153, CEP: 85.950-000, Bairro Jardim Dallas, Palotina - PR, Brazil c UFSC - Federal University of Santa Catarina. Trinity District CTC, Florianópolis, Santa Catarina, 88040-900, Brazil b

ARTICLE INFO

ABSTRACT

Keywords: Volatile acids Biohydrogen Bioenergy Environmental sanitation Gompertz

The objective of this work was to assess the anaerobic digestion of a cassava starch polymer (CSP) under different inoculum to substrate ratios, evaluating the material decomposition level and the energy production from the biogas generated in this process. The experiment was designed with 6 treatments in triplicate, which consisted of volatile solids (VS) of the inoculum to VS of the CSP ratios (VSI/VSCSP) of 0.04, 0.08, 0.20, 0.60, 1.00, and a control treatment without the addition of CSP. Five destructive reactors were built for each treatment to monitor the degradation kinetics in the digestion process. Higher total solids (TS) and volatile solids (VS) removal values were found in lower ratio treatments. The maximum VS removal values found were greater than 85% in the 0.08 treatment and greater than 90% in the 0.04 treatment. The digestion process yielded a product with a high biogas content, rich in methane and hydrogen. The treatment with ratio of 0.08 resulted in a production of 1384 mL biogas per gram of VS removed. The results of the biogas analysis showed up to 44% hydrogen content in the acidogenic phase and 87% methane after stabilization of the methanogenic phase. The data from the 0.04, 0.08, and 0.20 treatments were fitted to the bisigmoidal model, using an equation adapted from the Gompertz function. Specific production rate was estimated for each phase of the digestion process. The rapid dissolution in water and easy degradation of the sugars of CSP into volatile acids indicates the possibility of studying anaerobic digestion in reactors with acidic and methanogenic phases physically separated, thus, optimizing the parameters to obtain more hydrogen and methane.

1. Introduction Recycling of packaging can significantly reduce environmental impacts and large volumes of improperly discarded municipal waste. However, this process requires a high-energy expenditure, resulting, in most cases, in unsustainable economic scenarios (Da Cruz et al., 2014). Direct and indirect contaminations of food packages with the food itself make the reuse of these materials difficult or impossible. Conventional recycling facilities are not prepared to clean and decontaminate these compounds, causing them to be disposed in landfills. The current challenge concerns technologies in which the packaging can be discarded together with the food residue (Musiol et al., 2016); therefore, the use of biodegradable foams and plastics is considered a viable alternative to solving the problem of the accumulation of plastics, mainly in the food packaging sector (Dicastillo et al., 2016). Starch is a carbohydrate composed primarily of amylose and

⁎

amylopectin, whose proportions vary depending on the culture by which the starch is obtained (Dean et al., 2008). This polymer is considered one of the most promising raw materials to replace petrochemical plastics and foams (Shogren et al., 2002). Variations in amylose and amylopectin concentrations directly change physicochemical characteristics and properties of plastic materials made from starch (Ellis et al., 1998; Shimazu et al., 2007). Cassava starch is a carbohydrate that is metabolized by a wide range of microorganisms to obtain products such as ethanol, hydrogen, and methane (Amon et al., 2007; Bai, et al. 2008; Mohee et al., 2008; Kryvoruchko et al., 2009; Russo et al., 2009). In this context, the anaerobic digestion process is an interesting alternative for the degradation of the polymers formed by this starch, with the benefit of energy production from the use of the biogas generated in the process. However, specific and fundamental parameters, such as the inoculum activity, solids loading, and degradability time, have not yet

Corresponding author. Tel.: +55(44)99712-8711. E-mail address: [email protected] (P.A. Cremonez).

https://doi.org/10.1016/j.indcrop.2019.111709 Received 21 March 2019; Received in revised form 19 August 2019; Accepted 19 August 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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been fully studied (Guo et al., 2011), which reduces the reliability of the products with respect to technical and environmental issues. Thus, the objective of this study was to evaluate the influence of the inoculum to substrate ratio on the degradation of a cassava starch polymer and the biogas production in this process.

