3.2 Effect of hybrid (MW-H2O2)
pre-treatment in anaerobic digestibility of FVW in co-digestion:
3.2.1 Biogas production
The efficiency of hybrid (MW-H2O2) feed
sludge pre-treatment in enhancing anaerobic digestibility of
co-digestion (MWAS+FVW) was studied in two-stage anaerobic digesters
with the co-digestion ratio 3:1 (MWAS: FW). Microwave enhanced advanced
oxidation process is an effective method in improving sludge
solubilisation and consequently promotes biomethanation in waste
activated sludge 20. In the current study, hybrid (MW-
H2O2) pretreatment significantly
improved the biogas production and biogas quality in co-digestion (3:1).
The pretreated co-digestion (3:1) yielded 2.17-fold higher cumulative
methane yield compared to untreated co-digestion (3:1) at the end of 20
days (Figure 1a). 2.91-fold higher methane production was achieved in
thermophilic phase I of treated digester compared to untreated
co-digestion (3:1). Besides, the biogas quality achieved in pre-treated
sludge fed thermophilic digester was also significantly improved. On an
average, treated phase I digester maintained 0.73
CH4/CO2 ratios, whereas untreated
digester suffered a low 0.21 CH4/CO2ratio (Figure 1b). On average, the phase-II biogas quality was improved
with CH4/CO2 ratio of 1.55 in the
treated digester, whereas untreated digester maintained 1.05.
Literature reports suggest that pre-treatments enhance anaerobic
digestibility of FVW by accelerated hydrolyzation and breakdown of
macromolecules 3. Zhou et al, reported 115% increased
anaerobic digestion performance and a 2-fold increase in digestion rate
in thermally pre-treated co-digestion involving FVW compared to
untreated digestion 17. Quite recently, Shanthi et al,
reported a 31% increase in biomethane yield in anaerobic digestion of
FVW by combined (Dimethyl sulfoxide and ultrasonication) pretreatment16. Moreover, combined
(MW-H2O2) pretreatment achieved an
8.72-fold increase in biogas production in anaerobic digestion of dairy
wastewater 20. The study also reported that combined
(MW-H2O2-acid) pretreatment enhanced
biogas production by 9.78-fold, emphasizing the efficiency of combined
treatment under acidic conditions. In the current study, the hybrid
(MW-H2O2) pre-treated FVW co-digestion
outperformed untreated digestion in both phases, making it be an
appropriate pre-treatment technique for organic content-rich substrate.
The untreated digestion showed a lag phase in biogas production during
the thermophilic condition, whereas the treated digestion exhibited an
exponential increase due to the soluble substrate available for the
methanogenic population (Figure 1a). Most of the studies on pretreated
anaerobic digestion, suggests that enhancement in biogas production is
mostly due to the initial hydrolyzation achieved through pretreatment.
Besides, phase separation in anaerobic digestion of FVW has been highly
efficient due to the process stability and enhanced biomethanation31,39. Moreover, the improved methane production in
both phases in treated digestion could be due to hydrogenotrophic and
syntrophic acetate oxidizing microbes, as the latter has a significant
effect at high VFA (>1000 mg/l) concentration34.
3.2.2 Sludge solubilisation and digester performance
In the current study, the FVW was initially blended and homogenized to
facilitate anaerobic digestion. Consequently, the mixed co-digested
sludge achieved an initial COD solubilisation of 22% which was 2.2
folds higher than pure MWAS anaerobic digestion reported in our previous
study 22. Application of hybrid
(MW-H2O2) pre-treatment on mixed sludge
further enhanced COD solubilisation by 33% facilitating easily
available soluble organic content for subsequent utilization by
acidogenic microbial population (Figure 3 and 4). The microwave enhanced
oxidation process exhibits oxidative stress on anaerobic digestion by
the generation of reactive oxygen species (ROS), that oxidize organic
microbial fractions and causes cell wall breakdown 23.
The efficiency of hybrid (MW-H2O2)
pre-treatment on optimum sludge solubilisation of organic compounds
without the formation of recalcitrant products is very much dependent on
the dosage of hydrogen peroxide 24,40. An optimum
sludge solubilisation involves the disintegration of EPS matrix and
dissolution of proteins and carbohydrates fraction of activated sludge,
without the formation of melanoidins through Maillard reaction23. In the current research, the dissolution of
proteins and carbohydrates was consistent with the COD solubilisation as
per the equivalent relationships: 1.5 gCOD/ gProtein and 1.06 gCOD/
gCarbohydrate 41. Accordingly, the soluble protein was
observed to be 18.35% and 39.40% of sCOD in the untreated and treated
digester respectively; soluble carbohydrate constituted 40% and 42% of
sCOD in the untreated and treated digester, respectively. As the
feedstock constitutes 25% FVW, the solubilized fraction of
carbohydrates was observed to be significantly higher than the proteins
(Figure 4). Application of pretreatment enhanced protein solubilisation
by 177% and carbohydrate solubilisation by 18% (Figure 4). This
indicates that the application of
(MW-H2O2) pre-treatment affects EPS
disintegration more than the organic constituents of FVW, as EPS
constitutes more than 43% protein and 10-18% of carbohydrate in its
composition 40. The higher COD solubilisation and EPS
disintegration would accelerate the subsequent anaerobic digestion in
the two-stage digesters.
