Bioconversion of Apple Pomace to Meyerozyma guilliermondii and Scheffersomyces stipitis Biomass

Abstract

Poland is one of the leading apple-producing countries, both in Europe and around the world. One of the main byproducts of apple processing is pomace, which can account for 20–35% of the harvested apples. Pomace is a potential source of many valuable bioactive components and can also serve as a food ingredient, either directly or indirectly (after bioconversion with fodder yeast). This study aimed to evaluate the possibility of converting polysaccharides contained in apple pomace to yeast biomass. Meyerozyma guilliermondii and Scheffersomyces stipitis yeasts were grown in a medium prepared by pretreatment of the raw material with water or 2% sulphuric acid at 120 °C. Subsequently, enzymatic hydrolysis was performed using a Cellic CTec2 preparation at 30 °C or 50 °C. The resulting hydrolysates were enriched with ammonium salts, and shaken yeast cultures were incubated at 30 °C for 72 h. Based on the results, it can be concluded that acid pretreatment of apple pomace is more effective than water pretreatment under the same time and temperature conditions. The Meyerozyma guilliermondii strain grows in apple pomace hydrolysates more efficiently (16.29 g/L) than Scheffersomyces stipitis cells do (14.63 g/L).

1. Introduction

According to recent statistical data, apple production in European countries has been estimated at around 18 million tonnes. Around 4 million tonnes are Polish apples. One of the main byproducts of apple processing is pomace (AP), which can account for 20–35% of the harvested apples [1,2].

Apple pomace, a source of valuable bioactive components, can serve as a food ingredient either directly or indirectly (after bioconversion with fodder yeast), making it particularly valuable given the increasing demand for feed and dietary protein [3,4,5,6,7,8,9,10,11,12,13,14]. The war in Ukraine, given the country’s significant share of cereal and sunflower production, is causing a substantial economic impact worldwide. Consequently, a pressing global issue is determining what should be used to feed animals.

Soya has been a popular animal feed ingredient due to its high content of proteins and valuable amino and fatty acids. There are two challenges associated with the feed use of soya. The first is the competing use of soya for feed with its direct use as human food. The second is the relatively high price of soya [15].

The chemical composition of pomace can vary greatly depending on the variety of apples, the ripeness, the climate, and the soil properties of the region from which the apples originate. Compared to the spent biomass of other fruits, apple pomace is particularly notable for its high pectin content, a polysaccharide that is part of the plant’s cell wall. In addition, apple pomace is characterised by a high content of cellulose, hemicellulose, and lignin, and proper processing of pomace results in the hydrolysis of cellulose into monosaccharides that are more bioavailable to microorganisms. The pomace is also rich in organic acids, sugars, fatty substances, and vitamins. Compounds are also characterised by strong antioxidant activity—polyphenols [4,7,16,17].

The pomace produced directly as waste from the fruit and vegetable industry typically contains a high water content of around 30% of the raw material. As a result, fresh AP is highly susceptible to undesirable microorganisms. Its storage life can be significantly extended by ensiling or drying processes. Dried AP, which usually consists of 90–95% dry matter, can be directly incorporated into animal feed, thereby enriching it with essential nutrients. Another alternative use for AP is in the production of pectins. Pectins also possess important properties such as the ability to gel, stabilise, and thicken [17,18,19].

For this reason, the pectins in apple pomace, when adequately modified enzymatically, chemically, or thermally, are used as hydrocolloids in industry [4,17,20]. AP can also serve as a microbial substrate in propionate–acetic fermentation, employing the bacteria Propionibacterium freudenreichii to obtain acetic acid and propionic acid [21]. AP is reportedly used in various technologies and end-products, such as baking, extruded foods, meat products, confectionery and dairy products, alcoholic beverages, edible mushroom cultivation, anthocyanins, and flavonoids [17].

Cellulose, hemicellulose, and lignins form a complex that is an integral part of plant biomass. To use it for the production of biofuels or microbial feedstocks, it is necessary to break down this complex to release the sugars it contains. The cell wall of plant cells consists of microfibrils, with hemicellulose occupying the outer part interspersed with lignin, while cellulose is centrally located. Cellulose is the most abundant component in this complex, typically comprising 40–60% of plant biomass, followed by hemicellulose at 20–50% and lignin at 15–35%. Cellulose is a polysaccharide composed of D-glucose residues linked by β-1,4-glycosidic bonds with cellobiose (containing two glucose subunits) being the repairing unit in this chain. Consequently, efficiently breaking down lignocellulosic raw materials to glucose makes them an excellent source of sugar for yeast in culture media [22].

One of the most efficient methods for converting lignocellulosic raw materials to glucose, alongside non-organic acid thermohydrolysis, is bioconversion through enzymatic hydrolysis using cellulases. The rate and extent of cellulose degradation depend on various factors, including the enzyme dose, hydrolysis temperature, and cellulose structure. Cellulose from different sources may exhibit varying degrees of resistance to hydrolysis [23,24,25,26].

Single-cell protein (SCP) is commonly produced from agri-food industry waste containing simple sugars or polysaccharides. During the First and Second World Wars, yeast SCP was used on a large scale in Germany to prevent hunger and malnutrition problems [25]. In light of this information, developing research on cultivating valuable yeast biomass using apple pomace as the primary nutrient medium seems advisable.