sugar-rich materials, such as potato and beet. The stability in the process was maintained by adding 5.6 g L−1 of sodium bicarbonate to each treatment. This was done to provide an alkalinity reserve to the reactors based on preliminary tests with the polymer, thus, preventing collapse of the reactors due to a possible high acidification at the beginning of digestion (Cremonez et al., 2016). Three reactors were used (replicates) for each treatment (VSI/VSCSP of 0.04, 0.08, 0.20, 0.60, 1.00, and control), and each replicate was placed in 3 different greenhouses maintained under the same experimental conditions, totaling 18 reactors. Five destructive samples were used for each treatment to monitor the initial degradation process of the polymers, which were kept in a fourth greenhouse under the same experimental conditions as the other reactors. Samples were collected for analysis at 24, 48, 72, 96, and 144 h. An illustrative schematic of the experiment is shown in Fig. 1. The results of the dependent variables (removal of total solids, biogas production, and methane production) were determined, and the data were fitted to a regression model by analysis of variance (ANOVA) to correlate the response of the variables. The models were evaluated using the F test at 5% significance level and coefficient of determination (R2). The means of the biogas and methane productions by VS removal were compared by the Tukey's test. All data were tested following the ANOVA assumptions (normality, homoscedasticity, and independence).

2. Materials and methods 2.1. Substrate for digestion The cassava starch polymer (CSP; approximately 95% starch in the composition) was obtained from a company that produces biodegradable packaging (cups and trays). The polymer was ground to a maximum particle size of 1 cm³. The processed materials were packed in hermetically sealed flasks and protected from light and moisture. The inoculum used in the digestion process was obtained from a tubular digester operating in a semicontinuous feed regime in mesophilic stage used for treating wastewater from pig farms. The inoculum volume was determined as 20% of the useful volume of the reactor, according to the range used by Cho et al. (2011) and Elbeshbishy et al. (2012). The total useful volume of the reactors was completed with deionized water. 2.2. Reactors and experimental conditions

2.4. Evaluation of parameters

The reactors were built from polyvinylchloride (PVC), with dimensions of 500 mm in height and 100 mm in diameter, totaling a volume of 4.00 L. The useful volume determined for the experiment was 3.20 L, maintaining a dead volume of 20% of the reactor volume to avoid any reflux in the exit of the gas collector that may be caused by generation of foam or pressure in the reactor. The gasometers were built with the same material as the reactors, with dimensions of 300 mm in height and 100 mm in diameter and a useful volume of approximately 2.40 L. These gasometers were immersed in a saline acid solution containing sodium chloride and sulfuric acid to avoid the escape of the produced gas and to prevent the dissolution of the carbon dioxide present in the biogas. The initial degradation profile of the polymers in the evaluated treatments was monitored; reactors with destructive samples were made using polyethylene terephthalate (PET) containers with a total volume of 0.50 L, following the same proportions as the previous reactors, with 20% dead volume, thus, presenting a useful volume of 0.36 L. These destructive reactors contained a gas outlet hose immersed in an aqueous, bubbling the generated gas and preventing oxygen from entering the system. The experiment was conducted at a temperature within the mesophilic range. The reactors and destructive samples were placed in greenhouses with temperature maintained at 37.0 ± 1.0 °C. A 32-day period was determined as the hydraulic retention time, based on the period in which the biogas production in the methanogenic phase ceased.

2.4.1. Substrate and effluent The parameters and their respective references used to characterize the inoculum and the CSP before and after the digestion process are shown in Table 1. 2.4.2. Biogas volume and composition The volume of biogas generated in the digesters was quantified by measuring the vertical displacement of the gasometers and subsequently applying the correction for standard conditions of temperature and pressure (STP). The biogas composition was determined using gas aliquots collected with the aid of gasometric ampoules, directly from the hose connecting the gas outlet of the reactor to the gasometer. The constituents of the biogas (hydrogen, carbon dioxide, and methane) were determined by gas chromatography in a Shimadzu® 2010 system equipped with a Carboxen® 1010 PLOT capillary column (30 m × 0.53 mm ×0.30 μm). Argon was used as the carrier gas, with flow of 8 mL min−1. A sample of 500 μL of the gas was injected; the injector temperature was set to 200 °C. The detection was performed in a thermal conductivity detector (TCD) at temperature of 230 °C. The oven was ser to a starting temperature of 130 °C and heated to 135 °C at a rate of 46 °C min−1 for 6 min (Penteado et al., 2013). 2.4.3. Biochemical potential of the biogas (BPB) and methane (BPM) produced BPB and BPM were tested to compare the gas production potentials with the results obtained for variations in the inoculum to polymer ratios. The test was performed according to the recommendations of the VDI 4630 procedure (VDI, 2006), with incubation in 200-mL reactors coupled to eudiometer tubes under mesophilic conditions (37 °C); the biogas production was monitored until stabilization [(dV/dt)/ ΣV < 1%]. The methane content was determined by an infrared meter (BIOGAS5000, Landtec, USA).

