3.2 Methane production with BSG pretreated by hydrothermolysis, severity 4.2
The range of BSG hydrothermolysis temperature (150 to 210 °C) and time (10 to 20 minutes) according to the central composite design (CCD), showed maximum methane potential production (P) from 235.0 ± 10.1 to 411.6 ± 7.2 mL. g-1 STV under different pretreatment severities (from 2.4 to 4.5) (Figure S1 and Table S2, Supplementary Material). Optimal severity of 4.2 for P (411.6 ± 7.2 mL. g-1 STV) was confirmed in assay B2, with methane production rate (Rm) of 2.1 ± 0.16 mL.g-1 STV .h, the condition in which total carbohydrates and organic matter removal was verified (90.4% and 81.1%, respectively) (Figure S3 and S4, Supplementary Material). In B1 and B3, with BSG under lower hydrothermolysis severities (2.5 and 2.8, respectively), lower values of Rm (0.9 ± 0.06 and 0.8 ± 0.14 mL.g-1 STV. h, respectively), as well as of P (250.0 ± 9.9 and 235.0 ± 10.1 mL.g-1STV, respectively) were obtained, suggesting mild changes in the lignocellulosic matrix. Under severity of 4.5 (B4), P of 333.0 ± 11.3 mL. g-1 STV and Rm of 0.8 ± 0.03 mL.g-1 STV.h, were observed, highlighting that the increase in hydrothermolysis severity from 4.2 implied damage to BSG anaerobic digestion (Figure S4, Supplementary Material). Acetic acid consumption, verified in all assays and (54.2 to 92.0%) simultaneous to methane formation, evidences the predominance of acetoclastic methanogenesis in these conditions, where there was also consumption of butyric (43.0 to 96.6%) and propionic acids (3.8 to 69.6%) (Figure S2, Supplementary Material).
In light of this, it was decided to continue this study with pretreated BSG by hydrothermolysis under severity of 4.2 (210 °C for 10 min). When the hydrothermolysis effects on BSG structure was evaluated under the above-mentioned condition, there was no significant change on the lignin content of the material (29.1%) when compared to BSG in natura (26.5%). However, after the pretreatment, an increase of total carbohydrates solubilization from 33.5% (BSG in natura) to 53.5% (pretreated BSG) was confirmed, as well as morphological modification on the structure of BSG fibers, verified by Digital Scanning Microscope (DSM) (Figure S5, Supplementary Material).
Kinetic parameters P, Rm and λ were obtained (Table 2) under different conditions of operation temperature (30 to 60 °C), pretreated BSG concentration (7.3 to 20.7 g.L-1) and hydrolysate volume (0.6 to 12.4 mL). In relation to P, the greater results were obtained for C13 and C3, with 305.8 ± 7.79 and 286.6 ± 3.47 mL.g-1.STV, respectively. In these assays, the start time of methane production (λ) were similar, 16.5 ± 7.4 and 14.3 ± 7.83 hours, Rm of 1.7 ± 0.1 and 1.7 ± 0.2 mL. g-1 STV.h, respectively. In C13 and C3, lower hydrolysate volumes were added, 0.6 and 3.0 mL, respectively, supporting the negative effect of this variable.
The effect of hydrolysate addition on P can also be observed when comparing assays C13, C14, C15, C16 and C17, with 14 g BSG.L-1, operated at 45 °C and with different hydrolysate volumes. In C13, with 0.6 mL of hydrolysate, 305.8 ± 7.79 mL CH4.g-1.STV was produced. Concurrently to the increase of hydrolysate volume to 6.5 mL (C15, C16 and C17) and 12.4 mL (C14), there was a decrease of 32.4 and 38.3% in maximum methane production, respectively. Hydrolysate produced after hydrothermolysis may contain inhibiting compounds, like furfural and 5-HMF, therefore the microbial community requires a longer adaptation period [30], which may have compromised the BSG anaerobic digestion, causing lower methane production in C14, C15, C16 and C17 compared to C13.