Based on scientific predictions, the protein requirement for the estimated global population of 9.7 billion, assuming a minimum requirement of 1 g/kg of body weight per day, results in an annual minimum requirement (assuming 60 kg as the average body mass) of 212,430,000 tonnes of protein for human nutrition alone [27,28]. Additionally, animals consume at least a comparable amount of protein annually. Regarding the anticipated number of livestock in the next 20 years and their protein needs, there are no precise statistics, but current trends indicate continued growth. However, given the current global situation, it is difficult to realistically forecast the protein demand for this sector [28,29].

Agriculture, facing challenges from a changing climate [30,31,32,33], may struggle to fully meet these needs. Consequently, there is growing interest in alternative protein sources, such as microbial protein.

Yeast has been widely used by humans for thousands of years for alcoholic fermentation and dough rise but it also has the capability to produce valuable dyes, proteins, hormones, surfactants, and vitamins [34,35,36,37,38,39,40]. Yeast’s biomass, rich in exogenous proteins and vitamins, makes it an appealing intermediate product for producing protein hydrolysates and for use in feed. Maintaining yeast cultures on an industrial scale is more manageable compared to multicellular organisms, and its acquisition does not incur high costs [41,42,43,44,45,46]. Due to its high nucleic acid content, yeast protein intake should not exceed 16% of the recommended daily protein dose [47].

Scheffersomyces stipitis and Meyerozyma guilliermondii are well-tested strains that reproduce efficiently by budding at 25–37 °C. These yeasts are capable of fermenting various carbohydrates such as glucose, galactose, maltose, and xylose [34,48,49,50,51,52,53,54,55,56]. Meyerozyma guilliermondii ATCC 6260, used in our experiments, is known as one of the yeast strains accepted for obtaining fodder SCP [57], while the other strain—Scheffersomyces stipitis LOCK P0047—is unique in its properties to efficiently grow in the hydrolysates of agricultural wastes, what was previously presented [50,58,59]. M.guilliermondii or S. stipitis have been used for years to convert lignocellulosic substrates to ethanol, xylitol, and biomass [24,50,51,59,60,61,62,63,64,65]. Therefore, we have set out to investigate the conversion of apple pomace to biomass by these two strains. Since industrial processes for biomass synthesis for feedstuffs include a thermal treatment step aimed at thermolysing the cells to improve the digestibility of the yeast biomass, the microbiological safety of this product containing thermally inactivated yeast cells is also ensured [34].

Considering all the factors mentioned above, it can be assumed that cultivating S. stipitis and M. guilliermondii cells using apple-derived wastes may also be a favourable method of their biovalorisation, particularly in light of the current geopolitical situation. These factors motivated us to evaluate the possibility of converting apple pomace into assimilable sugars, followed by their conversion into yeast SCPs.

2. Materials and Methods

2.1. Raw Material

This study used dried apple pomace purchased from a local Polish manufacturer (Samfarm, Zascianki, Poland) as raw material.

2.2. Dry Matter Analysis in the Raw Material

The dry matter content was measured using a Radwag WPS-30S weighing dryer (Radwag, Radom, Poland). The drying time/temperature sequence was as follows: 50 °C/3 min–>70 °C/3 min–>120 °C until the mass change was less than 1 mg/60 s.

2.3. Cellulose, Hemicellulose, and Lignin Assay in the AP

The content of essential constituents (holocellulose (cellulose, hemicellulose) and lignin) of AP was determined using the NREL protocol [66].

2.4. Pretreatment and Enzymatic Hydrolysis of the AP

To convert the lignocellulosic substrate into a cultivation medium, a 2-stage hydrolysis was performed. Aqueous or acid pretreatment of the raw material was carried out in 2 L flasks. A total of 100 g of dried apple pomace was mixed with 950 mL of distilled water or 950 mL of 2% sulphuric acid. The samples were then placed in an autoclave at 120 °C for 1 h. Subsequently, the samples were cooled down to room temperature, and the pH was set to 5.4 ± 0.1 using a 30% NaOH solution. Aliquots of 50 mL of sample were collected for HPLC analysis. Next, enzyme hydrolysis of the pre-prepared apple pomace was performed using the Cellic CTec2 preparation (Novozymes, Bagsvrd, Denmark) at a dose of 15 FPU (filter paper units)/g of cellulose. The hydrolysis process was carried out for 48 h at 30 °C or 50 °C. Then, the hydrolysates were filtered through a 0.45 µm filter, and the obtained liquid fractions (750 mL from 100g of AP) were used as a fundamental component of the medium for yeast cultivation.

