Physicochemical characterisation of effluents
The physicochemical composition of the three studied agro-industrial residues (CSL, SDW, and CW) was different in terms of carbohydrates, organic acids, nitrogen, cations, and anions (Table 2). The expired refrigerant “Guaraná” was not characterised since it was not significant. The CW was the only waste that presented lactose in detectable quantities (4.55 g L− 1), while glucose, fructose, and maltose were undetectable. Similar results has been documented in whey used for hydrogen production by Escherichia coli, but with a higher content of lactose (15 g L− 1) [20, 21]. Both CSL and soft drink wastewater contained glucose and fructose; however, CSL exhibited much higher concentrations (> 35 g L− 1 for each carbohydrate, versus 0.7 and 3.98 g L− 1 of glucose and fructose, respectively, in SDW). The CSL used in this study is richer in the cited carbohydrates than the used by Martinez-Burgos et al. (2021b) for the production of bioH2. It is important to highlight that CSL also presented an elevated concentration of lactic acid (92.5 g L− 1) which can be used as a second carbon source for some lactic acid bacteria and species of the Clostridium genus [23, 24].
Table 2
Physicochemical characterisation of corn steep liquor, soft drink wastewater, and cheese whey.
Substance | CSL | SDW | CW | Substance | CSL | CW |
Glucose (g L-1) | 36.31 | 0.7 | - | Thr (µmol mL− 1) | 147 ± 12 | 75 ± 12.5 |
Fructose (g L-1) | 35.49 | 3.998 | - |
Maltose (g L-1) | 1.493 | - | - | Cys (µmol mL− 1) | 116.4 ± 18.0 | 35 ± 14.8 |
Lactose (g L-1) | - | - | 4.55 |
Lactic Acid (g L-1) | 92.55 | 0.5676 | - | Val (µmol mL− 1) | 135.3 ± 22.9 | 58.8 ± 5.4 |
Citric Acid (g L-1) | - | 1.182 | - |
Acetic Acid (g L-1) | 0.3617 | 0.3071 | - | Met (µmol mL− 1) | 211.8 ± 28.4 | 15 ± 7.4 |
Total nitrogen (g L-1) | 10.8 | 0.5 | 3.6 |
Phosphorus P2O5 (g L-1) | < 1.0 | < 1.0 | 12 | Ile (µmol mL− 1) | 69.5 ± 18 | 47.9 ± 8.9 |
Na(mg L-1) | 1.412 | 268.6 | 805 |
K (mg L− 1) | 38.00 | 38.3 | 1650 | Phe (µmol mL− 1) | 77.9 ± 22.2 | 25 ± 5.8 |
Ca (mg L-1) | 1.170 | 43 | 819 |
Mg (mg L-1) | 2.875 | 32.45 | 131 | His (µmol mL− 1) | 72.5 ± 15.5 | 17.89 ± 6.5 |
Fe (mg L-1) | - | 3 | - |
NH4 (mg L-1) | 1.412 | 5.85 | 217 | Trp (µmol mL− 1) | 49.1 ± 17.6 | 17.5 ± 8.7 |
NO3 (mg L-1) | - | 16.4 | - |
F (mg L-1) | 25.057 | - | - | Lys (µmol mL− 1) | 64.8 ± 12.7 | 100 ± 15.4 |
Cl (mg L-1) | 36.576 | 87.8 | - |
pH | 4.32 | 7.56 | 4.76 | Arg (µmol mL− 1) | 124.8 ± 19.7 | 17.35 ± 5.35 |
COD (g L-1) | 245.4 | 4.27 | 47.3 |
According to the organic matter composition, all effluents showed high COD. The COD of CW and CSL were similar to that described in the literature [25–28]. SDW, however, presented lower values for COD in comparison to the scientific literature [11, 29, 30] and was more similar to beverage wastewater [31].
The presence and concentration of Mg, Fe, Na, and K have a significant effect on hydrogen productivity, especially the first two [32]. Mg, Na, and K were observed in high concentrations in all wastewaters. The concentration of Mg observed in SDW, although lower than in CW and CSL, is enough to stimulate biohydrogen production metabolism. Fe was not detected, and its supplementation could enhance the productivity obtained in this study [33–35]. Ca was another cation observed at high concentrations for all the three wastewaters. Mn and Cu, which are widely recognised as enzymes cofactors, were not identified. Supplementation with Mn and Cu could be considered in future studies.