2.3. Experimental design Five treatments with different concentrations of CSP were chosen based on their ratios between volatile solids (VS) of the inoculum and VS of the CSP (VSI/VSCSP). In general, ratios less than 2.0, in the treatment of most residues, present solids removal and biogas production efficiencies greater than those at higher ratios. The ratios used were 0.04, 0.08, 0.2, 0.6, and 1; a treatment containing only the inoculum and deionized water was used as a control. These ratios were chosen based on the optimal ranges described by Lopes et al. (2004) and Boulanger et al. (2012) for different urban solid wastes; Reischwitz et al. (1998) and Ryan et al. (2017), who evaluated poly(hydroxybutyrate)-based bioplastic reactors; and Parawira et al. (2004), who evaluated processing residues of

2.4.4. Profile of organic acids The acetic, butyric, propionic, formic, and lactic organic acids were quantified by high-performance liquid chromatography (HPLC) in a Shimadzu® system equipped with an Aminex® HP-87H column (300 mm × 7.8 mm Bio-Rad), a CTO-20A oven at 64 °C, a CBM-20A 2

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Fig. 1. Illustrative schematic of the greenhouses containing the 3 replicates of each of the treatments and the greenhouse with the 30 destructive samples of the same treatments.

controller, a UV detector with an SPD-20A diode array at a wavelength of 208 nm, and an LC-20AT pump. The mobile phase consisted of an ultrapure Milli-Q water (Millipore®) acidified with 0.005 M H2SO4 at flow rate of 0.5 mL min−1 and injection volume of 20 μL (Lazaro et al., 2012; Penteado et al., 2013).

Gompertz equations and is shown in Equation 1: M (t ) = M1 exp

exp

µm1 exp (1) (1 M1

t) + 1

+ M2 exp

exp

µm2 exp (1) ( 2 M2

t) + 1

where M(t) is the volume of biogas produced over time t; M_1 and M_2 are the volumes of biogas at the end of the first and second phases, respectively; μ_m1 and μ_m2 are the maximum rates of biogas production in the first and second phases, respectively; and λ_1 and λ_2 are the adaptation times of the first and second phases, respectively. The meaning of each of the parameters in Equation 1 is shown in Fig. 2. The data were fitted to the model using the fit function of the MATLAB program. This function uses the nonlinear least squares method and the trust-region reflective Newton algorithm. The initial approximation of each parameter in the fitting process was defined from the experimental curves. The fit of the biogas production data to the model, presenting the

2.4.5. Modeling of biogas production Two phases of biogas production were evaluated in the experiment because of the rapid degradation of the reducing sugars present in the CSP. The first phase is dominated by a group of acidogenic microorganisms, with accelerated biogas production and a large amount of hydrogen and carbon dioxide; the second phase produces mostly methane by the consumption of these intermediates. This type of production kinetics can be described mathematically using the sum of two sigmoid equations (Planas et al., 2004; Vázquez et al., 2009). The model used in the present study was obtained by summing two Table 1 Methods employed in the determining of parameters. Parâmetro

Método

Hydrogenionic Potential (pH) Total Solids (TS) Volatile Solids (VS) Fixed Solids (FS) Volatile Acidity (VA) Total Alkalinity (TA) Parcial Alkalinity (PA) Intermediate Alkalinity (IA) Higher calorific value (HCV) Lower calorific value (LCV) Total Sugars