2.5. Yeast Strains, Media, and SCP Cultivations

SCP cultivations were performed with the use of the ATCC strain (American Type Culture Collection; Manassas, VA, USA) and LOCK collections (Lodz Pure Cultures Collection of Industrial Microorganisms LOCK 105, Lodz, Lodzkie, Poland): Meyerozyma guilliermondii (formerly Candida guilliermondii) ATCC 6260 and Scheffersomyces stipitis (formerly Pichia stipitis) LOCK P0047. The first strain was used as the reference strain, commonly known and accepted in the EU as a possible feed SCP source, while the second, from the LOCK 105 pure culture collection, is known as a highly potent strain known for its high biomass yield in various agri-wastes hydrolysates. The yeasts were activated in 150 mL of YPG broth (comprising 10 g/L yeast extract, 20 g/L bactopeptone, 20 g/L glucose, and pH adjusted to 5.2 ± 0.1) sterilised by autoclaving at 121 °C for 20 min. The culture was incubated in 1 L round-bottomed flasks filled with 150 mL of the medium at 32 ± 1 °C for 24 h on a reciprocal laboratory shaker operating at 140 oscillations per minute (Eberbach E5900, Belleville, NJ, USA). Following cultivation, the yeast cell suspension was centrifuged at 5000× g for 10 min and washed twice with a sterile 0.9% NaCl solution. The seeding yeast suspension’s dry matter (DM) content was determined using a spectrophotometer (Rayleigh Analytical Instruments, Beijing, China) at 540 nm, referencing a pre-established standard curve. The 1 L round-bottomed flasks with 150 mL of apple pomace hydrolysates enriched with 7 g/L of diammonium hydrogen phosphate ((NH₄)₂HPO₄) were inoculated using previously prepared seeding suspensions of Scheffersomyces stipitis or Meyerozymea guilliermondii at a dose of 1 g of cell DM per 1 L of medium. The initial pH was 5.2 ± 0.1. The flasks were then plugged with cotton wool. The cultivation was carried out at 32 ± 1 °C for 48 h on a reciprocal laboratory shaker operating at 140 oscillations per minute (Eberbach E5900, Belleville, NJ, USA). After culture, 7 mL of post-culture suspension was centrifuged at 5000× g for 10 min. The supernatant was collected for HPLC analysis.

2.6. Yeast Cell Biomass Concentration

The dry matter (DM) content of the yeast cells in the medium was determined using a spectrophotometer (Rayleigh Analytical Instruments, Beijing, China) at 540 nm, referencing a pre-established standard curve. The biomass yield Yx/s was expressed as DM g of biomass obtained per g of substrates (sum of sugars and galacturonic acid) present in the specific medium before hydrolysis

2.7. HPLC Analysis of Hydrolysates and Post-Cultivation Effluents

The concentrations of glucose (GLU), arabinose (ARA), xylose (XYL), cellobiose (CEL), galacturonic acid (GA), citric acid (CA), succinic acid (SA), lactic acid (LA), formic acid (FA), acetic acid (AA), glycerol (GOH), and ethanol (EOH) in the medium were determined using HPLC (Agilent 1260 Infinity, Agillent Technologies, Santa Clara, CA, USA) equipped with a Hi-Plex H column (7.7 mm × 300 mm, 8 μm) (Agilent Technologies, Santa Clara, CA, USA) and a refractive index detector (RID) working at 55 °C. The column temperature was maintained at 80 °C. HPLC-grade 0.005 M H₂SO₄ was used as the mobile phase with a flow rate of 0.7 mL/min and a sample volume of 20 μL. Prior to analysis, the samples were treated with ZnSO₄ at a 10% concentration to induce protein precipitation. The sediments were removed by centrifugation at 7000 rpm for 10 min. The samples were filtered through 0.45 μm Teflon membranes before analysis.

2.8. Number of Samples and Statistical Data Treatment

All assays were conducted in triplicate. Statistical analysis, including variance analysis, standard deviation determination, and Student’s t-test at a significance level of α = 0.05, was performed using Origin 7.5 software.

2.9. Sample Designations

• W—sample after aqueous pretreatment;
• A—sample after pretreatment with 2% sulphuric acid;
• WE50—sample after aqueous pretreatment and enzymatic hydrolysis at 50 °C;
• AE50—sample after pretreatment with 2% sulphuric acid and enzymatic hydrolysis at 50 °C;
• WE30—sample after aqueous pretreatment and enzymatic hydrolysis at 30 °C;
• AE30—sample after pretreatment with 2% sulphuric acid and enzymatic hydrolysis at 30 °C;
• MgWE50—effluent after M. guilliermondii cultivation in the hydrolysate after aqueous pretreatment and enzymatic hydrolysis at 50 °C;
• MgAE50—effluent after M. guilliermondii cultivation in the hydrolysate after pretreatment with 2% sulphuric acid and enzymatic hydrolysis at 50 °C;
• MgWE30—effluent after M. guilliermondii cultivation in the hydrolysate after aqueous pretreatment and enzymatic hydrolysis at 30 °C;
• MgAE30—effluent after M. guilliermondii cultivation in the hydrolysate after pretreatment with 2% sulphuric acid and enzymatic hydrolysis at 30 °C;
• SsWE50—effluent after S. stipitis yeast cultivation in the hydrolysate after aqueous pretreatment and enzymatic hydrolysis at 50 °C;
• SsAE50—effluent after S. stipitis yeast cultivation in the hydrolysate after pretreatment with 2% sulphuric acid and enzymatic hydrolysis at 50 °C;
• SsWE30—effluent after S. stipitis yeast cultivation in the hydrolysate after aqueous pretreatment and enzymatic hydrolysis at 30 °C;
• SsAE30—effluent after S. stipitis yeast cultivation in the hydrolysate after pretreatment with 2% sulphuric acid and enzymatic hydrolysis at 30 °C.

