Effect of acid pre-treatment on the anaerobic co-digestion of physicochemical sludge, poultry manure and sugarcane wastes (SCW) for biogas production

doi:10.21203/rs.3.rs-1735864/v1

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Effect of acid pre-treatment on the anaerobic co-digestion of physicochemical sludge, poultry manure and sugarcane wastes (SCW) for biogas production

https://doi.org/10.21203/rs.3.rs-1735864/v1

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In Mexico, productive activities related to poultry farming and the sugarcane industry generate large amounts of three byproducts: physicochemical residual sludge, poultry manure, and sugarcane wastes, which have high concentrations of organic matter and lignocellulosic material. Therefore, the objective of the present study was to evaluate the effect of acid pre-treatment, with different doses of acetic acid (2%, 3%, and 4% v.v^− 1) and exposure times of 30, 60, and 90 min on the solubilization of organic matter and biogas production in the anaerobic co-digestion of a mixture of agroindustrial wastes (residual sludge, chicken manure, and sugarcane wastes). The biodegradability and biogas production were evaluated by anaerobic digestion for 30 days. Finally, for 90 days semi-continuously, the increase in applied organic load of 1 kg VS m^− 3 d^− 1 was considered with Δ1 kg VS m^− 3 d^− 1 every 30 days until reaching 3 kg m^− 3 d^− 1. Acid pre-treatment with a dose of 4% acetic acid and an exposure time of 90 min improved the solubility and hydrolysis, considered the limiting stage of the anaerobic process, reducing the hydraulic retention time from 19 to 11 d, and increasing biogas yields (Y_bio=609 ± 11.7 to 1857.2 ± 7.5 L_bio gVS_rem⁻¹) and methane (Y_CH4 = 426.9 ± 8.19 to 1392.9 ± 5.65 L_CH4 gVS_rem⁻¹).

Acid pre-treatment

Agroindustrial wastes

Anaerobic co-digestion

Biogas

Solubilization degree.

Modern society uses a variety of energy sources to achieve its basic needs and improve the quality of life [1], a change that has increased the demand for natural, food, and energy resources; being the agroindustrial sector one of the most developed in recent decades and one of the largest generators of organic waste.

Activities related to the raising of fattening birds generate organic waste that is generated from broiler farms, which is a mixture of manure and bedding materials (wood shaving, rice hull, and straw) and has high Biochemical Methane Potential (BMP), which is comparable to sludge BMP potentials [2]. The composition of this mostly organic [3] is complex and depends on many factors such as bird feeding practices, different bird litter materials, brooding time, and others [4, 5]. Additionally, during the slaughter of birds, large volumes of water are used for washing and cleaning operations [6]. The discharge of untreated slaughterhouse wastewater (SWW) is a serious threat to public health and the environment [7, 8]. Wastewater and sludge generated in a slaughterhouse are organic by-products that are difficult to treat due to their high protein and lipid content [7]. The major organic streams that portrayed SWWs as recalcitrant include blood, dissolved particles and forms, feathers, viscera, soft tissues removed during clipping and cutting, bones, soil, and various cleaning and disinfecting compounds [9].

Alternatively, the sugarcane industry is one of the largest globally. It processes large volumes of biomass, where part of these residues was reused but with low value-added technologies. At the same time, a significant fraction that is not used continues to impact the environment [10]. The cane residues that are lost during cutting and harvesting (Sugarcane Wastes) vary according to the volume of biomass produced, crop management, and irrigation or temporary system [11]; it is estimated that for every 100 tons of total sugars reached from the field, 134 tons of agricultural residues are dispersed in the area, affecting the productivity of the sugarcane fields [5].

Additionally, the evolution of production systems must meet the current requirements of sustainable development and contribute to the transformation of current economies based on the use of fossil fuels to green economies based on the use of biomass, thus generating significant changes in the socio-economic, energy, technical and agricultural systems, by stimulating research and innovation, as well as the use of natural renewable resources [12, 13]; processes such as anaerobic digestion (AD) emerge as an alternative for treating and using these agroindustrial residues. Anaerobic digestion is a biological process that can be used to treat organic wastes. Digestion performance (biogas production, organic reduction, inhibition characteristics, and process stability) is highly dependent on feedstock characteristics [14, 15]; it has been found that AD becomes more stable when the variety of substrates applied at the same time increases, the most common situation is when a large amount of a bare primary substrate (for example, manure or sewage sludge) is mixed and digests together with small amounts of one or more additional substrates, this process is called Anaerobic Co-Digestion (ACD) [16].

However, the ACD of agroindustrial wastes is usually limited due their complex lignocellulosic composition that presents resistance to hydrolysis, biodegradability, and low biogas yield [17, 18]. For this reason, the use of treatments before AD has become a frequent practice [19], the variety of pre-treatment techniques can be classified as physical, chemical, biological, and the combination of these pre-treatments implemented at the laboratory or pilot level, with different levels of success [20–22]. Studies conducted by Nava [5] evaluated the effect of thermal pre-treatment at low temperatures (80°C, 85°C, and 90°C) on the anaerobic digestion of two different mixtures of physicochemical sludge, broiler excreta, and sugarcane wastes (SCW), using as main response variables the degree of solubilization of organic matter and methane yield, the results showed an increase in the degree of solubilization of biodegradable organic matter and a significant increase in the rate of degradation in anaerobic conditions applying a thermal pre-treatment (90°C and 90 min).

