1 Introduction

According to estimates, the EU produces a staggering 118 to 138 million tons of bio-waste every year, yet only 40% of this waste is sustainably recycled into valuable products like compost and biogas. Shockingly, 27% of the bio-waste is incinerated, while 24% ends up in landfills [1]. Both of these practices are incredibly harmful to the environment, leading to groundwater contamination, the release of greenhouse gases, and a high risk of spontaneous fires [2,3,4]. Given the significant negative impact of these practices, there is a growing demand for new processes that can support the circular economy and divert unsustainable practices. It is crucial to recognize the potential of OFMSW as a resource that can be harnessed to create high-value products, thereby promoting sustainability and reducing harm to the environment.

In comparison to other waste biomasses as traditional food waste and starch-rich wastes, OFMSW is more abundant and, to a large extent, not regionally limited, but as a consequence, complexity and heterogeneity are increased, making it challenging to standardize for bioprocessing on a large scale. Additionally, OFMSW has a relatively high content of lignocellulose which makes it more recalcitrant towards biological conversions and subsequently more expensive to process as high-cost enzymes are required [5]. In contrast, conventional food waste and starch wastes can be directly processed by producing microorganisms, making them more cost-effective [6, 7]. For that reason, minimizing the necessary enzyme quantity for OFMSW conversion to the lowest feasible level would notably enhance the economic viability of the whole process.

Nevertheless, OFMSW could be excellent second-generation feedstock due to its high content of sugars, which ranges from 30 to 69%, including starch, cellulose, and hemicellulose, followed by fats (10–40%) and protein (5–20%) [8, 9]. These compounds have the potential to be converted into green energy, such as biofuels and biogas, as well as bulk biochemicals, which would contribute to the green transition. One promising approach for obtaining biofuels is to ferment OFMSW into high-energy-dense molecules, such as alcohols, methane, and lipids. However, a significant challenge in this process is optimizing multiple technological applications. This typically involves using mechanical, physical, and biological processes to extract sugars, which can then be further converted into biofuels and/or bulk chemicals by fermentation [10] (Fig. 1).

Fig. 1
figure 1

Sustainable biowaste processing: a proposed alternative to harmful practices such as landfilling and incineration

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The main effect of thermal pretreatment is an enhancement of solubilization of organics, such as sugars and proteins, and the disruption of cell wall integrity [11]. Furthermore, the characterization studies conducted on OFMSW have shown that the starch content is relatively high, ranging from 10 to 23% of dry matter [12, 13]. Therefore, starch gelatinization, a type of heat pretreatment, could be necessary to make starch swelled and consequently accessible for enzymatic and/or microbial digestion. For example, Parchami et al. [14] were able to recover 83% of the starch in the brewer’s spent grain by subjecting it to 140°C for 4 h. Thermal pretreatment can also serve as a sterilization step. In fact, Chen et al. [15] found that treating food waste at 90°C for 30 min resulted in complete inactivation of Staphylococcus aureus and total coliforms, as well as almost complete inactivation of molds and yeasts. However, it should be noted that excessive thermal pretreatment at 100°C and higher degrade sugars into fermentation inhibitors, like furan derivatives and organic acids, and increases substrate recalcitrance, impeding microbial digestion [14, 16]. Thus, it is important to balance its benefits with potential drawbacks for optimal results.

Although commonly used chemical pretreatments may result in high production yields from OFMSW, generally, they pose many disadvantages, such as high volumes of reagents and subsequent high cost of process on an industrial scale [17], the potential safety and environmental issues, the requirement for neutralization after pretreatment [18], change of C/N ratio if base reagents rich in nitrogen are applied [19, 20], production of growth inhibitors during pretreatment such furfural and HMF [21, 22], and degradation of sugars [23]. Hence, biological pretreatments emerge as the preferred choice for OFMSW preprocessing over chemical alternatives. However, certain methods, such as enzymatic hydrolysis, might lead to a considerable reduction in cost-effectiveness during production.

Partial anaerobic digestion can be a promising pre-hydrolysis step for sugar recovery. During the initial hydrolysis phase of anaerobic digestion, complex biopolymers break down into soluble sugars, amino acids, and long-chain fatty acids through the action of microorganisms in the absence of oxygen. This is followed by the production of short-chain fatty acids by acidogenic microorganisms [24]. Optimizing the depolymerization rate while keeping acetogenesis at an early stage can lead to an effective biological pretreatment process that results in sugar recovery. According to Soomro et al. [25], the hydrolysis and acidogenic stages of OFMSW exhibit kinetics that begin at the start of incubation. However, the hydrolysis stage has higher rates than the acidogenic stage, allowing for partial preservation of hydrolyzed free sugars.

This study aims to explore the potential of psychrophilic anaerobic digestion as a novel form of biological pretreatment of OFMSW for sugar recovery.

Enzymatic hydrolysis is the most commonly used and extensively researched biological pretreatment method for OFMSW. However, due to the complexity, inconsistency, and heterogeneous nature of OFMSW, the enzymatic hydrolysis process requires the use of industrial hydrolytic enzyme cocktails composed of amylases, cellulases, hemicellulases, lipases, proteases, and other enzymes to release most of the fermentable sugars [23]. The majority of these sugars are stored in the form of starch, cellulose, and hemicellulose. Cellulases and hemicellulases are needed in the enzymatic cocktail for the efficient breakdown of these biopolymers, leading to the liquefaction of the feedstock and increased substrate homogeneity. This facilitates the saccharification of cellulose and hemicellulose, converting them into fermentable sugars. Additionally, amylolytic enzymes are essential for the saccharification of starch into glucose, while other enzymes, such as pectinases and proteases, can provide extra nutrients for targeted cell factories.