3. Results and Discussion

3.1. The Basic Parameters of the Apple Pomace

The basic parameters of the apple pomace used in our experiments are presented in

Table 1. Dry matter and basic components of AP used in our experiments.

	Value
Dry matter [%]	90.68 ± 2.7
Cellulose [% DM]	15.42 ± 0.3
Hemicellulose [% DM]	12.45 ± 0.09
Lignin [% DM]	15.23 ± 0.12

.

The apple pomace contains residual sugars from the original apples. However, the critical components for the overall bioconversion into SCP are the polymerised forms of sugars such as cellulose and hemicellulose. The resulting dry matter content of the raw material (90.68 ± 2.7%) is close to that reported in the literature [17,18,45,46]; however, it must be noted that these values are specific to the climate, soil, apple cultivar, processing method, and storage conditions. The cellulose and hemicellulose content in the AP used in our experiments was 15.42 ± 0.3 g/L and 12.45 ± 0.09 g/L, respectively. According to the literature [17,67,68], the cellulose content in apple pomace varies from 12.7% to 21.22%. In comparison, the hemicellulose content falls within the range of 7.2–14.74%, and these results support the data presented in Table 1. The lignin content obtained by us (15.23 ± 0.12%) is lower than reported by Gama et al. (19.8%) [69] but correlates with the values presented by others (15.2–23.5%) [67,68,70,71]. The sum of the assayed cellulose, hemicellulose, and lignin makes up less than half of the dry matter of the analysed apple pomace. Therefore, based on the literature sources mentioned earlier, it can be inferred that the remaining portion includes pectin and some simple sugars like fructose and glucose, as well as organic acids and mineral constituents from the apple tissue. These compounds were not specifically assayed at this stage of our experiment due to its design; however, they were analysed in more detail in the apple-pomace-derived hydrolysates (e.g., pectin derivative—galacturonic acid was assayed).

3.2. Sugars, Organic Acids, and Alcohols in the AP Hydrolysates

Table 2. Sugars and galacturonic acid content in the AP hydrolysates.

Sample	CEL	GLU	XYL	ARA	GA
	[g/L]
W	0.081 ± 0.003	0.06 ± 0.0032	0.21 ± 0.0052	0.131 ± 0.0038	0.315 ± 0.0029
A	0.91 ± 0.052	7.258 ± 0.1798	7.19 ± 0.3013	5.659 ± 0.1159	0.947 ± 0.0272
WE50	0.787 ± 0.0291	18.55 ± 0.6058	5.76 ± 0.1958	5.348 ± 0.0665	7.013 ± 0.3713
AE50	1.44 ± 0.0246	18.325 ± 0.8415	7.61 ± 0.2352	5.592 ± 0.17	6.23 ± 0.3561
WE30	1.228 ± 0.0365	19.311 ± 0.8692	4.96 ± 0.1071	5.15 ± 0.2943	6.785 ± 0.277
AE30	1.485 ± 0.0583	14.866 ± 0.6147	7.92 ± 0.2332	5.669 ± 0.3002	6.438 ± 0.2378

,

Table 3. Organic acid content in the AP hydrolysates.

Sample	CA	SA	LA	FA	AA
	[g/L]
W	0.185 ± 0.007	0.127 ± 0.0016	1.27 ± 0.0415	0.067 ± 0.0019	0.262 ± 0.0043
A	0.043 ± 0.0023	0.091 ± 0.0038	1.2 ± 0.0098	0.083 ± 0.001	1.115 ± 0.0374
WE50	0.184 ± 0.0082	0.054 ± 0.0011	1.18 ± 0.0096	0.042 ± 0.001	0.905 ± 0.0275
AE50	0.085 ± 0.0026	0.062 ± 0.0018	1.11 ± 0.024	0.056 ± 0.002	1.141 ± 0.0244
WE30	0.215 ± 0.0063	0.007 ± 0.0003	1.19 ± 0.0541	0.045 ± 0.0018	0.858 ± 0.0337
AE30	0.101 ± 0.003	0.09 ± 0.0015	1.24 ± 0.061	0.065 ± 0.0023	1.178 ± 0.0386

and

Table 4. Glycerol and ethanol content in the AP hydrolysates.

Sample	GOH	EOH
	[g/L]
W	0.679 ± 0.0249	0.036 ± 0.0013
A	0.678 ± 0.0206	0.011 ± 0.0004
WE50	0.742 ± 0.0061	0.01 ± 0.0003
AE50	0.74 ± 0.0255	0.012 ± 0.0005
WE30	0.687 ± 0.0216	0.022 ± 0.0009
AE30	0.775 ± 0.0249	0.056 ± 0.0015

show the content of selected simple sugars, organic acids, glycerol, and ethanol in the AP hydrolysates.