After performing assays with concentrations of 0.4, 0.8 and 2.0 g.L-1of furfural and 5-HMF, [17] reported total inhibition of methanogenesis with 2.0 g.L-1of both aldehydes, and partial inhibition with 0.8 g.L-1of 5-HMF, under mesophilic (35 °C) and thermophilic (55 °C) conditions. According to the authors, the thermophilic microbial consortium was more sensitive to the increase of intermediate compound concentrations released from the hydrolysis of lignocellulosic biomass when compared to mesophilic consortium, characterized by reduced methane production. These results corroborate those verified in this study, where assays with lower hydrolysate volumes (0.6 and 3.0 mL) and lower concentrations of compounds derived from BSG lignocellulosic disruption, were favorable conditions to anaerobic digestion, since there was greater methane production (305.8 ± 7.79 and 286.6 ± 3.47 mL.g-1.STV).
Table 2 Methane production (P), methane production rate (Rm) and time to start methane production (λ) from BSG pretreated under various methanogenic conditions
Assays
|
Conditions
|
Methane
|
|
T
(°C)
|
BSG
(g.L-1)
|
Hyd.
(mL)
|
P
(mL.g-1STV)
|
Rm
(mL.g-1STV.h)
|
λ
(h)
|
R2
|
C1
|
36
|
10
|
3
|
189.9 ± 3.2
|
4.1 ± 0.4
|
1.7 ± 2.2
|
0.97
|
C2
|
54
|
10
|
3
|
155.0 ± 2.2
|
1.8 ± 0.1
|
7.0 ± 2.6
|
0.99
|
C3
|
36
|
18
|
3
|
286.6 ± 3.5
|
1.7 ± 0.2
|
14.3 ± 7.4
|
0.98
|
C4
|
54
|
18
|
3
|
182.9 ± 2.4
|
1.8 ± 0.1
|
7.7 ± 2.3
|
0.99
|
C5
|
36
|
10
|
10
|
165.0 ± 4.0
|
1.8 ± 0.2
|
6.2 ± 4.5
|
0.99
|
C6
|
54
|
10
|
10
|
187.0 ± 5.0
|
1.5 ± 0.1
|
1.2 ± 4.6
|
0.97
|
C7
|
36
|
18
|
10
|
186.9 ± 5.4
|
1.5 ± 0.1
|
11.6 ± 4.4
|
0.98
|
C8
|
54
|
18
|
10
|
145.1 ± 5.2
|
0.8 ± 0.1
|
22.5 ± 5.2
|
0.98
|
C9
|
30
|
14
|
6.5
|
182.6 ± 3.5
|
2.0 ± 0.2
|
40.8 ± 3.6
|
0.98
|
C10
|
60
|
14
|
6.5
|
108.5 ± 2.0
|
1.5 ± 0.1
|
51.8 ± 2.9
|
0.99
|
C11
|
45
|
7.3
|
6.5
|
203.2 ± 1.8
|
2.1 ± 0.1
|
37.4 ± 1.9
|
0.99
|
C12
|
45
|
20.7
|
6.5
|
267.9 ± 3.2
|
2.3 ± 0.1
|
8.8 ± 2.5
|
0.99
|
C13
|
45
|
14
|
0.6
|
305.8 ± 1.8
|
1.7 ± 0.1
|
16.5 ± 7.8
|
0.99
|
C14
|
45
|
14
|
12.4
|
188.8 ± 3.3
|
2.2 ± 0.2
|
48.8 ± 3.5
|
0.97
|
C15
|
45
|
14
|
6.5
|
206.8 ± 3.3
|
2.7 ± 0.2
|
4.7 ± 3.2
|
0.98
|
C16
|
45
|
14
|
6.5
|
203.8 ± 3.0
|
2.4 ± 0.1
|
6.7 ± 2.6
|
0.99
|
C17
|
45
|
14
|
6.5
|
210.2 ± 2.6
|
3.5 ± 0.2
|
8.2 ± 2.0
|
0.99
|
*T: Temperature, BSG: Brewery spend grain concentration, Hyd: hydrolisate volume
Lower P was observed in C10 (108.5 ± 2.0 mL. g-1 STV), where 14 g BSG.L-1 and 6.5 mL of hydrolysate were added, operated at 60 °C, evidencing the negative effect of high temperature on microbial consortium. Additionally, higher λ was found in the assays, of 51.8± 2.9 hours and Rm of 1.50 ± 0.13 mL.g-1 STV.h, demonstrating the difficult microbiota adaptation to 60 °C. Similarly, Kim et al. [31] related the temperature influence on methane production from food wastes in reactors inoculated with mesophilic sludge, showing greater stability and methanogenic potential at 35 °C (230 mL.g-1 VS) when compared to 55 °C (170 mL.g-1 VS). For the same substrate, Zamanzadeh et al. [32] also observed better methane production (480 mL.g-1SV) at 37 °C when compared to reactors operated at 55 °C (448 mL.g-1SV). Various authors have reported superior performance of fermentative-methanogenic reactors conducted at mesophilic temperature in relation to thermophilic temperature, mainly when inoculated with a mesophilic microbial consortium, which may be related to the reduced adaptation ability of microbial groups to wide-ranging temperature variations [33, 34]. For Chen and Chang [35], the ideal temperature range for the methanogenesis stage is from 35 to 42 °C, and an increase to 55 °C can especially inhibit the activity of methanogenic archaea, even after the acclimatization of mesophilic inoculum at 55 °C for 13 days. Thus, in this study, the methanogenic potential of the inoculum from UASB operated at mesophilic temperature was affected in reactors conducted at thermophilic temperature, probably due to the greater abundance of protein denaturation of mesophilic bacteria in the inoculum.