CSL and CW showed up as important sources of nitrogen (10.5 and 3.6, respectively), values comparable to other research [21, 36]. This content is sufficient to support biomass production without the need to supplement with other nitrogen sources, such as yeast extract, meat extract, and ammonium sulphate [20, 37], which have a significant impact on the process economy. The amino acid composition of CSL and CW showed that L-cysteine, a recurring supplement for hydrogen production fermentations [35, 38, 39], is present in CW and CSL in significant amounts. The presence of methionine, alanine, histidine, and lysine in ranges from 1.0 to 10.0 g L− 1 may also have a positive impact on biohydrogen production. The synergetic effect of these amino acids with ferric oxide was described to increase the volumetric hydrogen produced 1.3 times [40, 41]. The higher amino acid concentrations found in this work were methionine and valine for CSL and histidine and arginine for CW. According to Hofer et al. (2018), phenylalanine and isoleucine should be primarily free in the effluents, while arginine, histidine, and lysine are essentially bound in proteins or peptides. Thus, arginine, histidine, and lysine concentrations in CW may be underestimated.
The initial pH is relevant for biohydrogen production. The optimum pH for Clostridium is approximately 6.0 [42, 43], while fermentation is severely impaired under pH 4.0 or over pH 12.0 [12]. Although all the fermentations were adjusted to pH 7.0, it is interesting to consider the amount of alkali necessary to correct the pH of CSL and CW. Despite the chemical composition of SDW not being remarkably interesting in terms of carbohydrates, cations, and anions, it could be used as a dilutant to facilitate pH correction.
Clostridium butyricum identification
According to the BLAST analysis and phylogenetic tree (Fig. 1), the isolate strain was identified as Clostridium butyricum with 99% similarity. The phylogenetic tree constructed with the neighbour-joining method revealed that the C. butyricum was included in the same subcluster with C. saccharoperbutylacetonicum, C. puniceum, C. saccharobutylicum, C. chromiireducens, C. diolis, C. beijerinckii, and C. neonatale.
Variable Selection, Optimisation, And Mathematical Models
The Pareto diagram (Figure not shown) showed that among the five variables tested, only %CSL and fermentation time significantly affected the production of hydrogen (p ≤ 0.05) for the isolated strain. For Clostridium beijerinckii, CW, %CSL, and fermentation time were significant (p ≤ 0.05). Previous works demonstrate that the concentration of CSL, whey, and the fermentation time affect hydrogen production via dark fermentation [22, 27]. The inoculum rate was not significant (p ≤ 0.05), which may have been because large inoculum volumes were tested; an inoculum rate of 10% was then used for all experiments.
It was expected that due to its large amount sugar the expired soft drink would be significant in all tests performed. However, this variable was not significant (p ≤ 0.05). Soft drinks were added to the medium in a concentration of 20%, and even though it was diluted, the antimicrobial properties of the finished product were still outstanding. According to Kregiel [44], soft drinks contain chemical preservatives and acids that play an important role in avoiding microbial growth. Sorbates and benzoates present in the soft drink act together to enhance antimicrobial effectiveness against bacteria and other microorganisms. Notwithstanding, the author still emphasises that this combination causes the inhibition of amino acid uptake and destroys the internal proton level of microbial cells.
On the other hand, the contour surface graphs (Figs. 2A and 2B) showed that the optimal hydrogen production is reached with 12–14% CSL, at 37.5–48 h time of fermentation and 38.5–45 % CW for Clostridium beijerinckii. In the case of Clostridium butyricum, the optimum hydrogen production was 10–14 % CSL and 36–52 h (Fig. 2C).
Mathematical Models
Two second-order polynomial models that simulate the production of biohydrogen under the conditions described are presented in equations 2 (Clostridium beijerinckii) and 3 (Clostridium butyricum). The mathematical models presented an R2 of 0.95 and 0.895 for equations 2 and 3, respectively, which indicates that 95% and 89.5% of hydrogen production is governed by the variables selected. According to Hye et al. and Martinez-Burgos et al. [23, 45], mathematical models with R2 ≥ 80 % are adequate to simulate the behaviour of the response variables.