Acid pre-treatment (APT) is one of the most efficient for lignocellulosic biomass because it makes holocellulose more accessible to enzymatic or bacterial attacks and increase the global biodegradability of lignocellulosic materials [23]. It breaks down the bond between polysaccharides and lignin to make cellulose and hemicelluloses more accessible to bacteria [24]. Inorganic acid pre-treatment can be divided into dilute or concentrated acids. It generally involves using inorganic acids (sulfuric, hydrochloric, and nitric) or organic acids (formic, acetic, propionic). The use of focused acids can apply high temperatures (around 100°C) for several hours (2–10 h), with some negative drawbacks, such as risks of corrosion, increased toxicity, and the need to dispose of and recycle chemical products, in addition, diluted acids are used as catalysts for the degradation of cellulose and hemicellulose at high temperatures (from 100 to 240°C) but for a shorter time than concentrated acids. Another significant disadvantage of using concentrated acids is in the production of inhibitory by-products, such as sulfuric acid, furfural, and hydroxymethylfurfural (HMF), and the loss of sugars due to the excessive degradation of substrates [25, 26].

For all the above, the objective of this study was to evaluate the effect of acid pre-treatment with doses of acetic acid (2%, 3%, and 4% v.v^− 1) on the solubilization of organic matter and biogas production in the co- anaerobic digestion of a mixture of agroindustrial wastes (physicochemical sludge, poultry manure, and SCW).

This study was conducted at the Tecnológico Nacional de México (TecNM), campus Orizaba (in Orizaba, Veracruz, México). It is located at 1230 m altitude; the climate of this area is classified as a temperate and humid mountain, with an average temperature of 22º C. and annual rainfall of approximately 1,977 mm. At this location, experimental data were collected in four experimental stages. In the first part, sampling of agroindustrial waste (sludge, chicken manure, and SCW) was carried out to prepare the waste mixture and its subsequent characterization. In the second stage, the effect of the acid pre-treatment on the solubilization of organic matter present in the waste mixture was evaluated according to the increase in the Soluble Chemical Oxygen Demand (sCOD). In the third stage, the acid pre-treatment in the biogas production and anaerobic biodegradability of the mixture of agroindustrial wastes through Biochemical tests of Methane Potential (BMP) in anaerobic reactors with a batch operation was evaluated. Finally, the fourth stage consisted of the semi-continuous anaerobic digestion of the agroindustrial wastes mixture with an increase in the organic load during long-term experiments; The percentage of organic matter removal, biogas production, and methane yield were monitored.

Sampling and characterization of agroindustrial wastes

Every week, physicochemical sludge samples were extracted from a bird trail wastewater treatment plant located in Fortín de las Flores Veracruz, Mexico. In poultry processing, water is primarily used for scalding in the plucking process, washing birds before and after evisceration, cooling, cleaning, sanitizing equipment and facilities, and cooling mechanical equipment such as compressors and pumps. The coagulation-flocculation process was used to treat wastewater; the physicochemical sludge obtained contains poultry food residues, blood, manure (urine and feces), animal fats, proteins, and high levels of pathogenic bacteria [5]. Once the samples were obtained, they were stored in 10 L bottles and transported to the laboratory for conservation (4°C), conditioning, and the subsequent use and characterization [27].

The excreta of the broilers were obtained from the bedding of the birds of a confinement station located in the town of Soledad de Doblado, Veracruz, Mexico; the sampling was carried out weekly. Mixing always occurs between the excreta of the broilers and the material used as litter for the chickens (sawdust, rice or soybean husks, soil, corn cobs, etc.). On the other hand, SCW were collected from six sugarcane agricultural areas in Lerdo de Tejada, Veracruz, Mexico. Said residue is composed of green leaves (8%), sheath and leaves (20%), and clean stems (72%).

In the case of solid waste, these were transported to the laboratory in plastic bags for subsequent conditioning, which consisted of: a size reduction to pass pores of 1 mm, providing a large surface area to absorb the substrate and increase anaerobic microbial activity and, therefore, increase biogas production [28, 29], once conditioned the samples were stored at 4 ± 1°C.

Once the agroindustrial wastes were characterized in terms of Total Solids (TS) and Volatile Solids (VS), they were mixed in the following proportion (60% physicochemical sludge, 20% broiler excreta and 20% SCW) based on of volatile solids, the percentage composition of the waste mixture was based on the results obtained by Nava [5], who reported the effect of thermal pre-treatment on two mixtures of agroindustrial wastes (physicochemical sludge, poultry manure, and SCW) with compositions of 70% physicochemical sludge, 15% poultry manure, and 15% SCW and the second mixture with 60% physicochemical sludge, 20% poultry manure and 20% SCW, mixture with which the highest percentages of organic matter removal and higher biogas production were obtained, in anaerobic digestion.

The equivalent volume percentages of each residue were obtained through a mass balance of the volatile solids in each one. Mixing was carried out at 115 rpm to ensure correct inclusion and sound mixing. Said mixture was stored in hermetically sealed polyethylene containers and refrigerated at 4°C for conservation.

Pre-treatment conditions

The use of low concentrations of strong mineral acids and high concentrations of organic acids considered weak acids, such as acetic acid, were conventionally used as pre-treatment for the solubilization of lignocellulosic material [30–32]. However, the use of concentrated acids is a toxic, corrosive, energy-intensive, and expensive process; unique materials are required to construct the reactor [33].