With regard to soluble metabolites in C1- C17, acetic acid concentrations were verified in the initial samples, from 35.0 to 301.1 mg.L-1 (Figure 1). Disruption of the lignocellulosic structure by hydrothermolysis contributed to the acetic acid content in the initial samples, since this acid is a product from hemicellulose degradation. In fact, it was confirmed that there was 93.0 ± 14.1 mg.L-1 of acetic acid in the hydrolysate from BSG hydrothermolysis, which surely contributed to the respective initial concentrations. Thus, the assays with higher acetic acid concentrations in the initial sample were C7 (279.0 mg.L-1) and C14 (301.1 mg.L-1) with 10 and 12.4 mL of hydrolysate, respectively.
During the exponential phase of methane production (Figure 1 II), there were expressive concentrations of propionic (165.4 to 847.3 mg.L-1), butyric (175.8 to 966.62 mg.L-1), valeric (43.0 to 255.8 mg.L-1), isovaleric (12.3 to 105.4 mg.L-1), isobutyric ( 13.2 to 53.3 mg.L-1) acids and, mainly, of acetic acid (158.2 to 2.075.3 mg.L-1).
The highest concentration of propionic acid (836.1 mg.L-1) in the intermediate phase was found in C10, the assay with the lowest methane production (108.5 ± 2.0 mL.g-1 STV). The accumulation of this acid indicates reactor disorder, since the overload is a consequence of the high partial pressure of hydrogen, which in turn suggests an unfavorable condition for acetogenesis, where a concentration between 900 and 2000 mg.L-1 is considered toxic to the process [36]. Therefore, at the end of the assays (Figure 1 III), in C10 there was an increase of propionic acid (947.4 mg.L-1) and decrease of acetic acid (599.2 mg.L-1), demonstrating the unfavorable condition of acetogenesis and methanogenesis at 60°C.
The organic matter data (Figure S6, Supplementary material) corroborates the decrease in total organic acids concentration from the exponential phase (II) to the final methane production phase (III). The highest concentration of total organic acids at the end of operation corresponded to assay C8 (5613 mg.L-1), where the increase of acetic acid (2909.8 mg.L-1) was verified. Probably, the low methane production observed in C8 (145.1 ± 5.2 mL.g-1 STV) occurred due to the inhibition of acetoclastic methanogenic activity, indicated by the acetic acid stock and caused due to the high temperature (54 °C) and hydrolysate volume (10 mL).
Depending on the condition, there was final pH ranging from 5.7 ± 0.07 to 7.2 ± 0.07. The lowest values were related to C8 (5.7 ± 0.07) and C10 (5.9 ± 0.10), where the high final concentrations of total organic matter were measured (5613.3 and 1639.0 g.L-1, respectively), which certainly contributed to the lower methane recovery (145.1 ± 5.2 and 108.5 ± 2.0 mL.g-1 STV , respectively). The assays C3, C12 and C13, operated with the highest P values (286.6 ± 3.5, 267.9 ± 3.2 and 305.8 ± 1.8, respectively) ended with pH of 6.7 and 6.8. From the co-digestion of rice husk and Salvinia molesta, Syaichurrozi et al. [37] verified that in assays with initial pH 6.9 and final pH of 6.8 ± 0.05, the methane content was 65.45%, similar values found for assays conducted with initial pH 7.0 and final pH of 7.0 ± 0.05 (62.64%). As in this study, the pH close to 6.8 favored the anaerobic digestion of lignocellulosic substrates.