Our experiment began with the pretreatment of apple pomace, conducted at a temperature of 121 °C for 1 h, using water or 2% H₂SO₄ as medium. Comparing the data in Table 2 between sample A (acid-assisted pretreatment) and sample W (water used as a medium), it is evident that pretreatment with 2% sulphuric acid released substantial amounts of free sugars from the raw material, particularly glucose (7.258 g/L), xylose (7.19 g/L) and arabinose (5.659 g/L). The sulphuric acid also catalysed the release of cellobiose from the lignocellulosic matrix; 0.91 g/L of this sugar was detected after acid pretreatment, while only 0.081g/L of cellobiose was found in the reference sample, where water was used as the medium. Additionally, significant amounts of galacturonic acid were observed in the pretreated samples: 0.315 g/L in the water-treated sample (W) and three times higher at 0.947 g/L in the acid-assisted pretreatment (sample A).

The aqueous pretreatment did not significantly affect the amount of sugars available. However, the primary purpose of pretreatment is to modify the structure of the raw material to enhance enzymatic accessibility. The release of sugars during pretreatment can sometimes be disadvantageous, as it hinders the removal of the reactants used in the pretreatment stage, leading to a loss of the already released monosaccharides.

Looking at the results for the WE and AE samples, obtained after enzymatic hydrolysis at 30 °C or 50 °C, an increase in the content of all sugars and galacturonic acid was observed compared to the W and A samples (before enzymatic hydrolysis). The cellobiose content at this stage ranged from 0.787 g/L to 1.485 g/L, with the lowest value in the WE50 sample, and the highest in the acid-pretreated sample hydrolysed at 30 °C. Glucose content ranged from 14.866 g/L (in the sample obtained after 2% acid pretreatment and enzymatic hydrolysis at 30 °C) to 19.311 g/L (WE30 sample). After enzymatic hydrolysis, the amount of xylose in the samples ranged from 4.96 g/L (sample WE30) to 7.92 g/L (AE30 sample). The amount of arabinose observed in the WE and AE samples ranged from 5.15 g/L for the WE30 sample to 5.669 g/L for the AE30 sample. It can be seen that 2% sulphuric acid significantly supported the decomposition of the apple pomaces matrix. However, we predicted that the general increase in the efficiency of enzymatic hydrolysis would be observed for samples pretreated with sulphuric acid instead of water (for the exact temperature); in the results of the statistical comparison (including the variance and p-value comparison) of WE30-AE30 and WE50-AE50, some of the results for specific sugars do not support such a general conclusion. For example, the glucose concentration in the WE30 sample was significantly higher p < 0.05 (19.311 ± 0.8692 g/L) than in the AE30 sample (14.866 ± 0.6147 g/L). For the same sugar, the concentrations in WE50 and AE50 were 18.55 ± 0.6058 g/L and 18.325 ± 0.8415 g/L, respectively, and the difference was not statistically significant (p > 0.05). However, the specific sugar concentrations are the results of the unique combination of substrate characteristics and the hydrolysis course; the considerable concentration, promising for yeast yield, was that the galacturonic acid, the derivative of pectins present in the AP, and reported also by others [69,70,71].

The purpose of conducting enzyme hydrolysis at a temperature of 30 °C, which is approximately 20 °C below the optimal for the preparation of Cellic CTec2 [72], was to investigate whether performing this stage at 50 °C is necessary for maximising hydrolysis efficiency and achieving the final SCP yield. These temperatures are not tolerated by yeast cells, making it impossible to implement a time- and energy-saving process of simultaneous saccharification and SCP cultivation. However, the results presented in Table 2 suggest promising prospects for this approach, as the sugar concentrations released during the hydrolysis of pretreated AP samples at 30 °C are comparable to those obtained at 50 °C.

The final concentrations of specific sugars after pretreatment and enzymatic hydrolysis are not only influenced by the efficiency of the enzymes but also by the substrate load ratio (SL) (substrate: working liquid) used. In our study, this parameter was close to 10% (w/v). Gama et al. reported a glucose content in the AP hydrolysate up to 4 g/L but for 2% SL (w/v), which is a lower value than 19.311 ± 0.8692 g/L of glucose obtained in our WE30 sample. However, Magyear et al. [73] reported glucose concentrations of up to 30 g/L in hydrolysates obtained with a mild sulphuric acid solution at SL ratios of 20–30%. Our findings regarding sugar content in hydrolysates are also supported by Vaez et al. [74]. In our studies, approximately 750 mL of liquid hydrolysate was obtained from 100 g of apple pomace (AP), indicating that approximately 133 g of AP was used to obtain 1 L of hydrolysate. Comparing this with the total sugars and galacturonic acid concentrations presented in Table 2, it appears that for the AE50 hydrolysate (39.197 g/L), approximately 29.47% of the AP was converted into the analysed sugars and galacturonic acid. In contrast, only about 0.6% of the raw material for the W sample was hydrolysed into the analysed components presented in Table 2.

The citric acid content in the samples after aqueous pretreatment (Table 3) was 0.188 g/L, which is more than four times higher than in the sample after acid pretreatment—0.043 g/L. Following enzyme treatment, the citric acid content ranged from 0.085 g/L to 0.215 g/L, depending on the enzymatic hydrolysis variant. The highest content of citric acid in the enzymatic hydrolysates was observed in the WE30 sample (0.215 g/L), while the lowest was in the AE50 sample (0.085 g/L). Additionally, it was noted that in samples subjected to acid pretreatment and enzyme hydrolysis, the citric acid content was at least twice as high as in the initial sample before enzyme treatment. For instance, in sample A, the citric acid concentration was 0.043 g/L, compared to 0.085 g/L in the AE50 hydrolysate and 0.101 g/L in the AE30 sample.