Overall, the use of industrial enzymatic cocktails has shown promising results for sugar recovery from OFMSW and subsequent fermentation yields. For instance, Izaguirre et al. [26] recovered 56% of sugars from OFMSW using a combination of Pentopan 500 BG, Celluclast BG, and Glucoamylase NS 22035 (Novozymes A/S). Molina-Peñate et al. [27] observed a 51% increase in reducing sugars with Viscozyme L® (Novozymes A/S) cocktail containing arabanase, cellulase, β-glucanase, hemicellulase, and xylanase. Li et al. [28] achieved 53% cellulose conversion using Trichoderma reesei cellulase (Sigma-Aldrich), while Haske-Cornelius et al. [29] attained 25% hydrolysis yield with StargenTM 002 (DuPont) amylolytic cocktail. Nwobi et al. [30] achieved 60% saccharification yield via cellulase complex, amylase, hemicellulase, pectate lyase, lipase, and protease (Novozymes A/S), reaching 140 g of glucose/L.

Furthermore, previously conducted studies have shown that heat pretreatment optimization can significantly increase saccharification yield in OFMSW. For instance, Ebrahimian et al. [31] found that treatment at 120°C for 60 min followed by enzymatic hydrolysis using Cellic® CTec2 (Novozymes A/S) and amylases (Novozymes A/S) resulted in a 20% higher glucose recovery compared to untreated samples, yielding 40.9 g/L of sugar. In another study, Mahmoodi et al. [32] reported a 23–72% increase in sugar yield after hydrothermal treatment at 100–160°C for 0–60 min with Cellic® CTec2 (Novozymes A/S) and Cellic® Htec2 (Novozymes A/S) compared to untreated OFMSW, with the highest yield achieved at 130°C for 60 min.

The main objective of this study was to enhance the understanding of enzymatic hydrolysis and heat pretreatment as effective methods for converting OFMSW into fermentable sugars in order to ultimately improve the yield of subsequent fermentation procedures. To assess the viability of enzymatic hydrolysates from OFMSW for fermentation, the choice was made to produce ethanol, given its established use as a biofuel, using Saccharomyces cerevisiae due to its resilience and strong resistance against natural inhibitors in OFMSW. Nevertheless, it is also important to explore the production of alternative energy sources, such as biobutanol [33], lipids [34], and hydrogen [35], as these possess substantial potential in the process of maximizing the value of OFMSW as a second-generation feedstock [5].

2 Materials and methods

2.1 Materials

The OFMSW was collected at Holsted municipality (55° 30′ 41″ N, 8° 54′ 59″ E), Denmark, and provided by Ragn-Sells Denmark A/S. The first batch was collected in March 2022, and the second batch was collected in October 2023. The OFMSW was stored at −80°C between uses. The enzymatic cocktail was based on Cellic® CTec3 (Novozymes A/S) as a source of cellulases and hemicellulases with specific enzymatic activity 214 FPU/mL and AMG® 300 L BrewQ (Novozymes A/S) as a source of glucoamylase with specific enzymatic activity 300 AGU/mL. Sugar, organic acids HMF, and furfural were analyzed through high-performance liquid chromatography (HPLC) (1260 Infinity II, Agilent Technologies).

2.2 Dry matter, lipid content, protein content, and CHNS elemental determination

The dry matter (DM), also known as total solid, was determined in heat-treated and untreated OFMSW samples based on the following analytical protocol by National Renewable Energy Laboratory (NREL) [36].

For the lipid extraction, the Soxhlet extraction method was used based on NREL technical protocol [37]. Lipid extraction was done for raw OFMSW samples. For the dried waste sample, 1.5 g of dried sample was put in an extraction thimble. This thimble was dried and kept in a desiccator prior to its use. The round bottom bottles were also weighed. Two hundred milliliters of hexane was added to each round bottom bottle, and the extraction was run for 4 h starting from the boiling time. After the extraction, hexane was removed with a rotating evaporator.

A CHNS elemental analyzer (Pekin Elmer 2400 series CHNS/O) was used to analyze the carbon, hydrogen, nitrogen, and sulfur content of the OFMSW samples. The samples were analyzed as a dried sample, where a 2–10-mg sample was weighed on small tin capsules and analyzed on the elemental analyzer.

The protein content was calculated based on the nitrogen content. To do this, a nitrogen-to-protein conversion factor (NPCF) was used. The NPCF, which is used, is 6.25, as it is assumed that all proteins have a 16% (w/w) content of nitrogen. The number, taken from the literature, is not an exact figure but represents a well-accepted, widely used approximation. Equation 1 used for the nitrogen-to-protein conversion is:

$$N\times \textrm{NPCF}=P$$
(1)

where N is nitrogen content % (w/w) in the dried sample, NPCF is the mass ratio of nitrogen to protein, 6.25, and P is protein content % (w/w) in the dried sample.

2.3 Heat pretreatment, anaerobic incubation, and enzymatic hydrolysis

For thermal pretreatment, OFMSW was defrosted at 4°C, and 100 g of sample was weighed into each 250-g bottle. Bottles were placed into preheated oil batch in an oven. The duration of pretreatment was 4 h, and for each hour sample was taken. The pretreatment temperature ranged from 70 to 160°C, with an increment of 10°C for each taken sample. Anaerobic incubation was made as follows: OFMSW was defrosted for 2 days at 4°C and incubated at 20°C. Every 24 h, 30 g of the sample was taken for free sugar analysis for a period of 3 days.