(4500-H* / APHA, 1995) (2540-B / APHA, 1995) (2540-E / APHA, 1995) (2540-E / APHA, 1995) (SILVA, 1977) (SILVA, 1977) (SILVA, 1977) (SILVA, 1977) ASTM 05865-12 ASTM 05865-12 DUBOIS et al., 1956

Fig. 2. Graphic description of the parameters M1, µm1, Equation 1. Adapted from Vázquez et al. (2009). 3

1,

M2 , µm2 and

2

of

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Fig. 3. Fit of the bisigmoidal model to the experimental data for the production of (a) biogas, (b) methane and (c) hydrogen.

acidogenic and methanogenic phases over the digestion, is shown in Fig. 3.

amenable to anaerobic digestion. The input parameters of the evaluated treatments are shown in Table 2. All treatments had solid contents lower than 2%, which guarantees the fluidity of the material and facilitates the access of the microorganisms to the substrate for digestion.

3. Results and discussion The inoculum obtained from a swine wastewater treatment had a slightly alkaline pH, and volatile acids to total acids ratio (VA/TA) within the ideal range established by Van Haandel (1994) and Borja et al. (2004), who found that ratios lower than 0.4 result in stability of the digestion process. Similarly, Correia and Del Bianchi (2008) found that the lower the VA/TA ratio, the better the stability of the digestion process. A high volatile acids (VS) content was found in the fraction of total solids of the cassava starch polymer (CSP). More than 93% of the VS were composed of total sugars, indicating that most of the compound is

3.1. Concentration of the volatile acids and pH The final pH after 32 days of digestion for the treatments with ratios of 1.0, 0.6, 0.2, 0.08, and 0.04 were 7.92 ± 0.03, 7.98 ± 0.08, 7.96 ± 0.06, 8.08 ± 0.03, and 7.95 ± 0.06, respectively; the final pH of the control treatment was 8.02 ± 0.04. Although near neutral, the effluents were slightly alkaline. Near-neutral pH values indicate that the methanogenic phase of the digestion process occurred efficiently, consuming the organic acids generated in previous stages. The digestion process is directly affected 4

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Table 2 Input parameters of studied treatments. Treatments (substrate + water + inoculum) Input ratios (VSI/ TS (g/L) VS (g/L) FS (g/L) VSCSP) Control 07.4123 2.6041 4.8082 0.04 15.1731 9.5034 5.6697 0.08 11.2927 6.0538 5.2389 0.2 8.9645 3.9839 4.9806 0.6 7.9297 3.0641 4.8656 1.0 7.7227 2.8801 4.8426 Inoculum CSP pH 8.12 pH (aqueous solution) TA 3196 mg/L TS (%) PA 621 mg/L VS (%) IA 2575 mg/L FS (%) VA 474 mg/L Total Sugars (%) Ratio AV/AT 0.15 HCV (mJ/kg) Added Baking Soda 5075 mg/L LCV (mJ/kg)

Content PCS (g/ L) 0.0000 6.8993 3.4497 1.3799 0.4510 0.2759 7.68 89.05 88.85 0.20 83.13 17.23 15.86

Fig. 5. Profile of the VA/TA ratio of the destructive samples of the treatments studied in the initial period of the digestion process.

by pH, which determines the microorganisms that develop more efficiently in the reaction medium. Regarding the overall digestion process, the ideal pH range is 6.8–7.4 (Mao et al., 2015). CSP has great potential for the generation of volatile acids because of high amylose and amylopectin concentrations and its fast dissolution in water. However, the pH did not show large variations in the first days of digestion, except for the treatment with the highest concentration (0.04 ratio). This occurred because of the addition of sodium bicarbonate to the reactors (based on previous tests), which provided stabilization of the pH by a chemical action, buffering the volatile acids. This buffering was also evident for the VA/TA ratio in the destructive samples. Treatments with ratios of 0.04 and 0.08 presented similar results, with an increase in the VA/TA ratio up to the ideal limit at day 4 due to the high concentration of CSP solids that can be converted to volatile acids. The addition of sodium bicarbonate or hydroxide to the digestors to provide alkalinity is a preventive practice used in several studies (Plaza et al., 1996; Hamzawi et al., 1998; Ağdağ and Sponza, 2005) and by industries. The pH and the VA/TA ratio results until the sixth day of the digestion of the destructive samples are shown in Figs. 4 and 5. The production of volatile acids in reactors operating in batch mode was high in the first days of the digestion process, decreasing according to the consumption of the available organic matter. The sodium bicarbonate demand in this step was enhanced because the activity of acidogenic bacteria and acid production were higher than its consumption by methanogenic bacteria, which can lead to the inhibition of the methanogenic phase and the consequent collapse of the reactor

