The evolution of the alkalinity, ammonium and pH with time during the different BM feeding strategies are presented in Figures 1b and 2. The production of organic acids that follow the DF is responsible for decreases in the culture pH. Figure 2 shows the alkalinity and ammonium concentration during different BM feeding strategies (CHW:BM ratios). Addition of BM to the reactor resulted in an increase in alkalinity, which stabilized the culture pH during the process around 4.8 to 5.0 (Figures 1b and 2). The simultaneous production of VFAs during the two initial experimental periods (Period I and II) resulted in a decrease in culture pH. Therefore, BM was introduced
along with CHW (with CHW:BM ratio equivalent to 1 g VS g VS-1). The total alkalinity
increased gradually during period III, which resulted in an increase in HPSI. Furthermore, increasing the CHW:BM ratio (from 1 to 4 g VS g VS-1) led to an increase
in HY and HPR with slight decrease in the total alkalinity. The results are in agreement with Marone et al. [13] and Tenca et al. [17], who obtained optimal H2 production under
the application of BM (25%-54% composition) with the readily degradable substrates, such as CHW and fruit and vegetable wastes.
Figure 2. CHW:BM ratio and total alkalinity, and ammonium concentration during
the different buffalo manure feeding strategies in the semi-continuous dark fermentation reactor.
The pH of the influent was not adjusted with any chemical buffering agent, therefore, the effluent pH can be related to the endogenous alkalinity provided by the BM. The presence of alkaline components in BM, such as bicarbonate and ammonium, may have contributed to its indigenous buffering capacity. The addition of BM containing ammonium enhances the buffering capacity of the system which can neutralize the VFAs produced [35]. The chemical reaction can be written as follows:
𝐶𝑥𝐻𝑦𝐶𝑂𝑂𝐻 ↔ 𝐶𝑥𝐻𝑦𝐶𝑂𝑂−+ 𝐻+ (2) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 20 40 60 80 100 C H W :B M ( gV S :g V S ) A m m on ia -N it ro ge n a n d T ot al A lk al in it y (m gN H4 +-N /L ; m g C aC O3 /L ) Time (Days)
Ammonia-Nitrogen Total Alkalinity CHW:BM (gVS:gVS)
74
𝑁𝐻3+ 𝐻2𝑂 ↔ 𝑁𝐻4++ 𝑂𝐻− (3)
where CxHyCOOH represents the VFAs. On combining Equations (2) and (3), Equation
(4) can be obtained as:
𝐶𝑥𝐻𝑦𝐶𝑂𝑂𝐻 + 𝑁𝐻3+ 𝐻2𝑂 ↔ 𝐶𝑥𝐻𝑦𝐶𝑂𝑂−+ 𝑁𝐻4++ 𝐻2𝑂 (4)
Therefore, more VFAs can be neutralized at higher ammonia concentrations allowing the stabilization of the pH. Similarly, the co-fermentation with BM allows production of VFAs without a sharp decrease in pH. The total organic acids (TAc, sum of all organic acids measured, i.e. lactic, acetic, propionic, butyric, valeric, iso-valeri, caproic, iso-caproic acids) to total alkalinity (TAk) ratio can indicate that the total acids production can be balanced by the alkaline species initially contained in the BM to maintain a suitable pH, and thus a stable H2 production. Figure 3 gives the relationship
between the TAc/TAk ratio with the HPSI. The higher HPSI was obtained at a TAc/TAk ratio of 3-4, suggesting a constant requirement of an alkalinity source to
maintain a stable culture pH and H2 production during the DF process. BM can act as
co-substrate in the DF of readily degradable substrate like CHW.
Some studies have linked the improvement in H2 production during the co-fermentation
of animal waste to the maintenance of the TAc and TAk balance during the DF process [13, 17, 25]. Nonetheless, the study done by Zhang et al. [36] attributed the increase in biogas production from sorghum stem upon supplying cow manure to maintaining a suitable Carbon to Nitrogen (C/N) ratio for anaerobic digestion. In another study, Perera
and Nirmalakhandan [37] reported that the H2 production can be enhanced by manure
supplementation due to the indigenous H2 producing microorganisms present in the
animal manure.
A limitation in the use of animal manure might be the need for physical or chemical
pre-treatment to inhibit methanogenic activities which consume the H2 [25, 34]. This
study did not consider any pre-treatment of the BM prior to its supplementation, in contrast to the study of Marone et al. [13], who sterilized the feed before the DF. The pretreatment can affect the physico-chemical properties of the substrates as well as impact the microbiology of the BM. Another limitation in the use of BM could be the
inhibition of the H2 production due to elevated ammonium concentrations in the BM if
used in a higher ratio in relation to the other co-substrate. Generally, animal manures such as swine, poultry and dairy manure have a low C/N ratio (C/N ratio of swine manure: 12.8) [38] and higher levels of ammoniacal nitrogen: cattle slurry contains
75 1040-1925 mg NH4+-N L-1 and poultry manure 7000-12.800 mg NH4+-N L-1 [39],
which might cause inhibition of the microbial activity. Cavinato et al. [40] reported the decrease in H2 production at a total ammoniacal nitrogen concentration higher than 2 g
N L-1. However, ammonium levels in this study were lower (80-390 mg NH
4+-N L-1)
than the inhibitory levels reported in the literature. Therefore, ammonium inhibition
will not be an issue for dark fermentative H2 production at higher CHW:BM ratios.