On the other hand, studies carried out by Taherzadeh [34] recommend treating biomass with low lignin content in acetic acid with a concentration of 9% for better enzymatic hydrolysis and sugar recovery. Acetic acid (C₂H₄O₂) was used at doses of 2.3 and 4% v.v^− 1 and exposure times of 30, 60, and 90 minutes. The agroindustrial waste mixture was gently stirred at 150 rpm during the pre-treatment time. Subsequently, the samples were neutralized with NaOH (4%) until reaching a pH of 7. APT was conducted in hermetically sealed reactors with a temperature controller and magnetic mixer. The operating volume of the laboratory scale reactor was 0.5 L Later, the samples were stored at 4°C. Each experiment was performed three times for all suggested pre-treatments and then analyzed.

To evaluate the solubilization degree (SD) of the APT, it was necessary to know the solubilization factor typified by the relation of the soluble chemical oxygen demand (sCOD) before and after the APT. For this purpose, the soluble fractions of the raw and pre-treatment samples were obtained by centrifugation at 12,000 rpm for 15 min and filtration of the supernatant with a 0.2-µm membrane.

The degree of solubilization was calculated with Eq. 1.

SD=(sCODT–sCODR)/ (TCODR)*100 (1)

where SD is the solubilization degree (%); sCODT is the soluble COD with pre-treatment (mg·sCOD L^− 1); sCODR is the soluble COD without pre-treatment (mg·sCOD L^− 1); and TCODR is the total COD of raw sample (mg·TCOD L^− 1).

Biochemical Methane Potential (BMP) Tests

Anaerobic reactors with a capacity of 1 L and a working volume of 0.7 L were used, with two sampling ports dedicated to extracting digestate and biogas outlet. The anaerobic reactors were fed with 60% substrate (mixture of 60% physicochemical sludge, 20% chicken manure, and 20% SCW, pre-treatment and raw) and 40% inoculum. The substrate/inoculum ratio (S/I) was determined for Nava [5]; however, it was shown that this S/I ratio allowed an acceptable behavior in the BMP tests and an adequate load capacity of the process.

The inoculum was obtained from the excess biomass of municipal wastewater treated in a mesophilic anaerobic reactor in Ixtaczoquitlán, Veracruz, Mexico. The essential characteristics of the inoculum were pH = 6.89, 2.9% TS, and 75% VS.

The reactors were hermetically sealed and kept anaerobic with a temperature range of 30 ± 4°C and intermittent agitation of 150 rpm for 35 days [35]. During this period, the operating parameters monitored were biogas production (mL of biogas), biogas yield (mL of biogas per gVS_removed), and concentration of organic matter expressed as grams of VS. The biogas produced was collected, and its volume was measured based on water displacement [36]. All assays were performed in triplicate.

Semicontinuous anaerobic digestion tests

After batch testing, recommended operating conditions in the semi-continuous flow of the acid pre-treatment followed by mesophilic anaerobic co-digestion were defined by selecting the best methane yield obtained in the BMP. Two identical anaerobic reactors were used and operated simultaneously for the mixture of agroindustrial residues with acid pre-treatment and a mixture of raw agroindustrial residues, with a capacity of 1 L and a working volume of 0.7 mL They were mixed at 150 rpm and operated at 30 ± 4°C. Two sampling ports were placed, dedicated to extracting digestate and biogas outlet. The reactor was fed semi-continuously every 24 h with an organic load (OLR) of 1 Kg VS. m^− 3. d^− 1. The increase in OLR was carried out every 30 days (1.2 and 3 Kg VS. m^− 3. d^− 1) [23, 37].

The OLR calculation was made in terms of TS concentration, not of flow rate, which was calculated with Eq. 2 proposed by López-Escobar [38]:

Q = A X (V/C) (2)

Were Q (L·d^− 1) is the flow rate, A is the OLR (g VS·L^− 1·d^− 1), V (L) is the reactor useful volume, and C (g VS·L^− 1) is the feed concentration.

Analyzed parameters were TS, VS, pH, biogas, and its methane content; from it, the substrate removal efficiency (% VSremoved) (Eq. 3), the substrate removed (VSremoved) (Eq. 4), and biogas yield (Ybiogas) (Eq. 5) were calculated [23, 38].

%VSremoved= (g substrate removed/g substrate fed) X100 (3)

VSremoved = substrate fed − g product (4)

Ybiogas = mL biogas / g substrate removed (5)

Analytical parameters

The physical and chemical characterization of the mixture of agroindustrial residues was carried out using the following parameters.

Total and volatile solids were determined by heating samples at 105°C for 24 h and 550° C for 2 h, respectively. These components were determined according to the Standard Methods [39]. The pH was determined according to the potentiometric method using a pH meter (Thermo Scientific TM Orion Star Model A1110).

The soluble fraction was obtained after centrifugation at 13,000 rpm for 15 min and subsequent filtration with a 0.45-mm nylon membrane filter. The total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (TCOD) were analyzed according to the closed reflux, colorimetric method (5220D) of the Standard Methods [39], using a spectrophotometer (Thermo Scientific ™, Genesys 20). Total Kjeldahl nitrogen (TKN) was obtained by digestion and oxidation. Carbohydrates were analyzed according to the Antrone method [39]. Besides, lipids were analyzed by Soxhlet extraction [39] (Table 1).