Prior to enzymatic hydrolysis, approximately 1 to 2 kg of OFMSW was defrosted at room temperature overnight and, if necessary, heat pretreated in an autoclave at 100°C for 4 h. The enzymatic hydrolysis was performed as follows: The enzymatic solution was prepared by weighing enzymes into a beaker on an analytical scale and adding Mili-Q water. The required amount of OFMSW, enzymatic solution, and Mili-Q water was added into 500-mL shaking flasks to achieve a final weight of 100 g (Table 1). The samples are then placed in an incubator for 24 h at 50°C and 150 RPM. Incubated samples are removed, and each hydrolysate is poured into two 50-mL falcon tubes, one of which was stored at −20°C. The samples are centrifuged (SL 16 Centrifuge, Thermo Fisher Scientific) at 5000 RPM for 20 min. Five milliliters of supernatant was carefully piped out into new 50-mL falcon tubes. The supernatant is diluted by adding 20 mL of Mili-Q water. The diluted hydrolysates are filtered using 0.22-μm syringe filters into HPLC vials and analyzed for ethanol, free sugar, and organic acid content. For storage, HPLC vials with samples were frozen at −20°C. All experiments were conducted using a 10% DM concentration and were diluted with Mili-Q water as necessary. Controls were also conducted under the same conditions but without the addition of enzymes.

Table 1 Reaction mixture for enzymatic hydrolysis of OFMSW with final DM of 10%
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2.4 Total starch content analysis and iodometric starch solubilization analysis

For total starch content determination, OFMSW samples were prepared by drying 100 g in an oven at 60°C overnight. Then, dried samples were powdered with a porcelain mortar and sieved with a 0.5-mm metal screen. The prepared samples were analyzed for starch content by Megazymes Total Starch HK Assay Kit (Megazyme Ltd.).

Solubilized starch was analyzed via iodometry as follows: For the preparation of the standard calibration curve, 100 mg of potato starch (Merck) was dissolved in 80 mL of Mili-Q water at 70–80°C under stirring on a heat block. After, the solution was transformed into a 100-mL volumetric flask and filled up with Mili-Q water. Obtained solution with a final concentration of 1 mg/mL was then used to prepare dilution series 0.3 mg/mL, 0.25 mg/mL, 0.2 mg/mL, 0.15 mg/mL, 0.1 mg/mL, and 0.05 mg/mL. Then each dilution was measured by adding 1 mL of the solution to the cuvette and 30 μL of iodine reagent (0.3 KI and 0.254 I2 g in 1 L of Mili-Q water). Prepared dilution samples were measured on a spectrophotometer (Agilent Technologies Cary 60 UV-Vis) at 600-nm wavelength. Obtained absorbances were used to create the standard calibration curve. For each gelatinization condition, 1.5 g of sample was measured into a 50-mL falcon tube on an analytical weight scale. Prepared falcons were filled with 35 mL of Mili-Q water and centrifuged (SL 16 Centrifuge, Thermo Fisher Scientific) for 15 min at 5000 RPM. The supernatant was transferred into a new 50-mL falcon tube and topped up with Mili-Q water to a total volume of 50 mL. Then, 1 mL of sample was added to the cuvette and 30 μL of iodine reagent (0.3 g of KI and 0.254 g of I2 in 1 L of Mili-Q water) and mixed well by pipetting down and up. Filled cuvettes were measured by spectrophotometer (Agilent Technologies Cary 60 UV-Vis) at 600-nm wavelength. Starch concentration was calculated with Eq. 2 as follows:

$${c}_{\textrm{s}}\ \left(\textrm{g}/\textrm{L}\right)=A/b$$
(2)

where cs is the starch concentration, A is the absorbance of the starch-iodine complex, and b is the regression coefficient of the linear regression equation based on the standard calibration curve. The described method was adapted from a protocol for potato starch analysis [38].

2.5 The analysis of sugars, organic acids, furfural, and hydroxymethylfurfural in OFMSW samples

OFMSW samples were transferred into 50-mL falcon tubes and centrifuged at 5000 RPM for 15 min. The supernatants were processed by weak acid hydrolysis (WAH) for the total sugar analysis in the liquid fraction and/or directly used for free sugar, organic acids, furfural, and hydroxymethylfurfural (HMF) analysis. The characterization of the liquid fraction was based on the analytical protocol by NREL [39]. The pellets were recovered into aluminum plates for drying in an oven at 60°C overnight and after being powdered with a porcelain mortar. Strong acid hydrolysis (SAH) was performed to identify the total sugar content in the solid fractions using the analytical protocol developed by NREL [40]. Produced acidic hydrolysates and supernatants were filtered by 0.22-μm syringe filters into HPLC vials and underwent HPLC analysis (Bio-Rad Aminex HPX-87H Column, Rio-Rad Laboratories Inc.), using H2SO4 mobile phase (0.005 M) and refractive index detector for sugar (glucose, cellobiose, xylose, and arabinose) and organic acids (malic acid, acetic acid, glycolic acid, formic acid, and succinic acid). The HPLC analysis of furfural and HMF was performed by InfinityLab Poroshell 120 EC-C18, Agilent Technologies, Inc., using a mixture of 20% acetonitrile and 80% H3PO4 (0.2%) (v/v) as mobile phase and a diode array detector. The mathematical conversion of HPLC results (g/L) into (g/100 g OFMSW DM) was performed as described in [30, 41]. A hydration factor of 0.9 was used for estimating glucan content from glucose concentration, while for the estimation of xylan and arabinan from pentoses, a hydration factor of 0.88 was applied.