Figure 3. Total acids/Total Alkalinity ratio and HPSI during the operational periods. 4.3.3. Effect of BM addition on metabolite production
Figure 4 shows the plot of correlation circles of the six major metabolites including hydrogen. The principal component analysis of co-relation circles presented in Figure
4 suggests that the H2 production was mainly due to the butyrate pathway (Equation
(6)), which co-related well with the HY. However, the presence of acetate not always
gives a good indication of H2 producing pathways, as homoacetogens can convert the
H2 and CO2 to acetate [41]. Unsurprisingly, other metabolites such as propionate,
lactate or ethanol, which normally do not represent H2 favorable pathways, were not
well correlated. These correlations can be supported by the following equations of DF with glucose as model substrate (Equations. (5)-(9)):
𝐶6𝐻12𝑂6+ 𝐻2𝑂 → 2𝐶𝐻3𝐶𝑂𝑂𝐻 + 2𝐶𝑂2+ 4𝐻2 (5) 𝐶6𝐻12𝑂6 → 𝐶𝐻3𝐶𝐻2𝐶𝐻2𝐶𝑂𝑂𝐻 + 2𝐶𝑂2+ 2𝐻2 (6) 𝐶6𝐻12𝑂6+ 2𝐻2 → 2𝐶𝐻3𝐶𝐻2𝐶𝑂𝑂𝐻 + 2𝐻2𝑂 (7) 𝐶6𝐻12𝑂6 → 2𝐶𝐻3𝐶𝐻2𝑂𝐻 + 2𝐶𝑂2 (8) 𝐶6𝐻12𝑂6 → 2𝐶𝐻3𝐶𝐻𝑂𝐻𝐶𝑂𝑂𝐻 (9) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 I II III IV V VI VII H P S I T A c /T A k Experimental Period
76 They respectively represent: acetate, butyrate, propionate, ethanol and lactate pathway derived from the theoretical glucose fermentation.
Table 4 gives the major metabolites, such as lactate, acetate, propionate, butyrate and ethanol produced during the different experimental periods. With considering the different concentrations of organic acids and alcohols present in the dark fermentation effluent, H2 production can be related to more than one biochemical pathway. The
culture pH has a profound impact on the selection and growth of fermentative microbial communities and thus their metabolic pathways (H2 production). Therefore, it is
necessary to maintain the culture pH in the DF process above the inhibitory levels of pH 4.5, which favours solventogenesis [22].
Figure 4. Correlation circle of six metabolites formed by the first two principle
components Dim1 and Dim 2, representing 26.56 and 23.65 % of the total variance, respectively.
The addition of BM at experimental period III seems to reduce the propionate yields, which is generally produced at low pH and regarded as H2 consuming pathway
(Equation (7)) (Table 4). Moreover, the presence of H2 consumers such as methanogens
and homoacetogens, which might be present in the BM, can utilize H2 and thus
negatively influence the HY. Nonetheless, the acidic culture pH range applied in this study (4.5-5.6) suggests that this pH range is sufficient to suppress the activity of methanogens, as methane production drops sharply at a pH lower than 6.3 [42]. The addition of BM may thus have influenced the metabolite production in three ways: i) a
-1.0 -0.5 0.0 0.5 1.0 -1. 0 -0. 5 0. 0 0. 5 1. 0
Variables factor map (PCA)
Dim 1 (26.56%) D im 2 ( 23 .65 %) Lactate Ethanol Acetate. Propionate Butyrate Hydrogen.
77 buffering action of BM that aided in maintaining a pH that shaped the microbial community of H2 producers, ii) directly influencing the microbial community in the DF
reactor through the microorganisms present in BM and iii) the addition of micro- nutrients (trace elements such as Fe, Zn, Ca) and macro-nutrients (such as Nitrogen-N, phosphorous-P, and potassium-K) that are beneficial to microbial growth and metabolism [10]. Thus, besides being used as buffering agent, BM might also be a source of nutrients for enhancing H2 production.
4.4. Conclusions
DF of substrates with an acidic pH is challenging due to the sharp pH decrease following the co-production of organic acids during the DF process. Along-term
continuous production of H2 has been demonstrated in this work using the addition of
BM to CHW.A HY of 152.2 (± 43.9) mL H2 g VS-1 was obtained with a HPSI of 0.71
when the CHW to BM ratio was 4 g VS g VS-1 (Experimental Period IV). BM
characterized by a higher alkalinity can be applied as co-substrate for maintaining an operable pH during the DF process around 4.8-5. Therefore, addition of BM can aid in providing stability of the continuous dark fermentative H2 production and removing the
dependency on chemical buffering agents. Hence, co-fermentation of CHW with BM or other locally available feedstock sources can give economic sustainability to scaled- up applications of DF processes.
78
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