(Gorris et al., 1989; Schievano et al., 2010; Pezzolla et al., 2017). Despite the control of the VA/TA ratio and the addition of bicarbonate, knowing the profile of volatile acids present in the reactor is fundamental to understand the stage and metabolic pathway of the microorganisms involved in the degradation process (Ye et al., 2013). In some cases, volatile acids can accumulate in the biomass boundary layers, generating local pH values that may inhibit the subsequent digestion process (Vavilin et al., 2008). The production profile of volatile acids in the first days of digestion of the destructive samples is shown in Fig. 6. The higher the starch content in the substrate, the faster the increase of the concentration of volatile acids produced during the digestion process (McInerney, 1998; Russo et al., 2009). The treatments with ratios of 0.04, 0.08, and 0.2 showed the highest concentrations of volatile acids from the first day of digestion, which were directly affected by the polymer load added to the treatments. Acetic acid and propionic acid were the most frequently found acids in all treatments. The acetic acid production is followed by generation of molecular hydrogen; in addition, when consumed, acetic acid can be converted directly to methane and CO2. However, from the degradation point of view, propionic acid levels higher than those of acetic acid indicate a slower degradation process and biogas production, because propionic acid must be converted to acetic acid before generating methane and CO2 (Fig. 7). However, Yokoi et al. (1998) state that the presence of propionic and butyric acids is positively related to the biohydrogen production. Moreover, the conversion of propionic acid is thermodynamically unfavorable, unless the hydrogen partial pressure in the reactor is maintained at an extremely low level (Ye et al., 2013). The presence of lactic acid also indicates system overload because lactic acid is a precursor of propionic acid in the anaerobic fermentation process (Fig. 7). Zhang et al. (2003) also reported increases in generation of propionic acid as a function of increases in starch concentration. According to Wellinger (1997) and Kryvoruchko et al. (2009), when the total volatile acids concentration exceeds 3000 mg L−1, inhibition of anaerobic digestion process can occur. Treatments with high ratios showed values that exceeded these limits in the first days of digestion. The increase in the alkalinity in the process ensured the no pH fluctuation. However, polymer concentrations higher than those tested in the present study would probably result in loss of efficiency of the process. The concentrations of volatile acids at the end of the digestion process were 125, 135, 165, 290, 370, and 165 mg L−1 for the 1.0, 0.6, 0.2, 0.08, 0.04, and control treatments, respectively. These results confirm the high VS removal in the treatments, especially in those with

Fig. 4. pH profile of the destructive samples of the treatments studied in the initial period of the digestion process. 5

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Fig. 6. Volatile acid profile. a) Control; b) 1.0; c) 0.6; d) 0.2; e) 0.08; f) 0.04.

higher concentrations, and the biogas production from the consumption of the organic acids. The low acid concentration at the end of the 32nd day indicates that the methanogenic phase occurred efficiently.

The data show that at the beginning of the digestion process the percentages of TS removal are increasing; similar results were found in all treatments with addition of CSP, which presented removal varying from 35% to 47%. Treatments with lower concentrations of CSP resulted in higher VS removal percentages until the sixth day of digestion; the treatments with ratios of 0.04 and 0.08 showed higher volatile acids production in the same period. This is because the absolute VS load of these treatments (Table 2) is substantially higher than that of the 0.6 and 1.0 treatments. VS represents the actual load of solids capable of being degraded by the microbiological community inside the reactor.

3.2. Removal of total solids (TS) and volatile solids (VS) One of the main parameters used to evaluate anaerobic digestion efficiency was the reduction of TS and VS in the reactors. The removal of TS and VS at the beginning of the digestion process of the destructive samples is shown in Figs. 8 and 9.