3.3 Statistical optimization of methane production with BSG pretreated by hydrothermolysis, severity 4.2
In the optimization of methane production from pretreated BSG by hydrothermolysis (severity 4.2), the effects of the variables operation temperature (x1), BSG concentration (x2) and hydrolysate volume (x3) were statistically analyzed. The methane production profiles (P) resulted from x1, x2 and x3 variations (shown in Table 3), which were delineated according to the central composite design (CCD) in Figure 2.
The statistical significance of the model was determined by ANOVA (Table 3) and the correlation coefficient calculated (R2) was 89.9%, describing the quality of the obtained adjustment. The second order polynomial equation (Equation 2) represents methane production as a function of significant variables (p > 0.10):

Where Y= predicted methane production (mL.g-1 STV), x1= temperature operation (°C), x2= BSG concentration (g.L-1) and x3= hydrolysate volume (mL).
For temperature (x1), a significant negative effect was verified (square and linear). BSG concentration variable (x2) had significant positive linear effect, and the variable hydrolysate volume (x3) presented significant negative linear effect. Significant effects were also observed for the interactions between factors (x1.x2, x2.x3 and x1.x3).
Table 3 ANOVA for the effects of temperature (x1), BSG concentration (x2) and hydrolysate volume (x3)
Variable
|
Sum of Squares
|
Degrees of Freedom
|
Mean Square
|
F-value
|
P-value
|
Factors
|
x1 (L)
|
5903.84
|
1
|
5903.84
|
14.33705
|
0.006835
|
S
|
x1 (Q)
|
8013.53
|
1
|
8013.53
|
19.46027
|
0.003114
|
S
|
x2 (L)
|
3308.05
|
1
|
3308.05
|
8.03337
|
0.025249
|
S
|
x2 (Q)
|
301.74
|
1
|
301.74
|
0.73274
|
0.166878
|
|
x3 (L)
|
7794.01
|
1
|
7794.01
|
18.92719
|
0.003353
|
S
|
x3 (Q)
|
979.67
|
1
|
979.67
|
2.37905
|
0.166878
|
|
x1 . x2
|
2166.47
|
1
|
2166.47
|
5.26110
|
0.055503
|
S
|
x1 . x3
|
1741.98
|
1
|
1741.98
|
4.23026
|
0.078741
|
S
|
x2 . x3
|
2647.37
|
1
|
2647.37
|
6.42895
|
0.038917
|
S
|
Error
|
21.2
|
7
|
411.780
|
|
|
|
Total
|
39178.2
|
16
|
|
|
|
|
*S: Significant effect.
The operation temperature (negative square effect) represented the greatest magnitude effect on methane production, followed by hydrolysate volume (negative linear effect) and BSG concentration (linear effect). Based on the significant factors, the response surface for maximum methane production was developed, evidencing the optimal points resulting from the interaction of variables (Figure 3).
Throughout the statistical model, the optimal predicted operation temperature was at 36 °C, with reduced hydrolysate volumes (lower than 3.0 mL), and 18 g.L-1 of BSG. In this conditions, optimal predicted methane production was 302.4 mL.g-1STV.
Triplicates of reactors (assay C18) were carried out under these optimal predicted conditions for the respective variables, to validate the statistical model. 274.2 ± 5.6 mL.g-1 STV of methane production, Rm of 1.91 ± 0.10 mL.g-1 STV, λ of 18.6 ± 3.9 hours and R2 of 0.99 were obtained. Therefore, there was 90.6% of accuracy in relation to the predicted values, thus, the data fit the model.
3.4 Taxonomy of microbial communities and potential BSG conversion pathways
The structure of microbial communities (Bacteria and Archaea domains) was performed based on two samples, one of them was from the hydrothermolysis optimization assay (B2), and the other from the optimization assay of fermentative-methanogenic activity (C18).