2.6 Ethanol fermentation

Each sugar-rich hydrolysate was supplemented with 0.20 g of Saccharomyces cerevisiae (commercial dry yeast, Malteserkors tørgær, De Danske Spritfabrikker A/S, Denmark) after enzymatic hydrolysis. Nitrogen was used to flush prepared 500-mL flasks for 10–15 s to eliminate air, and yeast locks containing 2 mL glycerol were added. The incubation was performed at 32°C with 130 RPM for 96 h in a shaking incubator (LS-Z shaker with Kelvin+, Kuhner). The weight of each flask was measured before incubation and subsequently at 4, 8, 20, 30, 44, 72, and 96 h into the incubation. Following incubation, each fermentation broth was poured into two 50-mL falcon tubes, one of which was stored at −20°C. Samples were centrifuged at 5000 RPM for 20 min, and 5 mL of supernatant was carefully pipetted into new 50-mL falcon tubes. The supernatant was diluted with 20 mL of Mili-Q water, and the diluted hydrolysates were filtered with 0.22-μm syringe filters into HPLC vials and assessed for ethanol, free sugar, and organic acid content. The samples were frozen at −20°C for storage. Calculation of ethanol production from gravimetric determination, where MW is the molecular weight, can be seen in Eq. 3. Gravimetric measurements were verified by ethanol analysis via HPLC.

$$\textrm{EtOH}\ \left(\textrm{g}\right)=\textrm{C}{\textrm{O}}_2\ \left(\textrm{g}\right)\times \left[\textrm{MW}\ \left(\textrm{EtOH}\right)/\textrm{MW}\ \left(\textrm{C}{\textrm{O}}_2\right)\right]$$
(3)

2.7 Statistical methods

All trials were performed at least as triplicates. All results are given as mean values with standard deviation. The one-way and two-way analyses of variance (ANOVA) were carried out on all results with one or more independent variables, and Multiple Linear Regression (MLR) was performed for all results with two or more correlated independent variables (regressors). Additionally, Tukey’s HSD tests were conducted as an extension of ANOVA. MLR models were used to create predictions, which were used to make 2D contour plots. R version 4.0.3 [42] and RStudio version 2022.07.2 Build 576 (Posit, PBC) were used for all statistical analyses.

3 Results

3.1 The compositional analysis of used OFMSW in experimental work

The composing elements of the OFMSW applied in this study are tabulated in Table 2. The DM for the March batch was 12.7% (w/w), and for the October batch was 14.4% (w/w).

Table 2 The composition of OFMSW is presented as g/100g OFMSW DM, and ethanol and organic acids are presented in grams per liter
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3.2 The optimization of thermal pretreatment

October batch underwent thermal pretreatment, and Fig. 2A illustrates an increase in solubilized starch with higher reaction time and temperature, except for the last three groups (140°C, 150°C, and 160°C), which does not demonstrate a significant change in released starch after 4 h, according to contour plot Fig. 2B. The highest gelatinization yield was obtained at 130°C for 4 h, resulting in 36.2%. However, the results suggest that there is only a minor variation in solubilized starch after 1 h of pretreatment for all temperatures.

Fig. 2
figure 2

A Starch solubilization after thermal pretreatment of the October batch of the OFMSW. B Contour plot of MLR model of starch gelatinization

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MLR analysis was performed to investigate how the amount of solubilized starch as a response variable is influenced by reaction time and temperature, which are independent variables that influence each other. The MLR model revealed that R2 = 0.83, which means that 83% of the variation in the amount of starch values can be explained by changing temperature and reaction time. The P-value was 2.2 × 10−16, and F-statistic was 189.1. Obtained regression coefficients showed that (1) if the temperature is increased by 1°C, on average, the amount of solubilized starch rises by 0.017 g per 100 g DM of OFMSW; (2) if the reaction time is increased by 1 h, on average, the amount of solubilized starch rises by 0.724 g per 100 g DM of OFMSW; (3) every time that temperature is increased by 1°C, the effect of reaction time is getting larger by 0.012 of solubilized starch/100 g DM of OFMSW per 1 h. The contour plot Fig. 2B illustrates how temperature and reaction time impact the starch gelatinization process, confirming that during the first hour of gelatinization, changes in temperature have a minimal impact on starch solubilization.

Furthermore, the ANOVA test confirmed that temperature and time are statistically significant for gelatinization, with a P-value of  2 × 10−10, an F-value of 53.2 for temperature, a P-value of 2 × 10−10, and an F-value of 340.3 for reaction time, respectively. The combined effect of temperature and time had a P-value of 2 × 10−10 and an F-value of 14.6, suggesting a strong interaction between factors. In addition, the Tukey HSD test confirmed a strong effect on starch solubilization from rising reaction time (shown in Table S1 in Supplementary Material). Also, significant differences in mean values were found between gelatinization temperatures of 100, 120, 130, 140, 150, and 160°C compared to 70, 80, and 90°C (shown in Table S2 in Supplementary Material).