Fig. 7. Conversion reactions of volatile acids in the anaerobic digestion process. (1) Conversion of acetic acid; (2) conversion of propionic acid; (3) conversion of lactic acid.

6

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Fig. 8. TS removal profile (%) for the tested treatments. Fig. 10. Removal of TS for the VSI/VSCSP ratios experimentally tested using an exponential regression model and an asymptotic function. Equation y = a - bcx, where a = 40.765, b = -40.897, and c = 7.578E−4; R² = 0.976; pvalue = 5.676E-5. Bars correspond to the standard deviation (three replicates were used for each treatment).

treatments with ratios of 0.04 and 0.08 resulted in much higher biogas production than the other treatments. The results were significant and fitted to the exponential regression model. The data showed that the smaller the inoculum to substrate ratio in VS, the greater the cumulative biogas production. Higher ratios tend to reduce biogas production, which is confirmed by the results of Boulanger et al. (2012). The biogas production data (Fig. 12) at the beginning of the process show an accelerated production of biogas (a), with high carbon dioxide and hydrogen contents. This stage is the acid phase of the digestion process. After conversion of most of the sugars to hydrogen, carbon dioxide, and organic acids, the digestion process performed by the microorganisms ceased (b). In contrast to the acidogenic phase, the growth of methanogenic microorganisms occurs slowly. As a result, the methanogenesis step requires a much higher retention time of the material in the reactor than that typically required for the degradation of these volatile acids and for methane production (Lee et al., 2014; Khan et al., 2016). Thus, the highest daily and cumulative biogas productions containing high

Fig. 9. VS removal profile (%) for the tested treatments.

The high solid degradation results in the first days of digestion are primarily related to the high solubility of the polymer and the high surface area for access to microorganisms and hydrolytic enzymes. The tendency for rapid degradation of the polymers is followed by a stabilization phase, in which the organic acids generated from the degradation of the polymer will be assimilated by acetogenic and methanogenic microorganisms in a step that occurs at a slower metabolism when compared to the initial stages of acidification. High carbohydrate removal in the first days of digestion was also found by Elbeshbishy and Nakhla (2012) when evaluating the co-digestion of proteins and carbohydrates, with a degradation of 95% of carbohydrates into particles, and much of the soluble carbohydrates degraded by the fifth day. Regarding the final TS removal, the highest removal percentages were found in the treatments with the lowest VSI to VSCSP. The data of TS fitted with statistical significance to an exponential regression model, showing that ratios higher than 1.0 do not present a trend of TS removal higher than 40%. However, the reactors with the higher ratio treatments had a fixed solid load proportionally higher than those with the lower ratio treatments. Considering the TS removal, the related values for the removal are closer, although significantly different. The TS removal in all treatments after 32 days of digestion is shown in Fig. 10.

Fig. 11. Cumulative biogas production for the (VSI/VSCSP) ratios experimentally tested using an exponential regression model and an asymptotic function. Equation y = a - bcx, where a = 350.41006, b = -11,071.525, and c = 5.965E−4; R² = 0.949; p-value = 5,313E-10. Bars correspond to the standard deviation (three replicates were used for each treatment).

3.3. Biogas production The cumulative biogas production is shown in Fig. 11. The 7

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g−1 (Fig. 14). The 0.08 treatment presented the highest biogas production by amounts of solids, with an average of approximately 1027 mL g−1, which was higher than the results of the BPB analysis. This shows that increasing the VSI/VSCSP strongly affected the biogas production and provided better results, following the same results of the lag phase. The results of biogas production are similar to those found in other studies that used digestive substrates rich in cassava and biopolymers. Mohee et al. (2008) evaluated a biodegradable material composed of 60% starch or derivatives and of approximately 40% biodegradable hydrophobic resin and found a biogas production of 286 mL g−1. Malina and Pohland (1992) found biogas productions by VS removal of 750 to 1000 mL g−1. Torres et al. (2009) evaluated digestion of wastewater from cassava processing and found VS conversions to biogas of 509 to 1642 mL g−1. Xiao et al. (2017) evaluated carbohydrate-rich food residues in a single-stage reactor and found biogas productions means of 931 to 1016 mL g−1. Guo et al. (2011) found methane productions of 264.1 to 293.7 mL g-1 by digesting packages based on starch biopolymers and polyvinyl alcohol in a digester operating in the mesophilic phase.

