The statistical methods include a principal component analysis (PCA)-

biplot used for correlating the samples under different conditions to their metabolic profile and a principal coordinate analysis (PCoA) used for correlating the diversity in microbial profile. All statistical methods are described in section 3.12.

The experimental plan is described in Table 4.2, along with the performance outcomes (HPR and CH4 production rate (MPR)).

4.3 Results

4.3.1 Continuous hydrogen and methane production using flocculated culture

Continuous H2 production was carried out in UASBRs fed 5 g glucose L-1 at pH

5.0. The data in Table 4.2 show that in the control cultures the HPR increased with decreasing HRTs from 48 h to 24 h. However, note a significant amount of CH4 was

detected for all of the HRTs examined in this study. An increase in HPR (≥ 90%) and decrease in MPR (85%) was observed in LA inhibited culture compared to the corresponding untreated control cultures operating at the same HRT (Table 4.2). In control cultures, the decrease in CH4 yield was 28% (from 1.01±0.13 to 0.72±0.10

mol mol-1 glucose) with a decrease in HRT suggested that CH4 production was not

suppressed even at pH 5.0 (Figure 4.1a). With pretreatment (inhibition by LA), the CH4 yield was reduced to 0.17±0.04 mol mol-1 glucose at the end of operating at a 48

h HRT (day 40) and the CH4 yields were negligible with further decreases in the

HRT. The H2 yield in the LA inhibited cultures increased from 1.24±0.14 mol mol-1

glucose and reached a peak of 3.16±0.22 mol mol-1 glucoseat 24 h HRT on day 57; however, the yield decreased to 2.28±0.20 mol H2 mol-1 glucose with further

operation (Figure 4.1a).

4.3.2 Continuous hydrogen and methane production using granulated culture

In granular cultures, increasing H2 production with decreasing the HRT was

similar to the trend observed for the flocculated cultures (Table 4.2, Figures 4a and

b). The maximum H2 yield obtained from untreated control (C) cultures was

1.50±0.07 mol mol-1 glucose at a 24 h HRT. A maximum H2 yield and HPR of

2.48±0.08 mol mol-1 glucose and 1.56±0.07 L L-1 d-1, respectively, were observed in LA treated cultures operateingd at a 24 h HRT (Figure 4b and Table 4.2). The CH4

yields (mol mol-1 glucose) in the control cultures decreased from 1.21±0.10 to 0.34±0.06 when the HRT decreased from 48 to 24 h (Figure 4b). In comparison to

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control cultures operating at 48 and 36 h, a 1.5 fold increase in the H2 yield was

observed in LA treated cultures operating at the same HRT conditions (Figure 4b). The CH4 yield observed in LA treated cultures ranged from 0.12 to 0.22 mol mol-1

glucose, which is approximately 35-60% less in comparison to the control cultures operating at similar HRTs.

Figure 4.1. Hydrogen and methane yields under different HRT for control (C) conditions and after treatment with linoleic acid (LA): a) flocculated culture b) granulated culture

Notes: The H2 and CH4 yields plotted shows average values for duplicate reactors R1

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4.3.3 Substrate utilization

The effectiveness of the substrate utilization is characterized from the residual level present in the effluent and the byproduct produced during the fermentation. In addition to the gaseous products produced, the soluble metabolite products (SMP) included volatile fatty acids such as acetic acid (HAc), propionic acid (HPr) and butyric acid (HBu) as well as alcohols such as ethanol (EtOH) and i-propanol (i- PrOH). In the current study, glucose conversion was more than 95±2% in both the granulated and flocculated cultures throughout the experiment (> 60 days of operation). This large conversion indicates that the reactor operation was suitable for microbial fermentation (data not shown).

4.3.4 Soluble metabolite profile and electron balance

The metabolite profile obtained during continuous dark fermentation using glucose as the substrate is shown in Figures 4.2a and b for flocculated and granulated culture, respectively. HAc and HBu were the major SMPs present throughout fermentation. The HAc and HBu levels, 4 to 15 mM, detected in the untreated flocculated culture were stable during operation at each HRT. With the addition of LA, the HAc levels increased gradually from 15.8±0.8 mM on day 43 to 41.2±3.2 mM on day 56 of reactor operation (Figure 4.2a). When the reactor operation was extended further, the HAc level slowly decreased to 26.9±1.4 mM on day 65. In addition, the HBu concentration decreased over this period with the level decreasing from 12.6±1.6 mM on day 43 to 4.5±0.2 mM on day 64 in LA inhibited flocculated cultures.