B2 assay was operated with 10 g.L-1 of pretreated BSG by hydrothermolysis (severity of 4.2) at 37 °C, while C18 was operated with 18 g.L-1of pretreated BSG by hydrothermolysis (severity of 4.2) plus 3 mL of hydrolysate, at 36 °C. Based on the taxonomic characterization of both samples, it was possible to infer the effect of substrate concentration increase of 80% from B2 to C18 and the hydrolysate (3 mL) addition in C18.
The Bacteria domain populations had organisms belonging to the phylum Bacteroidetes (28.7 and 31.8%), Spirochaetes (22.1 and 17.4%), Proteobacteria (14.1 and 6.8%), Firmicutes (10.4 and 27.6%), Cloacimonetes (8.2 and 7.0%), Chloroflexi (7.6 and 4.3%) and Synergistetes (5.5 and 3.5%) for B2 and C18, respectively (Figure S8, Supplementary Material). For the Archaea domain, the prevalence of the phylum Euryarchaeota was found in both samples, with 99.5 and 99.4% of relative abundance in B12 and C18, respectively (Figure S9, Supplementary Material).
Considering the relative abundance of the bacterial phylum, changes in bacterial dominance can be verified because of the operational differences between B2 (10 g.L-1 of BSG) and C18 (18 g.L-1 of BSG plus 3 mL of hydrolysate in C18). In B2, Bacteroidetes (28.7%) and Spirochaetes (22.1%) were the prevalent phylum, and in C18, Bacteroidetes (31.8%) and Firmicutes (37.6%).
Bacteroidetes and Firmicutes are widely found in mesophilic reactors and are related to acidogenesis and the ability to metabolize organic molecules, such as carbohydrates and proteins, to organic acids [38]. Paranhos et al. [39] reported predominance of the phylum Firmicutes (43%) and Bacteroidetes (37%) when characterizing poultry manure sample applied as inoculum in anaerobic reactors for lignocellulosic biomass co-digestion. In the present study, bacteria belonging to the phylum Bacteroidetes and Firmicutes may also have resulted from poultry manure, since the inoculum used was anaerobic sludge from UASB reactor fed with poultry slaughterhouse wastewater. Thus, in this study, members of this phylum probably converted complex organic molecules from BSG to soluble carbohydrates and organic acids.
Bacteria belonging to Spirochaetes are often found in anaerobic reactors and also in natural aerobic environments. They are associated to acetate, ethanol and lactic fermentation from sugars [40]. Lee et al. [41] related Spirochaetes to acetate oxidation, a thermodynamically unfavorable reaction that results in hydrogen production, through a syntropic relationship with hydrogenotrophic methanogenic archaea. This metabolic pathway probably occurred in B2 and C18, since hydrogenotrophic methanogenic archaea (Methanolinea, Methanoregula and Methanospirillum) were identified.
Members of the phylum Chloroflexi (7.6 and 4.3% in B2 and C18 samples, respectively) are able to metabolize carbohydrates and amino acids as well as oxidize hydrogen via homoacetogenesis [42]. Cloacimonetes (8.2 and 7.0%, in B2 and C18, respectively) are proteolytic and amino acids consumers, commonly found in wastewater treatment plants [43]. Additionally, it is reported that the phylum Firmicutes, Chloroflexi and Cloacimonetes are resistant to toxic compounds resulting from corn straw pyrolysis, such as phenolic compounds 5-HMF and furfural [44], like in this study, this phylum was identified in the assays where the substrate was subjected to extreme temperature and pressure conditions, which may have released 5-HMF and furfural in the reaction medium [12, 45].
The difference between the bacterial genera identified in B2 and C18 was even more significant (Figure 4). In B2, the main bacteria identified were similar to Treponema (Spirochaetes), Syntrophorhabdus (Proteobacteria), Macellibacteroides (Bacteroidetes), unidentified_Spirochaetaceae (Spirochaetes) and Sphaerochaeta (Spirochaetes) with relative abundance of 11.8, 7.5, 7.4 6.0 and 3.8%, respectively. In C18, Macellibacteroides (Bacteroidetes) were also among the main bacteria (15.9 %), in addition to Sphaerochaeta (Spirochaetes). Clostridium (Firmicutes), Hydrogenispora (Firmicutes), Pseudomonas (Proteobacteria), Bacteroides (Bacteroidetes), Petrimonas (Bacteroidetes), with relative abundance, respectively, of 14.7, 8.1, 4.7, 4.5, 4.4 and 3.2%.