Table 1

Physicochemical parameters and methods used
Physic-chemical parameter	Method
pH	Conductometric [39]
Total solids	Gravimetric (Standard Methods, 2017) [39]
Volatile solids	Gravimetric (Standard Methods, 2017) [39]
Chemical oxygen demand for total and soluble	Colorimetric (5220D, Standard Methods, 2017) [39]
Total Kjeldahl nitrogen	Digestion and oxidation (TKN) [39]
Carbohydrates	Antrone method [40]
Total lipid content	Soxhlet [39]

During the anaerobic digestion tests, the cumulative gas production was measured using the water replacement method. This is one of the most frequently used methods at a laboratory scale to quantify a low quantity of biogas because of its low experimental uncertainty [23, 41]. The volume of biogas is measured by the weight of the water replaced. The composition of the gas produced in each test (O₂, CH₄, CO₂, and N₂) was determined as the mean value of the gas in each gasholder employing an off-line gas chromatographic analysis device using an Ultra-Trace gas chromatograph Thermo Scientific equipped with a Heliflex AT-Mol Sieve column (30 m × 0.53 mm diameter), a Heliflex AT-Q (30 m ×0.53mmdiameter length) and a thermal conductivity detector. In the first column, O₂, N₂, CH₄, and CO₂ were analyzed, while in the second column CH₄, and CO₂. The gas used as a carrier was helium.

Statistical analysis

A completely randomized design was used to evaluate acid pre-treatments and the biodegradability parameters. There were three replicates for each treatment. All data were analyzed by one-way analysis of variance (ANOVA), and Tukey’s mean comparison test was carried out at p ≤ 0.05. Likewise, the standard deviation was determined to evaluate some physical and chemical parameters.

Physical and chemical characterization of agroindustrial wastes

Table 2 summarizes the initial characterization in terms of physical and chemical parameters of the agroindustrial residues obtained after sampling and conditioning of the samples.

The samples were conditioned at a concentration of TS between 2.5-4% to not clog the ducts in anaerobic reactors, which is recommended in specialized literature [42]. The organic material content is represented in the volatile solids since, during the incineration of VS, the residual products represent an inorganic or mineral phase that cannot volatilize; therefore, a high biodegradability can be inferred [43].

On the other hand, the pH in both the agroindustrial residues and the waste mixture is slightly acidic (5.9–6.56); however, Charalambous [44] indicate that the anaerobic biomass can acclimatize to a moderately low pH (5–6).

The concentration of total and soluble COD (5630.315 ± 145 and 4160 ± 71 g L^− 1 respectively) present in agroindustrial wastes is attributed to their organic nature. Each type of biomass contains various organic and inorganic elements that considerably affect the digestion process [45]. However, different authors have reported the presence of cellulose and lignin in agroindustrial residues [46–48], which can hardly be degraded by hydrolytic microorganisms [49].

The increase in biogas production depends directly on the substrates content [50], with carbohydrates, proteins, and lipids constituting a large part of the biomass destined for biogas production systems [51]. In this sense, the concentration of proteins in the mixture of agroindustrial residues reported was 19.57 ± 0.42%. According to Braun [52], they are not considered an ideal substrate for biogas generation due to the high production of toxic compounds and inhibitory effects. Even so, the availability of nitrogen (3.12 ± 0.27% N-total in the wastes mixture) under balanced conditions is fundamental for the generation of cells, producing equivalent moles of ammoniacal nitrogen, in addition to the alkalinity of bicarbonate [53].

Carbohydrates consisting of easily fermentable sugars present degradation and a high potential for biogas production in short periods; on the contrary, residues composed of lignocellulosic carbohydrates show slow degradability [45]. In this sense, the SCW (4761 ± 245 mg. L^− 1) presented the highest carbohydrate content in the three residues used, a residue considered to have lignocellulosic content. In general, carbohydrate-rich substrates tend to have low C/N ratios, impair cell formation, and reduce biogas production rates [54], so co-digestion processes are considered attractive to balance the properties of these substrates [50].

About the lipids concentration, the physicochemical residual sludge presented a higher concentration (4312 ± 015 mg. L^− 1) than poultry manure and SCW; However, lipids are among the compounds with the most significant potential for biogas production; they present a slow degradation in the digesters, often requiring previous treatments to accelerate the process [55, 56].

Table 2

Physical and chemical characterization of agroindustrial wastes
Parameters/ Waste	Physicochemical Sludge (PS)*	Excreta of Broiler Chicken (EBC)*	Sugar Cane Waste (SCW)*	Mixture (60% PS, 20% EBC, 20% SCW) *
Total solids (% m·v^− 1)	3.5 ± 0.5	3.98 ± 0.98	2.84 ± 0.43	3.44 ± 0.31
Volatile solids (% m·v^− 1)	85.42 ± 0.63	74.98 ± 0.46	87.37 ± 0.61	82.59 ± 0.83
pH	6.5 ± 0.36	6.56 ± 0.41	5.90 ± 0.46	6.32 ± 0.21
COD Total (mg. L^− 1)	8654 ± 114	3122 ± 098	3923 ± 102	5630.315 ± 145
COD Soluble (mg. L^− 1)	1324 ± 96	1852 ± 030	723 ± 26	4160 ± 71
Carbohydrates (mg. L^− 1)	2368 ± 422	3500 ± 125	4761 ± 245	862 ± 059
Lipids (mg. L^− 1)	4312 ± 015	3045 ± 090	1061 ± 043	2500 ± 082
N-Total (%)	3.82 ± 0.37	4 ± 0.40	1.19 ± 0.61	3.12 ± 0.27
Proteins (%)	20.02 ± 1.89	23.14 ± 4	6.98 ± 2.13	19.57 ± 0.42
*Data are given as mean ± SD, n = 5