Fig. 12. Profile of biogas production as a function of time.

methane content were found after day 13 (c). The cumulative biogas production data of the 0.04, 0.08, and 0.2 treatments fitted to the bisigmoidal model (Fig. 13 and Table 3). The model adequately described the phenomena, with R² higher than 0.99 in all cases. The 0.04 treatment had the largest cumulative biogas generation in the acidogenic phase (P1), which is explained by the large amount of sugars converted to volatile acids, resulting in the generation of and carbon dioxide and hydrogen in the cellular metabolism of the microorganisms. In the methanogenic phase, the highest cumulative production was found in the 0.08 treatment. The lowest lag phase times for the two phases ( 1 and 2) were found in the 0.08 treatment, with times of 0.54 and 10.93 days for the acid phase and methanogenic phase, respectively. These minimum latency periods indicate that the best cell and substrate proportions to initiate cell multiplication and biogas production were at the beginning of this treatment, when it is assumed that the pH presents no significant changes due to the preventive buffering. The 0.04 treatment showed an abrupt drop in pH between the first and second day of digestion (Fig. 4); this is directly related to the process stability, consequently affecting the time of the microorganism adaptation phase. The treatments with the highest biogas production rates (R1 and R2) were in decreasing order for the treatments 0.2 > 0.04 > 0.08, and in phase 2 for 0.04 > 0.08 > 0.2. This is because upstream reactions, such as hydrolysis of plastic compounds, depend on the amounts of hydrolytic organisms and the surface area of the residue bioavailable to hydrolysis. In some cases, the bioavailable surface is more limiting than the methanogenic reactions that use the volatile acids previously produced. The 0.6 and 1.0 treatments showed no sufficient points of biogas production to fit to the bisigmoidal model. The test of biochemical potential of biogas (BPB) showed a biogas production of 802 mL per gram of VS (mL g−1). It was similar to the biogas production of the 0.04 treatment, which produced 854 ± 61 mL

3.3.1. Biohydrogen production The generation of some volatile organic acids indicates the production of hydrogen gas. The production of hydrogen is an energetically attractive route because it does not consume much energy and can occur in the same methanogenic reactor or in parallel to the primary process. Similar to biomethane production, biohydrogen production contributes directly to wastewater treatment by reducing the organic load and solid contents (Arantes et al., 2017), although at a lower extent than the biomethane production. In the first 48 h of digestion, the 0.04, 0.08, 0.2, and 0.6 treatments achieved a maximum hydrogen content of approximately 19.5%, 44.7%, 46.4%, and 15.65% in the biogas, respectively. Only the 1.0 treatment did not produce hydrogen at the beginning of the digestion process. The total cumulative biogas contents varied between 5% and 10% in the treatments with the highest concentrations. These results indicate the rapid digestion of the reducing sugars present in the substrate for the formation of organic acids during the first 24 h of fermentation (Fig. 6). Biohydrogen can be produced or consumed in the anaerobic digestion during the synthesis or decomposition of several organic acids inside the reactor by the action of different anaerobic microorganisms. The different routes of glucose degradation for the formation of organic acids and production or consumption of hydrogen are shown in Fig. 15. Some routes, such as the production of acetic acid and butyric acid, favor the hydrogen production and have the highest yields of molecular hydrogen in the process. The large amounts of hydrogen produced in the 0.04, 0.08, and 0.2 treatments are from the high levels of acetic acid produced on the first day of digestion. The formation of propionic acid on the second day supports this route because its formation requires the consumption of molecular hydrogen, and the lactic acid present in the

Fig. 13. Adjustment of the bisigmoidal model to the experimental biogas production data for the treatments a) 0.04; b) 0.08; and c) 0.2. 8

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Table 3 Gompertz model parameters optimized from experimental treatments. Coefficients

Ratio 0.04

Ratio 0.08

Ratio 0.2

P1 (mL) P2 (mL) 1 2 R1 R2 R²

3152 (2651, 3654) 4827 (4106, 5547) 0.8021 (0.5802, 1.024) 14.29 (12.98, 15.6) 3448 (1993, 4904) 619.9 (475.8, 764.1) 0.995