Treponema, dominant genus in B2 (11.8%), use a wide variety of carbohydrates and amino acids as carbon source and can metabolize short and long organic acids chains [46]. Furthermore, they accomplish homoacetogenesis, through which hydrogen and carbonic gas consumption occurs with acetate production [47]. This metabolic characteristic is probably associated with the low relative abundance of methanogenic hydrogenotrophic archaea, since hydrogen was converted to acetate and methane by homoacetogenesis - acetoclastic methanogenesis.
Syntrophorhabdus (7.5% in B2) allows the breakdown of compounds such as phenols, p-cresol, 4-hydroxybenzoate, isophthalate and benzoate in syntrophy with hydrogenotrophic methanogenic archaea [48]. Probably, this organism is involved in the conversion of phenolic compounds released from BSG lignocellulosic degradation, which favored the methanogenic activity in B2, the highest methane production (411.6 ± 7.2 mL. g-1 STV) in assays B1 to B7.
Macellibacteroides and Sphaerochaeta, belonging to the Spirochaetaceae family, are fermenters identified in both reactors. Spirochaetaceae (6.0% in B2 and 2.1% in C18) convert carbohydrates and amino acids into organic acids, mainly into acetic and butyric acids [49]. Usually found in mesophilic anaerobic reactors, bacteria similar to Macellibacteroides (7.4% in B2 and 15.9% in C18) have optimal growth at 35 - 40 °C and pH 5.0 - 8.5, which can convert sugar monomers primarily into lactate, acetate, butyrate and isobutyrate [50]. Similarly, Sphaerochaeta (3.8% in B12 and 14.7% in C18) can consume carbohydrates and produce, predominantly, acetate, formate and ethanol [51]. Regarding C18, it is likely that the sugar concentrations for BSG hydrolysis and hydrolysate favored Macellibacteroides and Sphaerochaeta, which were predominant in the reactor.
Under C18 conditions, Pelotomaculum were verified (2.3%), a acidogenic bacteria which are strictly anaerobic and metabolize a limited number of compounds, including propionate, primary alcohols, low molecular weight aromatics and lactate [52]. Both Hydrogenispora and Lutispora are endospore-forming bacteria, found in C18 (4.7 and 1.1%, respectively). Lutispora consume, primarily, amino acids and release amino acids into iso-butyrate, propionate and iso-valerate [53]. Hydrogenispora grows on medium rich in sugars, starch and yeast extract, forming acetic acid, ethanol and hydrogen, and are found in both anaerobic sludge and vegetables [54, 55]. Thus, in this study, Hydrogenispora may have resulted from both BSG or anaerobic sludge.
Clostridium, identified in C18 (8.1%) and in B2 (1%) has been used as inoculum in reactors fed with lignocellulosic biomass due to its cellulolytic-fermentative ability, resulting in an increase of substrate degradability and, consequently, also due to the intermediates availability for methanogenesis [56]. Rabelo et al. [57] identified great quantities of Clostridium in reactors fed with in natura sugarcane bagasse (19%), as well as in reactors with the bagasse after hydrothermolysis (12.2%), which can metabolize organic compounds, such as carbohydrates and peptones, into diverse organic acids, acetones and alcohols. Moreover, Clostridium species are known for their resistance to high furan concentrations, as well as their ability to degrade them [30, 58]. In this study, the bacteria of the genus Clostridium probably degraded these compounds, whose presence is undeniable because of the hydrothermolysis severity applied to the BSG (4.2).