3.3 Investigation of sugar, organic acids, furfural, and HMF content in thermally pretreated OFMSW

Based on results from starch solubilization, samples treated at 100 to 130°C for 4 h were chosen for further analysis, which included analysis of sugars, organic acids, furfural, and HMF.

The content of monomeric glucose, xylose, arabinose, and dimeric cellobiose, referred to as free sugars, was investigated in liquid fractions of raw and heat pretreated OFMSW from the October batch. In order to analyze the total content of solubilized sugars in liquid fractions, weak acid hydrolysis was conducted. WAH was used to depolymerize oligosaccharides and polysaccharides such as starch, cellulose, and hemicellulose. The measurement of complex sugars in the liquid fraction was determined by subtracting the content of free sugars in the liquid fraction from the total sugars present in the same fraction.

Figure 3 demonstrates that all pretreatment temperatures yielded higher sugar content in OFMSW samples than the initial expectations based on the raw samples. This discrepancy may arise due to the inherent recalcitrance of the raw material during the initial analysis. Consequently, not all sugar fibers are effectively broken down during the acidic hydrolysis process, leading to an underestimation of the measured sugar content.

Fig. 3
figure 3

The analysis of free sugars and complex sugars in liquid fractions and complex sugars in solid fractions of untreated and heat pretreated samples of the OFMSW

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The highest amount of free sugars was obtained under the condition of 100°C, as depicted in Fig. 3 and Table 3. All heat pretreatments, except for 100°C, resulted in a lower amount of sugars than untreated OFMSW. Glucose content decreased as the reaction temperature increased, indicating that glucose in monomeric form degraded during the treatment. The breakdown of lignocellulose fibers during treatment was suggested by comparing the results of xylose and cellobiose in untreated and in 100°C treated samples, which resulted in the liberation of xylose, cellobiose, and possibly glucose. With an increase in reaction temperature, the content of cellobiose and xylose decreased, possibly indicating that xylose and cellobiose underwent undesired chemical reactions.

Table 3 The sugar composition in liquid and solid fractions of untreated and thermally pretreated OFMSW. Results are pretreated as g/100g OFMSW DM
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The results presented in Fig. 3 and Table 3 indicate a significant increase in the total sugar content in liquid fractions, primarily glucose, for all pretreatment conditions tested. This glucose is likely derived from sources such as starch, cellulose, and other oligosaccharides. Interestingly, there was no statistically significant difference between the various pretreatment conditions, with all resulting in comparable total sugar content in liquid fractions, as shown in Table 3. Additionally, there was a noticeable trend of decreasing glucose and increasing xylose content with increasing pretreatment temperatures (Table 3). Nevertheless, when the total sugar content in the liquid fractions is further analyzed and categorized into free sugars and complex sugars, a new trend emerges. As shown in Fig. 3, an increase in reaction temperature corresponds to a decrease in the amount of free sugars present in the liquid fraction, while simultaneously, an increase in the amount of complex sugars is observed. This observation supports the hypothesis that monomeric sugars undergo degradation at higher temperatures. At the same time, it suggests that sugar-rich fibers, such as lignocellulose and starch, are liberated and subsequently broken down during the heat pretreatment process. The findings were further confirmed with one-way ANOVA (Table 4). Further, Tukey’s HSD test (shown in Fig. S1, Fig. S2, and Fig. S3 in Supplementary Material) was performed, confirming the assumption that variation in temperature is not statistically significant for total sugar content in liquid fraction, but it is significant for the amount of free sugars and complex sugars in samples with increasing differences in reaction temperature.

Table 4 The statistical values of the one-way ANOVA test for sugar content in each fraction of thermally pretreated samples of OFMSW
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The solid fractions underwent strong acid hydrolysis to analyze the sugar profile remaining in the solid fractions after heat pretreatment. Figure 3 illustrates that the total amount of remaining sugars decreases with increasing temperature. Raw samples have the highest amount of remaining sugars, while samples treated at 130°C have the lowest amount. The main reduction in sugar is observed in glucose content (Table 3). These findings align with the fact that higher reaction temperatures lead to the breakdown of sugar-rich fibers, which become solubilized in the liquid fraction of the OFMSW. The outcomes of Tukey’s HSD analysis (shown in Fig. S4 in Supplementary Material) indicated that the sugar content within solid fractions subjected to treatments at 100, 110, and 120°C did not exhibit any statistically significant distinctions. However, as the reaction temperature escalated to 130°C, a statistically significant variance in sugar content emerged.

The examination of organic acids, as depicted in Fig. 4, reveals a decline in the concentration of succinic/glycolic acids following heat pretreatment at temperatures above 100°C. It is important to note that the similar retention times of succinic acid and glycolic acid make it difficult to distinguish between the two. Thus, the results represent a combination of both acids. Additionally, the concentration of acetic acid increases as the reaction temperature rises. This can be attributed to the degradation of sugars, as acetic acid is one of the byproducts of this process. Notably, there were no considerable variations observed for malic acid across different temperatures. Additionally, based on the relatively high content of organic acids, it can be suggested that the OFMSW undergoes pre-fermentation by present microorganisms during storage.

Fig. 4
figure 4

The analysis of organic acids, HMF, and furfural in liquid fractions of untreated and thermally treated samples of the OFMSW

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The analysis of furfural and HMF in Fig. 4 reveals that the amount of furfural increases with rising reaction temperature, while HMF has a substantial increase between 100 and 110°C but decreases at higher temperatures compared to 110°C. Furfural and HMF were not detected in the raw OFMSW samples. Both HMF and furfural are produced by sugar degradation, with furfural originating from pentoses such as xylose and arabinose and HMF originating from hexoses such as glucose and fructose. Severe pretreatment, particularly in an acidic environment, tends to convert saccharides in the OFMSW into HMF and furfural. Both compounds are known to inhibit microbial growth and are generally toxic. Furfural and HMF were not detected in untreated samples of OFMSW.