Effect of acid pre-treatment on the solubilization of organic matter in the mixture of agroindustrial wastes

Figure 1 shows the percentages of the degree of solubilization (% SD) obtained consecutively from acid pre-treatments. In general, the rate of solubilization increased with the increase in the application of the dose of acetic acid and exposure time, showing a linear behavior. Values of 8%, 11.8%, and 15.3% SD were obtained by applying a dose of 4% and exposure times of 30, 60, and 90 min, respectively, results that obey the equation %SD = 0.1652 T + 1.3398 where T is the exposure time (R² = 0.9534) (Fig. 1c). Likewise, when applying a dose of 3% acetic acid, the results could be described by the equation %SD = 0.143 T + 1.2733 where T and R² = 0.9445 (Fig. 1b), the reported average values of %SD were 7.2%, 10.3%, and 13.3 % for times o 30, 60 and 90 min respectively. Regarding the 2% dose, the average values of %SD for the 30, 60, and 90 exposure times reported were 4%, 6.7%, and 9.4%, respectively, a linear behavior that is described by the equation %SD = 0.1032 T + 0.3904 where T and R² = 0.9894 (Fig. 1c).

The statistical analysis revealed that the acid pre-treatment, the exposure time, and the interaction between both factors had a significant effect (p ≤ 0.05) on the percentage of solubilization of organic matter present in the mixture of agroindustrial residues.

Furthermore, a higher dose of acetic acid and a longer exposure time will increase the percentage of the degree of solubilization, an effect that can be attributed to the fact that acetic acid breaks the bonds and transforms non-biodegradable materials into biodegradable compounds that can be used as a substrate in anaerobic digestion, emphasizing the effect on substrates with lignocellulosic composition [57], extracellular polymeric substances may also become more bioavailable [58], contributing to the conversion of slowly biodegradable particulate organic materials into readily biodegradable low molecular weight compounds [59].

The influence of the pre-treatment on the biodegradability of the raw material varies according to the pre-treatment and the type of substrate [60], Devlin [61] indicate that an increase in soluble COD suggests that the acid pre-treatment contributes to the decomposition of the polymers present in the substrates into monomers or oligomers by breaking the bonds and that it can be observed in the soluble COD values obtained in this study (Table 3).

Table 3

Summary of values soluble COD and percent solubilization degree APT
Acid pre-treatment	%SD*	sCOD mg sCOD.L^− 1 *
4% 90 min	15.3 ± 0.75	5020 ± 42.4
4% 60 min	11.8 ± 0.25	4825 ± 14.1
4% 30 min	8 ± 0.56	4612 ± 31.8
3% 90 min	13.3 ± .19	4907 ± 10.6
3% 60 min	10.3 ± 0.31	4724 ± 17.7
3% 30 min	7.2 ± 0.19	4567 ± 10
2% 90 min	9.4 ± 0.31	4692 ± 17.7
2% 60 min	6.7 ± 0.17	4535 ± 86.1
2% 30 min	4 ± 0.31	4387 ± 17.7
Raw agroindustrial wastes	0	4160 ± 23.1
*Data are given as mean ± SD, n = 3

Biochemical Methane Potential (BMP) Tests

Anaerobic biodegradability tests were carried out with the mixture of pretreated and raw agroindustrial residues in batch operation to evaluate the effect of acid pre-treatment on VS removal, biogas production, and biogas and methane yield.

The experimental results of the accumulated production of biogas during the batch tests are shown in Fig. 2, in which it is observed that the reactors fed with the raw agroindustrial waste mixture were the ones that generated the lowest volume of biogas (744 ± 5.6 mL). At the same time, the pre-treatment with a dose of 4% acetic acid and a contact time of 90 min was the one that generated a greater volume of accumulated biogas (2990 ± 14.4 mL). The statistical analysis revealed that applying the acid pre-treatment, the exposure time, and the interaction between both factors had a significant effect (p ≤ 0.05) on the accumulated biogas production.

Based on the above, it is verified that applying acidic pre-treatments in the mixture of agroindustrial residues (physicochemical sludge, poultry manure, and SCW) favors the accumulated biogas production is attributed to the fact that the pre-treatment improves the degradation long-chain and/or lignocellulosic compounds present in agroindustrial wastes, this fraction being decreasing and consequently increasing the soluble contents available for the microorganisms in the reactors to biogas produce [62]; in addition, previous studies have reported that the use of co-digestion processes favors the biogas production. The effect of pretreatment and anaerobic co-digestion of food waste (FW) and waste activated sludge (WAS) was assessed by the reductions of total suspended solids (TSS), volatile suspended solids (VSS), COD removal and methane production. Thermal treatment and anaerobic digestion reduced VSS up to 43.4% (FW) and 43.1% (FW þ WAS) by increasing the solubilization of the feedstock. Methane yields of ultrasonic treatment reached 206.4 (FW) and 326.3 (FW + WAS) mL CH₄ g^− 1 VSS removed which were 50.5 and 56.2% higher than that of the control. Results showed that each pre-treatment gave distinctive effect on different feedstocks due to dissimilar composition. Co-digestion conferred superior result than mono digestion with FW or WAS [63].