\({H}_{2}=11.96+3.7CSL+2.58t{-1.29CSL}^{2}-{2.74t}^{2}+2.54CSL*t+0.13CW\) (Eq. 2)
\({H}_{2}=20.76+4.68CSL+5.13t{-4.76CSL}^{2}\) (Eq. 3)
According to the mathematical model (Eq. 2), the optimal conditions for the production of biohydrogen with Clostridium beijerinckii were CSL = 12%, t = 48 h, and CW = 40%. The experimental and predicted hydrogen volumes at the optimum condition were 18.5 ± 1.68 mL and 16.87 mL, respectively. In the case of Clostridium butyricum, the conditions were CSL = 12 % and t = 48 h. The experimental and predicted biohydrogen volumes for this strains were 27.4 ± 1.84 mL and 26.5 mL, respectively. In both cases, there was no significant difference between the experimental and modelled volumes.
Evaluation Of Biohydrogen Production Under Optimum Conditions
Under the optimum conditions selected, the maximum volume of hydrogen produced by the bacteria Clostridium butyricum and Clostridium beijerinckii was 27.47 ± 1.8 mL and 19.05 ± 0.70 mL, respectively (Fig. 3A). It was also shown that after 32 h and 48 h for C. butyricum and C. beijerinckii, respectively, the production of hydrogen did not improve so the fermentation process could be stopped at this time. It is noteworthy that the yield of hydrogen produced with C. butyricum (310 mL H2/g COD removed) was approximately 40% greater than that obtained as C. beijerinckii (235 mL H2/g COD removed). These yields are higher than those reported in other studies that had been supplemented with some compounds. [46] supplemented the effluent with Fe2+, peptone, and Na2HPO4 + 2H2O and obtained a yield of 215 mL H2/g CODremoved. Zampol et al. [47] supplemented the medium with KH2PO4, K2HPO4, MgSO4, NaCl, CaCl2, yeast extract, iron citrate and reported a maximum yield of 137 mL H2/g COD removed. Ozkan et al. [48] supplemented the effluent with Basal medium and obtained a yield of 115.6 mL H2/g COD removed.
On the other hand, it was observed that for both microorganisms studied no significant difference (p-value ≤ 0.05) between the hydrogen production between the cysteine-HCl-supplemented and not supplemented media (Fig. 3B). This indicates that the CSL can supply the amino acid demands of the microorganisms and its use could decrease the costs of the culture media destined for the production of hydrogen via biological production.
In both cases, the main carbon sources such as glucose and fructose were consumed almost completely (Figs. 3C and 3D). In addition, it was observed that the microorganisms could use lactic acid as an alternative carbon source. The metabolites produced in the fermentation were mainly butyric and acetic acid, with predominance of the first. It is noteworthy that the production of the latter was greater with C. butyricum which could be an explanation of the greater production of hydrogen with this strain, acetate generates 2× more H2 than butyrate [49]. Also, it was observed that part of the acetic acid and the butyric acid produced was consumed in the same fermentation, as these can also be used as alternative carbon sources. [23, 50] showed that acetic and butyric acid can be used as carbon sources by some microorganisms in dark fermentation. Likewise, both microorganisms produced traces of propionic acid. However, the production of this metabolite is not desirable in dark fermentation because stoichiometrically hydrogen is needed for its production [8, 9].
The amino acid profile during the C. butyricum fermentation (Fig. 3E) show a slight consumption in the amino acids cysteine and threonine available in the medium. The methionine and valine concentrations were maintained above the concentration of the other amino acids, and most of them showed similar concentrations in the beginning and end of the fermentation time. Sharma and Melkania [40, 41] tested the effect of methionine, alanine, histidine, cysteine and lysine in the production of hydrogen by co-culture of E. coli and Enterobacter aerogenes in municipal solid waste. The best productivities were with the addition of alanine and cysteine, and the butyrate concentration was increased during the fermentation, favouring the butyrate hydrogenogenic pathway. A similar increase in the butyric acid occurred in this work, possibly explaining why cysteine was consumed. Several authors indicate that cysteine supplementation in the medium enhance biohydrogen production [34, 35, 38, 39]. The bioavailability of each amino acid also indicates which can be used and directed for the metabolic pathways. Hofer et al. [24] found that the content of threonine, tryptophan, and phenylalanine were more than 60% free in CSL, while cysteine and valine were around 35% free in the solution. The cysteine and threonine found in this work were also likely available for C. butyricum dark fermentation.