Based on the one-way ANOVA shown in Table 5, the varying temperature is significant for all measured compounds. The highest effect was seen for acetic acid, succinic/glycolic acids, and furfural.

Table 5 The statistical values of the one-way ANOVA test for organic acids, HMF, and furfural for thermally pretreated samples of OFMSW
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3.4 Effect of anaerobic incubation on natural depolymerization of present sugars in OFMSW

During experimental work, it was discovered that the amount of free sugars in raw samples of the OFMSW increases if samples are incubated at room temperature (20°C) in an anaerobic environment. Based on this knowledge, sugar production is investigated in Fig. 5. The aim was to find how anaerobic incubation influences glucan conversion into glucose and cellobiose and xylan to xylose. The experimental work was performed on the October batch of OFMSW.

Fig. 5
figure 5

The sugar analysis of the OFMSW samples incubated at room temperature for 3 days. The F-value is 617.5, and P-value is 8.4 × 10−10

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Figure 5 displays that the anaerobic incubation of the OFMSW at room temperature results in the production of sugars, which could be attributed to the hydrolytic activity of microorganisms present in the sample. The highest sugar content was observed in samples incubated for 3 days, while the lowest was observed in non-incubated samples. When comparing the results of non-incubated samples to incubated samples, it can be observed that cellobiose is formed, and there is a negligible amount of xylose present. Since cellobiose and xylose are derived from lignocellulose, it can be inferred that microorganisms present in the sample possess both cellulolytic and hemicellulolytic activities. Thus, it can be suggested that anaerobic incubation can positively contribute to the enzymatic hydrolysis of OFMSW as it pre-digests the organic material and makes it easier to hydrolyze.

3.5 Investigation of effects of the enzymatic dosage and type of pretreatment on sugar recovery from OFMSW

The influence of enzymatic dosage and type of pretreatment method of OFMSW, which included heat pretreated OFMSW (100°C for 4 h), untreated OFMSW, untreated OFMSW incubated at room temperature for 3 days, and heat pretreated OFMSW (100°C for 4 h) after 3 days incubation at room temperature, was investigated. The main goal was to find the enzymatic mixture and type of pretreatment that results in the highest hydrolysis yield, also referred to as sugar recovery. The total sugar content of the original untreated October batch was 47 g of sugar/100 g DM OFMSW. In the following results, Cellic® CTec3 (Novozymes A/S) is referred to as Ctec3, and AMG® 300 L BrewQ (Novozymes A/S) is referred to as AMG.

Figure 6 illustrates an increasing trend in hydrolysis yield for all OFMSW samples as the enzymatic dosage increases from 1 FPU Ctec3 + 0.5 AGU AMG to 10 FPU Ctec3 + 5 AGU AMG per g OFMSW DM. However, there were no considerable differences in yields based on enzymatic dosages or the type of pretreated OFMSW. The highest sugar recovery of 54% was achieved using 10 FPU Ctec3 + 5 AGU AMG for both heat-treated OFMSW with and without anaerobic incubation, while the lowest saccharification of 38% was observed using 1 FPU Ctec3 + 0.5 AGU AMG for untreated OFMSW after anaerobic incubation. Furthermore, based on the results, the enzymatic dosage had the lowest effect on heat pretreated samples without 3 days of anaerobic incubation, as the sugar recovery showed the least steep increase in yield. Additionally, 51% of saccharification was achieved with 1 FPU Ctec3 + 0.5 AGU AMG, making it an attractive condition for further processes.

Fig. 6
figure 6

The analysis of hydrolysates of heat pretreated OFMSW (orange), untreated OFMSW (red), untreated OFMSW anaerobically incubated at room temperature for 3 days (blue), and heat pretreated OFMSW after 3 days of anaerobic incubation at room temperature (green). Control samples underwent the same procedure without the addition of the enzymes

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Although the differences in yields are not substantial, if differences in enzymatic dosage are considered, the two-way ANOVA test shown in Table 6 indicated that Ctec3 dosage, AMG dosage, and type of OFMSW are statistically significant for sugar yield. Additionally, Tukey’s HSD test (shown in Fig. S5 in Supplementary Material) revealed that thermal pretreatment has a positive effect, and anaerobic incubation has a negative effect on enzymatic saccharification. The statistical analysis of the sugar yield of thermally treated samples without anaerobic incubation revealed no significance of AMG and Ctec3 dosage for sugar yield. It could be suggested that heat pretreatment reduced the recalcitrance of OFMSW towards enzymatic decomposition, resulting in higher efficiency of lower dosages.

Table 6 The statistical values after the two-way ANOVA test for EH of various pretreated OFMSW
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It is suggested that the pre-digestion of OFMSW with anaerobic incubation does provide a negative effect based on a comparison of results for OFMSW with and without anaerobic incubation. In conclusion, the results indicate that heat pretreatment has a beneficial impact on enzymatic hydrolysis (EH) when comparing heat-treated and untreated OFMSW samples. Furthermore, the increase in dosages of Ctec3 and AMG enzymes demonstrated a significant positive influence on the hydrolysis yield. It can be inferred that employing higher enzymatic dosages in conjunction with thermal pretreatment may lead to improved outcomes in terms of hydrolysis efficiency.