Gosh [64] evaluated the potential of co-digestion for utilizing the Organic fraction of Municipal Solid Waste (OFMSW) and sewage sludge (SS) for enhanced biogas production. Metagenomic analysis was performed to identify the dominant bacteria, archaea, and fungi, changes in their communities with time, and their functional roles during anaerobic digestion (AD). The cumulative biogas yield of 586.2 mL biogas g VS-1 with the highest methane concentration of 69.5% was observed under an optimum ratio of OFMSW: SS (40:60 w.w^− 1).

The effect of mechanical, chemical, thermal, and hybrid pre-treatment on anaerobic digestion of fruit-juice industrial waste (FW) co-digested with municipal sewage sludge (MSS). The pre-treatment of the substrates with ultrasonication, microwave, weak alkali-acid caused an increase in cumulative biogas production of approximately 20.9, 14.9, 8.1, and 5.2%, respectively. Beside this, thermal and strong acid-alkali pre-treatment reduced biogas production. The highest cumulative biogas and methane yield was increased with hybrid pre- treatment which contains ultrasonication (US) and alkali (AL) pretreatment by 36% and 49%, respectively. Also, compared to untreated mixture, the soluble COD, carbohydrate, and protein removal efficiencies were increased from 42.6–65.6%, 65.1–86.6%, and 17.3–62.4%, respectively for US-AL pretreatment [65].

For all the above, pre-treatments in the anaerobic co-digestion process are attractive for generating energy from substrates such as sewage sludge, organic waste, and agro-industrial wastes.

The main components of biogas are methane and carbon dioxide, nitrogen, hydrogen, oxygen, and hydrogen sulfur, water, and saturated hydrocarbons such as propane and ethane in small quantities, with methane being the gas present in the mixture of most significant interest due to its caloric potential [66]. Therefore, the importance of determining biogas yields and methane, the BMP tests results are shown in Table 4. According to the statistical analysis, there was a significant effect on the yield of biogas and methane (p ≤ 0.05) when applying acid pre-treatments and increasing the time of exposure, noting the most significant impact with the pre-treatment with a dose of 4% acetic acid and a contact time of 90 min. In anaerobic digestion processes, incorporating a pre-treatment enhances the removal of organic material and reduces the lag phase of methanogenesis, leading to increased CH₄ production [66].

AD has been considered one of the appropriate processes for the valuation of waste based on the syntrophic mechanisms of microbial communities [67]. The biogas production through AD from organic waste such as primary sludge, waste activated sludge [68], sewage sludge with mixed cafeteria food waste [67], waste from food alone [69], organic fraction of municipal solid waste [50], bird litter [4], among others, have been investigated. Favorable results have been obtained for co-digesting said substrate and/or applying some pre-treatment. The biogas produced from AD can be converted into biomethane (CH₄) and used in heat/power generation [70], and the digestate residue can be used as fertilizer [71–73].

Table 4

Results anaerobic digestion batch APT
Acid pre-treatment	Y biogas * L bio g VS_rem⁻¹	Y CH₄ * L CH₄ g VS_rem⁻¹	Accumulative biogas * mL	VS removed* (g)	VS reduction efficiency* (%)	Hidraulic Retention Time (HRT)* (d)
4% 90 min	1857.2 ± 7.5	1392.9 ± 5.65	2990 ± 14.4	1.61 ± 0.01	57.50	11
4% 60 min	1842.6 ± 25.9	1382 ± 19.41	2755 ± 77.78	1.50 ± 0.02	53.39	11
4% 30 min	1670.8 ± 44.3	1253.1 ± 33.25	2364 ± 50.91	1.42 ± 0.01	50.54	13
3% 90 min	1691.7 ± 73.5	1218 ± 52.91	2697.5 ± 81.32	1.60 ± 0.02	59.96	11
3% 60 min	1679.2 ± 39.9	1209.1 ± 28.75	2485 ± 35.36	1.48 ± 0.01	52.86	13
3% 30 min	1531 ± 10.3	1102.3 ± 14.04	2197 ± 11.31	1.44 ± 0.03	51.25	15
2% 90 min	1236.3 ± 14.8	877.8 ± 10.52	1910 ± 14.14	1.55 ± 0.01	55.18	11
2% 60 min	1099.7 ± 24.09	780.8 ± 17.10	1611 ± 19.80	1.47 ± 0.05	52.32	13
2% 30 min	675.5 ± 4.3	479.6 ± 3.07	939 ± 15.56	1.38 ± 0.04	47.86	17
Raw agroindustrial wastes	609.9 ± 11.7	426.9 ± 8.19	744 ± 5.66	1.22 ± 0.01	43.57	19
*Data are given as mean ± SD, n = 3

The reduction of VS (Fig. 3) is another factor determining the efficiency of pre-treatment methods for biogas production from co-digestion processes [74]. In this study, VS removal and HRT were selected, which was obtained after meeting the 38% VS removal criteria for US EPA [72] vector attraction reduction requirements.

The removal efficiency of VS from the mixture of crude agroindustrial residues was 43.57% with an HRT of 19 days, which was exceeded with the application of acid pre-treatments in the proposed exposure times, a fact that is verified through the analysis statistical, which reflected the significant effect (p ≤ 0.05) of the acid pre-treatment in the removal of VS. Importantly, acid pre-treatment decreased HRT from 2d (Pre-treatment 2% 30 min) to 8d (in most pre-treatments). Generally, a HRT (approx. 15–20 days) is needed to reach an acceptable level of VS destruction and biogas production [73]. Likewise, the enhanced killing of VS in a shorter HRT is likely due to the acidic pre-treatment killing cells and extracellular polymeric substances releasing intracellular and extracellular constituents [74].