The amount of succinic acid in the samples after pretreatment varied; for aqueous pretreatment (W sample), it was highest at 0.127 g/L, and for acid pretreatment (A), highest at 0.091 g/L. After enzymatic hydrolysis, the succinic acid content of the samples ranged from 0.007 g/L to 0.09 g/L. The lowest content of this compound in the enzymatic hydrolysates was observed in the WE30 sample and the highest in the AE30 sample. In the case of succinic acid, it was noted that, with aqueous pretreatment, the presence of this acid is always lower in the subsequent enzymatic hydrolysate, regardless of the enzymatic hydrolysis temperature.

After pretreatment of AP with water (W) and acid (A), 1.27 g/L and 1.20 g/L of lactic acid were obtained in the medium, respectively. Enzymatic hydrolysis slightly affected the presence of this acid in the samples, and its concentration ranged from 1.11 g/L (AE50 sample) to 1.24 g/L (AE30).

The formic acid (FA) content in the pretreatment samples was, for the aqueous treatment, equal to 0.067 g/L, and for the acid treatment, it was 0.083 g/L. In the samples after enzymatic hydrolysis, the amount of this acid decreased and fluctuated between 0.042 g/L and 0.065 g/L. As formic acid is also a product of the decomposition of sugars during acid-thermal treatment, higher contents of this compound were observed for samples after such treatment. The dependence of FA concentrations between the hydrolysate samples was as follows: AE30 > AE50 > WE40 > WE50. The raw materials and products derived from apples are rich in organic acids; therefore, the results of Table 2 are supported by the other data [8,75,76,77,78].

In the aqueous pretreatment sample, an acetic acid content of 0.262 g/L was recorded, more than five times lower than that in the acid pretreatment sample of 1.115 g/L. In the samples after enzymatic hydrolysis, the citric acid content ranged from 0.858 g/L (WE30 sample) to 1.141 g/L for the AE50 hydrolysate. Higher values of more than 1 g/L were observed for samples after acid pretreatment: AE30—1.178 g/L and AE50—1.141 g/L. The literature states that the elevated temperature of the lignocellulosic matrix treatment favours some organic acid formation, such as acetic or formic acid; our experiments also observed this phenomenon. The negative effect of elevated organic acids concentration, usually greater than 1 g/L, on yeast metabolism was also widely reported [79,80,81,82,83,84,85,86].

Nonsignificant differences (p > 0.05) in glycerol contents (Table 4) were observed for the W and A samples of pretreated AP (0.668 ± 0.0206 g/l and 0.679 ± 0.0249 g/L, respectively). After the enzymatic hydrolysis stage, the highest glycerol content of 0.775 g/L was determined in the sample after sequential pretreatment with 2% sulphuric acid and enzymatic hydrolysis at 30 °C. In enzymatic hydrolysates, the glycerol content increased from initial values close to 0.678 g/L by approximately 0.06–0.1 g/L.

The ethanol content results (Table 4) show trace amounts of this compound at all stages of hydrolysate preparation. In the pretreatment stage with both water and acid, by enzymatic hydrolysis, the ethanol content of the samples oscillated between 0.011 g/L and 0.036 g/L, with the highest value reaching sample (A) after aqueous pretreatment of the raw material.

3.3. Sugars, Organic Acids, and Alcohols in the Post-Cultivation Effluents

Table 5. Sugars and galacturonic acid content in the post-cultivation effluents.

Sample	CEL	GLU	XYL	ARA	GA
	[g/L]
MgWE30	0.411 ± 0.0218	0.01 ± 0.0002	0.04 ± 0.0018	0.433 ± 0.0073	3.307 ± 0.0722
MgAE30	0.685 ± 0.0117	n.d.	0.04 ± 0.0016	0.376 ± 0.0142	6.506 ± 0.1486
MgWE50	0.358 ± 0.0119	0.01 ± 0.0002	0.04 ± 0.001	0.634 ± 0.0157	5.733 ± 0.1525
MgAE50	0.061 ± 0.0027	0.131 ± 0.0032	0.11 ± 0.0036	0.04 ± 0.0013	1.707 ± 0.0725
SsAE30	0.821 ± 0.0213	n.d.	0.01 ± 0.0002	0.01 ± 0.0004	6.166 ± 0.0897
SsWE30	0.118 ± 0.0043	0.111 ± 0.0029	0.06 ± 0.0027	0.02 ± 0.0007	3.141 ± 0.0916
SsWE50	0.02 ± 0.0001	0.151 ± 0.0025	0.05 ± 0.002	0.02 ± 0.0005	1.972 ± 0.06
SsAE50	0.427 ± 0.0177	0.149 ± 0.0037	n.d.	0.04 ± 0.0009	1.366 ± 0.0627

n.d.—not detected.

,

Table 6. Organic acid content in the post-cultivation effluents.