3.6 SSF of OFMSW with varying enzymatic dosage

Simultaneous saccharification and fermentation (SSF) was performed on thermally pretreated (100°C for 4 h) October batch samples without anaerobic incubation, as it resulted in the highest yield of saccharification. The pH corresponded to 4.02 and was not adjusted before and during the fermentation process. The hydrolysates were prepared with the same enzymes and enzymatic dosages. The sugar conversion to ethanol was attributed to the amount of glucose in hydrolysates, as used Saccharomyces cerevisiae strain was able to convert only the hexose sugars, such as glucose and mannose, into ethanol and CO2.

Figure 7 illustrates the results based on the gravimetric determination of CO2 release. The increasing ethanol yields correspond to increasing enzymatic dosages. The highest glucose conversion of 67% was achieved with 10 FPU Ctec3 + 5 AGU AMG after 96 h of fermentation. The lowest dosage of 1 FPU Ctec3 + 0.5 AGU AMG reached 56% glucose conversion to ethanol, which is significant if the amount of used enzymes is considered. Furthermore, based on CO2 release, it is predicted that 57% ethanol yield was obtained after 20 h of incubation with 10 FPU Ctec3 + 5 AGU AMG, corresponding to 29 g/L of ethanol concentration.

Fig. 7
figure 7

Gravimetric analysis of ethanol fermentation of the heat pretreated OFMSW hydrolysates. The F-value is 42.1, and P-value is 0.00019 for Ctec3. The F-value is 19.2, and P-value is 0.0023 for AMG

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Additionally, organic acids were investigated post-fermentation (Fig. 8), revealing that there are variations in metabolites for all conditions. Overall, the total content of organic acid increases with the increasing dosage of enzymes, especially succinic and malic acids. The concentration of acetic acid has increased in fermented samples compared to untreated ones. On the other hand, glycolic acid decreased in fermented samples compared to untreated ones. A slight fluctuation of pH from 4 to 4.1 was observed. Furthermore, residual amounts of formic acid were detected, varying between 0.15 and 0.3 g per 100 g DM OFMSW.

Fig. 8
figure 8

HPLC analysis of organic acids and ethanol before and after fermentation of the thermally pretreated OFMSW hydrolysates

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Ethanol analysis via HPLC after fermentation, as shown in Fig. 8, showed that ethanol content has a decreasing pattern with increasing enzymatic dosage, which contradicts to results obtained from gravimetric analysis, probably due to other gas-producing microorganisms metabolizing residual and C5 sugars. The highest obtained amount was observed for 1 FPU Ctec3 + 5 AGU AMG, and the lowest 10 FPU Ctec3 + 5 AGU AMG, 33 g and 30 g of ethanol per 100 g DM OFMSW, respectively. In general, there are no significant differences between the results obtained from gravimetric analysis (Fig. 7) and HPLC analysis (Fig. 8). The only notable distinction is observed in the amount of ethanol detected for lower dosages, specifically 1 FPU Ctec3 + 0.5 AGU AMG and 1 FPU Ctec3 + 0.5. However, these differences cannot be considered significant as they fall within the range of standard deviation.

Based on the statistical analysis results presented in Table 7, it can be concluded that the enzymatic dosage of Ctec3 has no significant impact on the levels of any compounds. However, the dosage of AMG does show a significant influence on malic acid and succinic acid. It is worth noting that the significance is somewhat debatable since it is close to the cutoff P-value of 0.05. Therefore, further investigation may be needed to determine the precise relationship between organic acids and ethanol and enzymatic dosage.

Table 7 The statistical values of the two-way ANOVA test for fermented samples of enzymatically hydrolyzed OFMSW (organic acids and ethanol)
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4 Discussion

The compositional analysis revealed that that are no significant differences between the two batches of OFMSW, although they have been obtained in different seasons. This is desirable as the similar composition makes it easier to establish bioprocessing steps for OFMSW feedstock. The high sugar content of 46.5±1.0 g/100 g DM for the March batch and 42.8±2.1 g/100 g DM for the October batch supports the idea of using OFMSW as a second-generation feedstock. In addition, the high protein content makes it a good source of nitrogen for fermentation processes, which is proven by successfully conducted ethanol fermentation without extra N-source additives.

Furthermore, the high content of organic acids and ethanol was observed, which points to the fact that OFMSW is digested during storage and easily accessible sugars are already fermented. The high concentration of organic acids and ethanol brings multiple negative effects. Iyer et al. [43] reported that a 30 g/L concentration of lactic acid inhibited cellulase saccharification by one-fourth. Tagaki [44] showed 50% inhibition of cellulase with ≥70 g/L concentration of lactic acid, acetic acid, succinic acid, and other organics. Further, he reported 50% cellulolytic activity inhibition with 26 g/L of ethanol and 25 g/L of glucose. Jing et al. [45] achieved one-third inhibition of saccharification with a 25 g/L concentration of acetic acid. It could be assumed that based on the compositional characteristics of the OFMSW, it will significantly reduce enzymatic activity and correspondingly influence sugar recovery. On the other hand, organic acids and ethanol are known as excellent biopreservatives, resulting in contamination control during storage, pretreatment, and biological conversion [46]. However, at high concentrations, these compounds can inhibit the growth of producing strains, such as Saccharomyces cerevisiae [47].