For all the above and based on the results obtained, it was decided to carry out the semi-continuous operation of the reactor fed with the mixture of agroindustrial waste (sludge, poultry manure, and SCW) pre-treated with a dose of 4% acetic acid and an exposure time of 90 min, in the same way, a comparison was made with a control test.

Semicontinuous anaerobic digestion tests

Figure 3 shows the behavior in biogas production before applying the acid pre-treatment (dose of acetic acid 4% 90 min) and the control test during the semi-continuous anaerobic digestion using different OLR.

The daily production of biogas increased as the OLR increased, for the reactor fed with the mixture of raw agroindustrial residues, the average biogas production was 403.8 ± 148, 909.6 ± 224.4 and 1675.4 ± 265.3 mL d^− 1 for OLR of 1 .2 and 3 Kg VS m^− 3. d^− 1 respectively, maintaining an average daily production of 995.6 ± 212.56 mL d^− 1 during the 90 days of operation, however, biogas production was exceeded by the reactor fed with the mixture of agroindustrial wastes pre-treated with doses of acid. 4% acetic acid of 90 min exposure time, obtaining average biogas productions of 610 ± 191.1, 1181 ± 323.6 and 2341 ± 413.5 mL d^− 1 for OLR of 1,2 and 3 Kg VS m^− 3. d^− 1 respectively, the average production during the 90 days of operation was 1450 ± 780 mL d^− 1, exceeding by 45.6% the average production of the reactor fed with the mixture of raw agroindustrial residues, likewise, the statistical analysis revealed that there is a significant effect (p ≤ 0.05) when applying an acid pre-treatment on biogas production.

On the other hand, the biogas yield (Table 5) reported was 0.70 ± 0.12, 0.74 ± 0.11, and 0.85 ± 0.07 L_biogas gVS_rem⁻¹ for OLR of 1,2 and 3 Kg VS m^− 3. d^− 1 in the reactor fed with a mixture of raw agroindustrial waste, while the reactor used for the digestion of the mixture of agroindustrial waste pretreated with a dose of 4% acetic acid and an exposure time of 90 min obtained biogas yields of 0.88 ± 0.21, 0.92 ± 0.15 and 0.99 ± 0.09 L_biogas gVS_rem⁻¹ for OLR of 1,2 and 3 Kg VS m^− 3. d^− 1, being more significant than those of the crude mixture. In general, these positive results can be attributed to the chemical properties of the components of the raw material and to the concentrations of nutrients present in the mixture of agroindustrial wastes, which is advantageous to provide a combined food for the activation and adequate function in the AD of methanogenic microorganisms [48, 75–76].

Similarly, the acid pre-treatment resulted in a higher conversion of organic components to methane. The methane yield in the two digesters (fed with the raw mixture and pretreated) was statistically significant (p ≤ 0.05), observing an effect on the increase in methane yield as the organic load in both reactors increased. In the first period of operation with OLR of 1 Kg VS m^− 3. d^− 1, the effect of the acid pre-treatment became visible by reporting 26.5% more methane yield than the control test, while for the periods of operation where OLR of 2 and 3 Kg VS m^− 3 d^− 1were applied, the effect of the pre-treatment in combination with the increase in OLR was reflected in 38.4% and 23.3%, respectively, higher than the control test.

Thermal, chemical, enzymatic, and combined pre-treatments have been reported to improve the methane production of lignocellulosic materials [46, 79–80]. Biomass pre-treatments are studied mainly to increase the concentration of compounds with higher biodegradability [81]. However, while the pre-treatments could improve the accessibility of organic matter and its conversion to methane compared to non-pretreated substrates [82], the excessive solubilization of organic matter could negatively affect the methane yields [83].

Table 5

Accumulated biogas, biogas, and methane yield, in the semicontinuous AD
OLR (Kg VS m^− 3.d^− 1)	Parameter	Raw agroindustrial wastes	ATP (4% 90 min)
1	Daily biogas production* (mL d^− 1)	403.8 ± 148	610 ± 191.1
	Biogas yield* (L_biogas gVS_rem⁻¹)	0.70 ± 0.12	0.88 ± 0.21
	Methane yield* (L_CH4 gVS_rem⁻¹)	0.49 ± 0.14	0.62 ± 0.07
2	Daily biogas production* (mL d^− 1)	909.6 ± 224.4	1181 ± 323.6
	Biogas yield* (L_biogas gVS_rem⁻¹)	0.74 ± 0.11	0.92 ± 0.15
	Methane yield* (L_CH4 gVS_rem⁻¹)	0.52 ± 0.09	0.72 ± 0.11
3	Daily biogas production* (mL d^− 1)	1675.4 ± 265.3	2341 ± 413.5
	Biogas yield* (L_biogas gVS_rem⁻¹)	0.85 ± 0.07	0.99 ± 0.09
	Methane yield* (L_CH4 gVS_rem⁻¹)	0.60 ± 0.14	0.74 ± 0.08
*Data are given as mean ± SD, n = 3

Organic matter reduction is an important parameter used to assess the efficiency of anaerobic digesters. This study evaluated the percentage of SV removal efficiency in the acid pre-treatment and the control test for three organic load indices. The VS fluctuations observed during the 90 days of AD are shown in Fig. 4.