1686 (1236, 2136) 5745 (4912, 6577) 0.542 (0.1582, 0.9258) 10.93 (9.351, 12.51) 2018 (164, 3872) 402.2 (334.8, 469.7) 0.9962

537.7 (423.9, 651.6) 2009 (1816, 2202) 1.214 (0.7449, 1.683) 14.98 (14.15, 15.82) 4215 (-5.028e+04, 5.871e+04) 294.3 (229.1, 359.5) 0.9936

P=Accumulated biogas production observed; R = Biogas production rate obtained;

Fig. 14. Biogas production by removal of volatile solids (VS) from the evaluated treatments. Means followed by the same letter do not differ significantly by the Tukey' test at 5% probability level. Coefficient of variation = 20.06%; pvalue = 0.0000.

= Time of Lag phase. Phase 1=acid, Phase 2= methanogic.

Fig. 16. Methane production by VS removal from the studied treatments. Means followed by the same letter do not differ significantly by the Tukey' test at 5% probability level. Coefficient of variation = 26.52%; p-value = 0.0000.

when evaluating the degradation of a poly(caprolactone)-starch blend and a poly(butylene succinate) biodegradable polymer under anaerobic conditions, obtaining a methane production of 554 mL g−1. Kryvoruchko et al. (2009) found methane productions of 430 to 481 mL g−1 after 28 days of digestion of sugar beet byproducts, and 323 to 377 mL g−1 when evaluating the degradation of cassava processing byproducts after 38 days of digestion. The cumulative methane production is shown in Fig. 17. The cumulative production data fitted to the proposed regression model, showing an exponential trend for the parameters: solids removal, cumulative biogas production, and cumulative hydrogen production. The

Fig. 15. Degradation reactions in glucose in organic acids. Production of 1) acetic acid; 2) butyric acid; 3) and 4) propionic acid.

process is a precursor of propionic acid (Fig. 7). Although the propionic acid concentrations increased in the ensuing days, the balance of hydrogen production remained higher than its consumption, considering the stoichiometry of chemical reactions in the acetic acid formation. Hydrogen is also generated during the degradation of propionic acid into acetic acid, which contributes positively to the hydrogen balance in the reactor. 3.4. Methane production There are 2 primary groups of methanogenic microorganisms that produce methane in the digestion process. The acetotrophic group transforms the acetic acid produced in the acid phases into methane and carbon dioxide; and the hydrogenotrophic group produces methane from the conversion of hydrogen and carbon dioxide. In the digestion process, 65% to 95% of the methane is produced by acetic acid degradation (Yu et al., 2016; Andre et al., 2016). The experimental test of biochemical potential showed a methane production of 414 mL g−1. Similar results were found for the 0.04 treatment. The 0.08 treatment produced more biogas than that of the potential test, following the same proportions found and discussed for the biogas production (Fig. 16). Intermediary values for test cases were found by Cho et al. (2011)

Fig. 17. Cumulative methane production for the ratios (VSIn/VSPol) experimentally tested using an exponential regression model and an asymptotic function. Equation y = a - bcx, where a = 126.356, b = -5,927.170, and c = 8.809E−4. R² = 0.941; p-value = 0.01316. Bars correspond to the standard deviation (three replicates were used for each treatment). 9

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cumulative methane production increased as the CSP load was increased or the inoculum to substrate ratio was reduced. The methane production was low from the beginning of the process, in all treatments, exception for the 1.0 treatment. The methane contents found in the biogas of the 0.04, 0.08, 0.2, and 0.6 treatments were 16.86%, 13.92%, 22.41%, and 14.59%, respectively, after 24 h of digestion. This was probably due to the conversion of acetic acid to methane, because significant concentrations of acetic acid were already present at this stage of digestion (Fig. 6). The methanogenic microorganisms stabilized after 13 days of digestion process, when the methanogenic phase began, achieving the highest rates of methane production. The maximum methane concentrations in the 0.04, 0.08, 0.2, 0.6, and 1.0 treatments were 87.09%, 84.48%, 81.45%, 74.97%, and 65.83%, respectively.

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