The solubilization of starch was enhanced by thermal pretreatment, with a gradual increase in the amount of solubilized starch observed up to 130°C for 4 h of reaction time, leading to 36.2% starch gelatinization. At 4 h of reaction time, the solubilized starch decreased with a further increase in reaction temperature, but at 3 h, there was still a positive effect observed with increasing reaction temperature up to 160°C. It could be expected that starch starts to degrade into formic acid, furfural, HMF, acetic acid, and levulinic acid at high temperatures with a long reaction period. Parchami et al. [14] observed a gradual increase in sugar degradation products at 98°C, 121°C, 140°C, and 180°C thermal pretreatment. Moreover, varying pretreatment temperatures at a 1-h reaction time did not show considerable differences in starch solubilization, resulting in 6–13% starch gelatinization. The thermal stability of starch in OFMSW could be explained by the high content of resistant starch, which is expected as resistant starch is commonly present in peels and seeds [48], as well as in retrograded starch with high resistance from post-cooked starchy food [49].

The highest quantity of free sugars in the liquid fraction was obtained at 100°C, but there were no statistically significant differences in total sugar content across all investigated temperatures. It is suggested that monomeric sugars, such as glucose and xylose, degraded at temperatures ≥100°C, resulting in a decrease in their concentration in the liquid fraction and, simultaneously, sugar-rich fibers breakdown, increasing content of dissolved oligosaccharides and polysaccharides in the liquid fraction. This hypothesis is supported by the increasing levels of furfural, HMF, and acetic acid, which are products of C5 and C6 sugar degradation, as well as the rising amount of complex sugars in the liquid fraction with increasing reaction temperature further supports this hypothesis. These findings align with the results obtained from the starch solubilization process conducted between 100 to 130°C. Additionally, the increase in xylose content from 100 to 130°C observed in the total sugar analysis of liquid fractions supports this idea, as xylose originates from lignocellulosic fibers. As the reaction temperature increased from 100 to 130°C, the investigation of sugars in solid fractions revealed a gradual decrease in the amount of sugar. Therefore, based on the results, thermal pretreatment at 100°C is considered the most attractive option as it is less severe, results in the highest sugar content in all fractions, produces trace amounts of furfural and HMF, and still achieves 25% of starch gelatinization.

Results from EH showed that thermal pretreatment increased the saccharification yield from 4 to 11% compared to untreated OFMSW. Interestingly the lowest tested dosage, 1 FPU Ctec3 + 0.5 AGU AMG per g of OFMSW DM, yielded the highest difference. Furthermore, it was observed that anaerobic incubation did not provide any advantages in terms of OFMSW saccharification. In fact, when comparing thermally pretreated OFMSW with and without anaerobic incubation, it was found that the hydrolysis yield decreased by 4–8% with anaerobic incubation. This decrease in sugar recovery could be anticipated, as some of the sugars are consumed during the anaerobic incubation process, even though it results in further sugar hydrolysis by native microorganisms. Based on the obtained results, it was determined that pretreatment at 100°C for 4 h represents the optimal condition for EH. This specific condition yielded an 11% rise in sugar yield at the lowest enzyme dosage employed. The outcome was a 51% saccharification yield, achieved with the utilization of only 1 FPU Ctec3 + 0.5 AGU AMG per g of OFMSW DM. This outcome holds significant value, as enzymes frequently constitute a major expense in the production process.

The sugar-rich hydrolysates of OFMSW underwent ethanol fermentation, and the gravimetric analysis indicated a gradual decrease in mass as the enzymatic dosage increased. This mass loss was attributed to the release of CO2 during ethanol fermentation. However, HPLC analysis revealed no significant difference in ethanol concentration with increasing enzyme dosage. Furthermore, the concentration of organic acids was examined. Overall, there was no discernible impact of enzymatic dosage on the concentration of organic acids in samples measured after fermentation. Additionally, it appears that the low pH of 4 and the relatively large amounts of organic acids did not influence ethanol production. The ethanol production from enzymatic hydrolysates of OFMSW of the present study was compared to previously reported studies in Table 8. Based on the comparison, it could be concluded that the present work achieved considerable production of ethanol at a remarkably low dosage of enzymes.

Table 8 Comparison of bioethanol production from enzymatic hydrolysates of OFMSW
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5 Conclusion

Thermal pretreatment at 100°C for 4 h was found to be optimal for solubilizing starch, while higher temperatures led to sugar degradation. Monomeric sugars degraded at temperatures ≥100°C, resulting in a decrease in their concentration and an overall drop in sugar content. Thermal pretreatment at 100°C was preferable due to milder conditions, higher total sugar content, and low concentration of furfural and HMF. EH of thermally pretreated OFMSW increased saccharification yield, with the lowest enzyme dosage showing the highest difference. Anaerobic incubation did not improve saccharification. Optimal conditions for enzymatic hydrolysis were pretreatment at 100°C for 4 h. During ethanol fermentation of sugar-rich hydrolysates, increasing enzymatic dosage did not lead to a significant increase in ethanol concentration. The results suggest that the optimal enzymatic dosage for ethanol fermentation from hydrolysates of thermally pretreated OFMSW is the lowest dosage of 1 FPU Ctec3 + 0.5 AGU AMG. This dosage yielded 31 g of ethanol per 100 g OFMSW DM, corresponding to 80% of total glucose conversion to ethanol. In conclusion, OFMSW has promising characteristics for bioconversion processes, but careful optimization of process parameters is necessary to maximize sugar recovery and ethanol production.