Sample	CA	SA	LA	FA	AA
	[g/L]
MgWE30	0.022 ± 0.0008	0.012 ± 0.0004	0.006 ± 0.0002	0.074 ± 0.0027	0.002 ± 0.0001
MgAE30	0.051 ± 0.0021	0.094 ± 0.0035	0.011 ± 0.0004	0.045 ± 0.002	0.004 ± 0.0001
MgWE50	n.d.	0.01 ± 0.0003	0.029 ± 0.0014	0.127 ± 0.0057	0.004 ± 0.0002
MgAE50	0.007 ± 0.0002	0.053 ± 0.0017	0.03 ± 0.0009	0.09 ± 0.0037	0.006 ± 0.0001
SsAE30	0.066 ± 0.0016	0.057 ± 0.0023	0.021 ± 0.001	0.102 ± 0.0042	0.002 ± 0.0001
SsWE30	0.058 ± 0.0026	0.049 ± 0.0004	0.024 ± 0.0006	0.097 ± 0.0039	0.009 ± 0.0004
SsWE50	0.054 ± 0.0205	0.045 ± 0.0015	0.009 ± 0.0002	0.057 ± 0.0012	0.023 ± 0.0006
SsAE50	0.013 ± 0.0002	0.031 ± 0.0004	0.032 ± 0.0011	0.134 ± 0.0039	0.012 ± 0.0005

n.d.—not detected.

and

Table 7. Glycerol and ethanol content in the post-cultivation effluents.

Sample	GOH	EOH
	[g/L]
MgWE30	n.d.	n.d.
MgAE30	n.d.	0.021 ± 0.0006
MgWE50	n.d.	0.014 ± 0.0005
MgAE50	n.d.	0.061 ± 0.0023
SsAE30	n.d.	0.02 ± 0.0008
SsWE30	0.008 ± 0.0005	0.019 ± 0.0009
SsWE50	0.024 ± 0.001	0.062 ± 0.0011
SsAE50	n.d.	0.113 ± 0.0019

n.d.—not detected.

show the content of selected simple sugars, organic acids, glycerol, and ethanol in the AP hydrolysates.

Considering the presence of the acids analysed in the post-culturing effluents from the yeast cultures M. guilliermondii and S. stipitis (Table 6), only trace amounts of citric acid were observed in the range of 0.000–0.066 g/L. The succinic acid concentration oscillated between 0.01 g/L (MgWE50 sample) and 0.094 g/L for the MgAE30 sample. The presence of lactic acid in the culture leachate was at a level of 0.006 g/L (MgWE30)–0.032 g/L (SsAE50). The formic acid content varied considerably in the post-culturing leachates, ranging from 0.045 g/L in the MgAE30 sample to 0.134 g/L for the SsAE50 effluent. Only traces of acetic acid between 0.002 g/L and 0.023 g/L were determined in culture leachates, compared to the 0.858–1.178 g/L observed for the AE and WE hydrolysates before cultivation.

Analysis of the acid concentration results before and after cultivation (Table 6) showed that citric, lactic, and acetic acid concentrations decreased during cultivation. In contrast, such consistency was not observed for succinic and formic acid. To evaluate the organic acid balance before and after cultivation, it must be remembered that the final concentration of these acids results from the possible activity of yeast strains and the undesired bacteria from the possible contaminations.

Samples after the cultivation of the yeast Meyerozyma guilliermondii and Scheffersomyces stipitis contained trace amounts of glycerol (0.024 g/L or less)—Table 7. After the culture of Meyerozyma guilliermondii cells, the highest ethanol concentration was recorded in sample MgAE50 (0.061 g/L). After the culture of yeast Scheffersomyces stipitis, the highest alcohol content was obtained in sample SsAE50 (0.113 g/L). The low ethanol results obtained in the post-culture effluents may suggest that the yeast carried out the expected process of efficient biomass culture rather than alcoholic fermentation. The observation of a reduction in glycerol concentration during culture is also promising, probably because it was assimilated by the yeast cells and used for energy and building cell mass.

3.4. The Results of the Yeast SCP Cultivation in the AP Hydrolysates

The last but most important results of our experiment are presented in

Table 8. Yeast biomass concentration.

Sample	Pretreatment Method	Temperature of Enzymatic Hydrolysis [°C]	Yeast Strain	Biomass Concentration [g DM/L]
MgWE50	water	50	Meyerozyma guilliermondii	8.55 ± 0.28
MgAE50	2% acid	50		16.29 ± 0.65
MgWE30	water	30		9.12 ± 0.48
MgAE30	2% acid	30		12.69 ± 0.53
SsWE50	water	50	Scheffersomyces stipitis	10.71 ± 0.23
SsAE50	2% acid	50		14.63 ± 0.36
SsAE30	2% acid	30		9.82 ± 0.14
SsWE30	water	30		13.03 ± 0.19

, where the biomass concentration after yeast cell cultivation in apple pomace hydrolysates is shown.