The digester performance of the two-stage anaerobic digestion with and
without hybrid treatment was compared with VFA accumulation and FOS/TAC
ratio (Figure 2). VFAs are common inhibitors of anaerobic digestion and
their accumulation within the digesters lead to process instability. In
the current research, no significant difference in the VFA accumulation
was observed in the initial thermophilic phase of both two-stage systems
(Figure 2a). However, the FOS/TAC ratio was 77% higher in the untreated
digester compared to the treated digester, during this phase (Figure
2b). The improved digester performance in the thermophilic phase of
treated digester could be due to the buffering capability of microbial
intracellular compounds released during the hybrid
(MW-H2O2) pretreatment. Zhou et al,
reported a similar phenomenon, wherein despite an increase in VFA
accumulation by thermal hydrolysis pre-treatment, the digester
maintained stable pH and VFA/alkalinity levels compared to the untreated
digester, indicating the buffering ability achieved during the
pretreatment 17. Besides, the increased nitrogen
mineralization with co-digestion (3:1) along with the pre-treatment
could also increase the buffering capability of the system42. The mesophilic phase II anaerobic digestion
significantly improved FOS/TAC ratio in both the systems with the
treated digester achieving an average of 0.26, which is well within the
acceptable range of stable anaerobic digestion. Although, Di Maria et
al, reported stable anaerobic digestion with FOS/TAC value
<0.1 in a co-digestion of FVW with sludge under mesophilic
conditions 8.
3.2.3 Oxidative status and sludge activity
Microwave enhanced advanced oxidation process is known for the
destruction of microbial cell walls and oxidation of numerous
recalcitrant organic pollutants present in wastewater through the
formation of hydroxyl radicals [OH]43. An optimum concentration of
H2O2 pretreatment leads to the formation
of [OH] radicals, the highly reactive and strongest
oxidant among H2O2 derivatives; higher
concentration of H2O2 might lead to the
formation of perhydroxyl radicals [\(\text{HO}_{2\ }]\),
which is less reactive and decreases the release of soluble organics20 according to equation 4.
\(H_{2}O_{2}+\text{OH}\leftrightarrow\ \text{HO}_{2\ }+H_{2}\text{O\ }\)equation (4) Apart from the ’Fenton’- driven hydroxyl radicals, there is
another short-living oxidative intermediate, namely, superoxide
[\(O_{2}^{-}]\) that are prominent in oxidation reactions
involving hydrogen peroxide 23.
The importance of anaerobic free radical formation has been
underestimated in the field of wastewater treatment. Anaerobic
microorganisms can encounter free radical formation even under strictly
anaerobic/microaerobic conditions, and they have developed radical
scavenging mechanisms (i.e, SOR; superoxide oxidoreductases) to
neutralize such radicals 44. In the current study, the
oxidative stress induced by hybrid
(MW-H2O2) pretreatment was analyzed by
the measurement of superoxide and the corresponding sludge bioactivity
by the measurement of TTC-DHA activity (Figure 5). TTC-DHA activity
helps to identify the biological activity of anaerobic digestion,
through the respiratory activity of the active microbial population.
Surprisingly, on day 1, the intracellular superoxide
s[\(O_{2}^{-}]\) levels of the untreated digester were observed
to be 81% higher than the hybrid
(MW-H2O2) pre-treated digester (Figure
5a). A plausible explanation of this phenomenon could be the formation
of more reactive hydroxyl radicals [OH] through
Haber-Weiss type reaction (equation 5), in the treated digester that
could account for the decrease in superoxide levels45.
\(O_{2}^{-}+H_{2}O_{2}\ \rightarrow\ \text{OH}+HO^{-}+O_{2}\)equation (5)
The mechanism of Haber-Weiss reaction makes use of Fenton chemistry and
the catalysis requires the presence of ferric ion in sludge. Mixed
sludge being a source of various indigenous metal ions becomes a
suitable target for Haber-Weiss reaction and accelerates the production
of the highly reactive OH radicals 46.
The high s[\(O_{2}^{-}]\) levels in untreated digester in day 1
could be due to the oxidative stress experienced by aerobic living cells
of activated sludge when they are exposed to anoxic conditions. In the
subsequent days, s[\(O_{2}^{-}]\) decreases in untreated
digester as the anaerobic microbial population takes over the entire
digestion. A delayed increase in s[\(O_{2}^{-}]\) levels was
observed in day 3, in the treated digester similar to our previous
reported results 22, which confirms that the oxidative
stress exerted by hybrid (MW-H2O2)
pre-treatment has a prolonged effect in anaerobic digestion (Figure 5a).
On comparison of the TTC-DHA activity, the treated digester exhibited
127% higher bioactivity during the initial thermophilic phase, which is
concurrent with the increased methane production and process stability
achieved in the treated digester (Figure 5b, 1 and 2a). On transfer to
the mesophilic anaerobic digester, an improvement in methanogenic
activity is observed in both the systems (Figure 5b). And
s[\(O_{2}^{-}]\) levels are diminished during this peak
methanogenesis (day 6 to 12) period in both the systems (Figure 5a).
Moreover, an increasing s[\(O_{2}^{-}]\) trend is observed as
the digestion comes to completion which is marked by decreasing TTC-DHA
activity (Figure 5). Briefly, the generation of s[\(O_{2}^{-}]\)radicals are downregulated during active methanogenesis and observed to
increase at dormant sludge activity.