In flocculated cultures, the EtOH levels reached 9±2 mM in the untreated control samples and were reduced by approximately 75% after adding LA. The EtOH levels in the control flocculated cultures on an average increased to 10±1 mM (between days 23-27) and in the LA treated cultures operating at 24 h HRT the EtOH levels decreased to 4.1±0.4 mM (between days 63-68). The other byproducts observed such as HPr and i-PrOH were found to decrease following LA treatment when compared to the control conditions under the same HRT. The HPr levels in the LA treated flocculated cultures was in the range of 0.5±0.0 to 2.0±0.2 mM, which is approximately 30% less compared to the HPr levels observed in the untreated

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flocculated culture. With the addition of LA, the i-PrOH levels reduced initially and stabilized to 2.6 ± 0.2 mM during 61-68 days of the operation.

In the case of granulated culture (shown in Figure 4.2b), consistent increases in the HAc levels were observed as HRT was reduced for the control conditions. Following LA treatment, the HAc levels remained stable until day 40 when HRT was reduced to 36 h, after which there was a sweeping increase in HAc levels over a period of 10 days, from 12.5±0.8 mM on day 41 to 36.5±2.0 mM on day 53. The HAc level then stabilized (between the 54th and 63rd day of the operation and at a 24 h HRT) in the range of 32.9 to 35.3 mM. There was no evident trend observed in the HBu levels detected in untreated granular culture as HRT was reduced. The HBu levels in control samples was in the range of 3.6±0.5 mM to 7.1±0.7 mM, whereas in the LA inhibited granulated culture, HBu was detected primarily in the range of 7.1±1.0 mM to 12.1±1.2 mM. These levels were higher than the levels observed in LA inhibited flocculated culture. The HPr levels were found to vary from 2.6±0.3 mM to 5.8±0.7 mM in both untreated and LA inhibited granular cultures at different HRTs examined in this study.

The EtOH and i-PrOH levels in the granular control cultures increased when the HRT was decreased from approximately 3 mM to 7 mM. In the LA treated granular cultures, the alcohol levels reached a maximum of approximately 3.2 mM.

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Figure 4.2 Soluble metabolite distribution during anaerobic fermentation at different HRTs with: (a) flocculated culture (b) granulated culture

Notes:

1. The operating conditions at each stage are specified over the arrow, where the #s represent the HRT and the letters ‘C’ and ‘LA’ correspond to untreated control and LA-treated culture.

2. Acetic acid = HAc; propionic acid = HPr; butyric acid = HBu; iso-propanol = i- PrOH and ethanol = EtOH.

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The electron-equivalent balance as the percent electron equivalents (e- equiv) distributed from glucose fermentation under the various experimental conditions is shown in Figure 4.3. The total e- equiv balance for the fermentation byproducts ranged from 89±7% to 104±8% for all the conditions examined in this experiment. The fs value (fraction of e- equiv from glucose to biomass) was assumed to be constant as 10% of the e- equiv from the initial glucose (Rittmann and McCarty, 2001). In the untreated flocculated and granular cultures, the largest electron sink was in CH4, followed by HBu and HAc, which each accumulated 10-15% of e- equiv on

average. The electron sink in the HPr was smaller and was measured at levels varying from 5-7%, while i-PrOH and EtOH accounted for 13-20% of e- equiv.

Following treatment with LA, differences in the electron distribution to metabolites were observed in both the granular and flocculated cultures. In the case of LA treated flocculated culture, a large fraction of the electrons was transferred to H2

at 36 h and 24 h HRT with the HAc levels varying between 13 and 35%. This result support the idea that HAc-type of fermentation is associated with higher H2 yield.

Initially, the major electron sink observed was HBu was with 30% of the available e- equiv derived from glucose; however, then the HAc increased in a linear fashion with a corresponding decrease in the HBu e- equiv to 18% (Figure 4.3). This suggests that acid (HAc through acetyl-CoA and HBu) formation is favorable for H2 production,

whereas alcohol production is an electron consuming reaction involving NADH (see