Other groups characterized by cellulolytic-fermentative activity were also identified, such as Pseudomonas, Bacteroides, Gracilibacter, Ruminofilibacter, Ruminoclostridium. Pseudomonas were identified in C18 (4.5%) and also in B2 (1.1%), a bacterial group characterized by cellulolytic activity to degrade cellulose, hemicellulose and lignin, in addition to consuming biphenols [59]. In addition to BSG polysaccharide degradation, Pseudomonas were probably involved in the conversion of long chain organic acids in the reactor, making substrates available for fermentative bacteria [60]. Synergistaceae (2.0% in B2 and 1.7% in C18), although belonging to the phylum with lower relative abundance in both reactors (Synergistes), probably acted on amino acids metabolization producing short chain organic acids, such as acetic and butyric acids, releasing substrate for methanogenesis. Bacteroides (4.4% in C18) are found in the intestinal gut of animals [38] and, therefore, their probable presence in the sludge used as inoculum and collected from a poultry slaughterhouse wastewater reactor. Members of Bacteroides are related to carbohydrate hydrolysis and proteins and lipid degradation, becoming the available acetic, isovaleric, isobutyric and succinic acids [38, 61].
Gracilibacter, Ruminifilibacter, Ruminoclostridium and Petrimonas were observed in lower relative abundance (lower than 3.2%). Gracilibacter are saccharolytic and produces acetate, lactate and ethanol [62]. Ruminofilibacter and Ruminoclostridium are cellulolytic and available hydrogen, carbonic gas, ethanol, acetate as fermentation products, readily consumed in the following anaerobic digestion steps [63]. Ruminoclostridium are generally related to enriched microbial culture with potential for cellulose degradation and hydrogen production [63]. Petrimonas (3.2% in C18 and 1.4% in B2) ferments a large number of monomers, with acetate and propionate as the main products, in addition to low levels of succinate [64].
Other bacterial groups with fermentative and acidogenic function were found with relative abundance lower than 2%, such as Gelria, Aminivibrio, Comamonas, Syntrophomonas and Syntrophus. Gelria (1.2% in B2) are strictly anaerobic, chemo-organotrophic, saccharolytic and hydrogen-producing bacteria [65]. Aminivibrio (1.7% in B2) are related to amino acids fermentation and organic acids oxidation [66]. Comamonas (2% in B2) can degrade acetic, butyric and propionic acids [67]. Syntrophomonas (1.6% in C18 and 1.7% in B2) and Syntrophus (1% in B2) convert propionic and butyric acids into acetic acid and hydrogen, and Syntrophus can also oxidize aromatic compounds and benzoate [48].
Concerning the Archaea domain, the predominance of acetoclastic methanogenesis pathway is reiterated by the high relative abundance of the genus Methanosaeta (with 87.2 and 80.4%, respectively, in B2 and C18) (Figure S9, Supplementary Material). These archaea are consumers of electrons derived from propionate and butyrate oxidized into acetate [68], which may have been favored to more expressive concentrations of certain metabolites, such as acetic acid.
This configuration with a predominant acetoclastic pathway and less significant hydrogenotrophic methanogenic activity was previously described by Leclerc et al. [69] as the minimum microbiota necessary for the stability of anaerobic reactor. Stability was verified for both conditions, with methane production of 411 ± 7.2 and 274.2 ± 5.6 mL.g-1 STV in B2 and C18, respectively. Archaea similar to Methanolinea (6.2 and 5.5% in B2 and C18, respectively), Methanoregula (5.4 and 8.3% in B2 and C18, respectively) and Methanospirilum (2.4% in C18) were identified at lower relative abundance. These are hydrogenotrophic methanogenic archaea, which reduce carbonic gas into methane using hydrogen molecules as electrons donors.
The degradation of recalcitrant compounds, such as lignocellulosic materials, occurs through various biochemical process and microbial interactions, and based on metabolic characteristics reported in the literature and on the results presented in this study, it was possible to correlate the metabolic pathways that appeared in the BSG anaerobic digestion (Figure 5).
Briefly, the BSG hydrothermolysis (severity 4.2) caused the rupture of BSG fibers, which favored substrate hydrolysis by Clostridium and Pseudomonas. BSG fermentation was also favored due to the higher release of soluble sugars which were converted into organic acids, mainly acetic acid, by Macellibacteroides, Bacteroides, Sphaerochaeta and Clostridium. Phenolic compounds resulting from hydrolysate and pretreated BSG did not compromise the fermentative-methanogenic process, since they were degraded by Syntrophorhabdus. Finally, acetic acid resulting from hydrolysate, released from the fermentation of soluble sugars, and formed from homoacetogenesis by Treponema, was converted into methane by Methanosaeta.