The final biomass concentration varied between 8.55 g/L and 16.29 g/L, depending on the hydrolysate and strain. For all samples, for yeast S. stipitis and M. guilliermondii, more than 25% higher biomass multiplication was observed for hydrolysates obtained after acid pretreatment. It was observed that the M. guilliermondii strain multiplied in the hydrolysed pomace more favourably than the S. stipitis cells did. The highest biomass content was recorded for the MgAE50 sample (16.29 yeast g DM/L) when the M. guilliermondii strain was cultivated in the medium prepared from apple pomace pretreated with 2% sulphuric acid and the subsequent enzymatic hydrolysis at 50 °C. This method of media preparation was also the most successful option for S. stipitis cells, where the highest concentration of the biomass was observed in the sample SsAE50 at 14.63 g DM/L. However, while the use of 2% acid is considered acceptable from the eco-point of view, the result obtained for AP pretreated only with water and Scheffersomyces stipitis strain (SsWE30) 13.03 ± 0.19 g/L seems a very promising perspective toward the development of industrial-scale technology not based on the acid use and the subsequent need for its neutralisation and salt-containing post-cultivation effluent generation. Our results confirm enzymatic hydrolysis as desirable from a process efficiency point of view but disadvantageous from an economic point of view. Unfortunately, the combination of efficient enzymatic hydrolysis and economic viability has been the bottleneck of industrial implementations of biomass and ethanol production for decades.

It must be mentioned that even if the assimilation patterns of the basic compound are similar for both strains, the final concentrations of biomass depend on many parameters such as strain characteristics, media composition (i.e., initial raw material, sugar content, inhibitors presence, pH, vitamins), the temperature, and the cultivation period; thus, the final concentration of the biomass is subject to significant fluctuations [60,61,87]. When comparing literature data, it should also be recognised that within individual species, different strain varieties may differ from the base strain in assimilating particular compounds and resistance to inhibitors present in lignocellulosic hydrolysates. All this can translate into substantial differences in biomass and metabolite production.

Over 30 g/L of sugars were present in the hydrolysates obtained from AP; however, the final biomass yield differed significantly depending on the hydrolysis method. Lower yields were observed for the hydrolysates obtained after pretreatment with only water. This may be the result of the possible presence of non-estimated inhibitors. Values of biomass yield expressed as the amount of biomass per consumed sugars varied from 0.274 g/g for the MgWE30 sample to 0.441 g/g for the MgAE30 sample (Figure 1); this indicates that at least 40% of accessible sugars were converted into SCP DM, which in light of literature data for lignocellulosic hydrolysates and the similarity to our strains (Scheffersomyces sp., Candida sp.) [53,54], may be considered a high and promising result. What is noteworthy is that in today’s yeast factories, this value typically does not exceed 50% (0.5 g/g) [47]. During M. guilliermondii cultivation in sugar cane bagasse hydrolysate, Molwitz et al. [62] noted 24.4 g/L of yeast DM in a medium containing around 60 g/L of xylose and glucose. Roberto et al. [63], and after cultivating the same genus strain in rice straw hydrolysates containing about 60 g/L of assimilable sugar, 7 g/L of yeast biomass was observed. Similar values were reported by Matos et al. [88] (Y_x/s yield 0.52 g/g corresponding to 25.26 DM g/L for M. guilliermondii isolated from Amazonian termites) and Da Silva et al. [64], corresponding to 22 g/L after cultivation of M. guilliermondii strain in sugarcane bagasse hydrolysate. For the S. stipitis strain cultivated in brewers’ spent grain hydrolysate, a Y_x/s equal to 0.46 g/g was noted by Duarte et al. [89]. After converting potato peels and sugar beet bagasse, 8.6 g/L and 8–10 g/L of S. stipitis biomass were obtained by Patelski and collaborators [50,58]. The results of the conversion of sugars to biomass obtained within the framework of the present work are, from an economic point of view, promisingly higher than those presented by Joshi and Bhusan [90] for the S. cerevisiae strain cultured in apple pomace hydrolysate (0.23–0.35 g/g). Papini et collaborators [60] reported for aerobic batch cultivations in minimal media containing 20 g/L of glucose, that the sugar-to-biomass conversion ratio was 0.17 g/g for the S. cerevisiae strain and 0.55 g/g for the S. stipitis strain. A Y_x/s exceeding 0.5 g/g should be considered very high since, except for biomass creation, the sugars assimilated are also used for energy creation, and a wide variety of metabolic by-products are also formed. For bagasse hydrolysate and M. guilliermondii strain, Martini et al. [65] noted a 0.08–0.17 g/g conversion ratio for glucose and 0.13–0.19 g/g for arabinose during 60 h of the cultivation process.

4. Summary and Conclusions

The objective of this study was to evaluate the conversion of polysaccharides in apple pomace into components suitable for yeast culture media and their subsequent conversion into yeast biomass. The results obtained demonstrate that aqueous or acid pretreatment followed by enzymatic hydrolysis using a commercially available enzyme preparation effectively released significant amounts of monosaccharides from the raw material, facilitating yeast cell multiplication. The sugar-to-biomass conversion ratio exceeded 0.4 g/g for Meyerozyma guilliermondii. Acid pretreatment proved more efficient than water treatment under identical time and temperature conditions for AP. Meyerozyma guilliermondii cells exhibited more efficient multiplication in AP hydrolysates compared to S. stipitis cells. Enzymatic treatment significantly enhanced monosaccharide release from both water- and acid-pretreated apple AP. Overall, our process converted up to 12.24% of AP mass into yeast biomass. These findings suggest that large quantities of apple pomace can be efficiently converted into single-cell protein (SCP) using Meyerozyma guilliermondii and Scheffersomyces stipitis yeast strains.