In this study, the SV removal efficiency percentage was 30.7 ± 9.1, 35.2 ± 4.7, and 36.5 ± 5.4% for OLR of 1, 2 and 3 Kg SV m^− 3. d^− 1 respectively for the control test, while, for the digester fed with a mixture of pretreated agroindustrial residues, the % removal efficiency of VS reported for OLR of 1, 2 and 3 Kg VS m^− 3. d^− 1 were 33.4 ± 8.8, 38.3 ± 4.9, and 40.5 ± 5.3%, respectively. The most significant elimination of VS was observed when operating with an OLR of 3 Kg VS m^− 3. d^− 1 and with the mixture of agroindustrial residues pretreated with acetic acid.

The application of the acid pre-treatment allowed HRT reduction due to the release of easily biodegradable organic components under anaerobic conditions [23]. This parameter is one of the most critical factors in the AD process; Conventional anaerobic digestion decreased HRT as OLR increased. This behavior was observed in the same way in the reactors with pre-treatment. However, when operating with the maximum OLR, a decrease of 5 days in the HRT was achieved when operating the semi-continuous anaerobic process with a pre-treated agroindustrial waste mixture, compared to the control test, reached an HRT of 19 days (Table 6). The decrease in HRT was of great importance because prolonged RHT can cause the death of microorganisms due to the lack of nutrients; in contrast, short HRTs can result in cell intoxication or low methane yield. VFA accumulation can also result from short HRT [42]. However, the pH values (Table 6) were maintained during the 90 days of operation of the anaerobic reactors, ranging between 6.98–7.78 and maintaining the necessary conditions for the operation of the anaerobic system.

Table 6

SV removal, HRT, and pH in the semicontinuous AD
OLR (Kg VS m^− 3 d^− 1)	Raw agroindustrial wastes			ATP (4%,90 min)
OLR (Kg VS m^− 3 d^− 1)	SV removal efficiency (%)	HRT	pH	SV removal efficiency (%)	HRT	pH
1	30.7 ± 9.1	20	6.98	33.4 ± 8.8	16	7.07
2	35.2 ± 4.7	19	7.03	38.3 ± 4.9	15	7.78
3	36.5 ± 5.4	19	7.25	40.5 ± 5.3	15	7.67

*Data are given as mean ± SD, n = 3

Biogas production can be improved by integrating acid pre-treatment into anaerobic co-digestion of physicochemical sludge, poultry manure, and SCW. Acid pre-treatment promoted COD solubilization for higher methane recovery in AD. Likewise, it favored the removal of VS and allowed an increase in the organic load fed to the anaerobic reactor under semi-continuous operating conditions. In addition to this, reductions in HRT were obtained, making the AD process fast and efficient. Anaerobic digestion improvement strategy allows energy to be obtained from a more significant amount of poorly biodegradable waste without increasing reactor volumes and providing added value to lignocellulosic waste that is seldom helpful for sustainable purposes and contributes to CO₂ emissions when burned at generation sites.

The synergy between the three substrates allows a more outstanding balance in the anaerobic process. It takes advantage of the advantages in terms of each waste physical and chemical composition, allowing to propose a viable option for their use and treatment. The acetic acid used as a preliminary treatment helps to inactivate competitive microorganisms for the intermediate substrates that lead to biogas production and, due to its biodegradable organic nature, also contributes to the production of methane.

ACKNOWLEDGMENTS

The authors acknowledge support from Tecnológico Nacional de México Campus Orizaba.

FUNDING

Not applicable.

AUTHOR INFORMATION

Affiliations

Tecnológico Nacional de México, Instituto Tecnológico de Orizaba Oriente 9, Colonia Emiliano Zapata, 94320 Orizaba, Veracruz, México.

Corresponding author: Juan Manuel Méndez Contreras

ORCID 0000-0002-9745-4628

Alejandro Alvarado Lassman

ORCID 0000-0001-9818-4300

Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C., Av. Normalistas 800 Colinas de La Normal 44270 Guadalajara, Jalisco, México.

Noemi Nava Valente

ORCID 0000-0002-1598-5821

Universidad Veracruzana, Facultad de Ciencias Químicas. Prolongación de Oriente 6, 1009. Colonia Rafael Alvarado, 94340, Orizaba, Veracruz, México.

Eduardo Aguilar Hernández

ORCID

0000-0003-2009-4051

Author contributions

All authors contributed to the study conception and design, material preparation, data collection and analysis, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

ETHICS DECLARATIONS

Conflicts of Interest

The authors declare no conflict of interest.

Competing interests

The authors declare that they have no competing interests.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

All authors read the manuscript and expressed their consent for publication.

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Effect of acid pre-treatment on the anaerobic co-digestion of physicochemical sludge, poultry manure and sugarcane wastes (SCW) for biogas production

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Figures

Introduction

Methodology

Sampling and characterization of agroindustrial wastes

Pre-treatment conditions

Biochemical Methane Potential (BMP) Tests

Semicontinuous anaerobic digestion tests

Analytical parameters

Statistical analysis

Results And Discussion

Physical and chemical characterization of agroindustrial wastes

Biochemical Methane Potential (BMP) Tests

Semicontinuous anaerobic digestion tests

Conclusions

Declarations

References

Status:

Version 1