3.1 CSTR experiments
3.1.1 Fermentative type
As shown in Fig. 2a, when pH was maintained at 4.0, the total concentration of fermentative products gradually increased in the first 14 day and then remained relatively stable from day 16 to 28, which was similar to prior findings with fruit and vegetable waste as substrate [19]. The average total COD concentration of fermentative products (tCODp) was 11.2 g COD/L and acidogenic rate was 35.0%. And thus the yield rate of total fermentative products was 3.73 gCOD/(L.d). Between days 16 and 28, the average concentrations of ethanol, acetate and lactate were 2.1 g/L, 1.8 g/L and 4.5 g/L, respectively. Carbon dioxide was the main gas produced (98.7%) in addition to a minor amount of H2 (1.3%).
In order to examine the metabolic mechanisms, high throughput pyrosequencing analysis of sludge samples was conducted (Fig.2b). After acclimation, the numbers of microbial genera found on day 12 and day 24 decreased compared with those found in the inoculums. There were two and three identified with proportion of more than 0.5% at day 12 and day 24. Lactobacillus was the dominant bacteria representing more than 80% of the genera identified. Bifidobacterium represented 4.2% of genera at day 12, but its proportion increased to 14.6% at day 24. And thus lactic acid bacteria including Lactobacillus and Bifidobacterium were the dominant bacteria.
The reported different kinds of lactate fermentation (heterolactic fermentation and homolactic fermentation) and other fermentation reactions for ethanol and acetate production are summarized in Table 2 [27-31]. Saccharomyces and Zymomonas can ferment glucose to ethanol and CO2 [27]. Ethanol-type hydrogen fermentation was shown by Ren et al. [32] with acetate, ethanol and hydrogen gas as fermentative products at pH of 4.2–4.5.
From day 16 to day 28, the molar ratio of ethanol: acetate: lactate was 1.00:0.67:1.07. Therefore, the heterolactic fermentation surely contributed a lot. In addition, it was most likely fermentation pathways were a combination of heterolacttic fermentation and ethanol fermentation. Because the concentration of H2 was very low, it was unlikely that ethanol-type hydrogen gas fermentation existed. And it was also unlikely that ethanol was produced by Zymomonas because Zymomonas did not exist at samples of day 12 and 24. However, the Saccharomyces as a kind of fungus could not be detected by 16s rRNA high throughput pyrosequencing. And thus it could be concluded that lactate fermentation and ethanol fermentation coexist, however, ethanol-type fermentation from Saccharomyces or others unknown reactions could not be sure.
3.1.2 Stability of acdiogenic fermentation
In order to investigate the stability of fermentative type, the pH was increased to 5.0 from day 29 to day 42 (Fig. 2a). The average COD concentration of total fermentative products (tCODp) was 20.5 g COD/L, and the acidogenic rate increased from 35.0% at pH 4.0 to 64.0% at pH 5.0. There was a slight increase in lactate from day 30 to day 36, however, from day 38 to 42, the proportion of lactate decreased as the COD proportion of acetate and butyrate increased to more than 70%. Between day 38 and day 42, the proportion of hydrogen gas increased to 26.3% and the average concentrations of ethanol, acetate, lactate and butyrate were 5.6, 4.6, 1.9 and 1.8 g/L, respectively. It was obvious that the composition of fermentative products at pH 5.0 was different from that at pH 4.0.
The pH was controlled at 4.0 from day 43 to day 62 and the concentration of all fermentative products decreased (Fig. 2a). The average total COD concentration of fermentative products (tCODp) was 7.1 g COD/L. The butyrate concentration went down from 5.3 g/L at day 44 day to 0.1 g/L at day 62. Lactate, ethanol and acetate were the main fermentative products, with average concentrations of 2.2, 0.8 and 1.7 g/L, respectively.
Thus, pH has a key influence on the fermentative type. At pH 4.0, lactate, ethanol and acetate were dominant fermentative products; however, acidogenic fermentation rate was low. Raising the pH to 5.0 resulted in different fermentation products and a higher acidogenic fermentation rate.
For sludge samples of day 38, there were eleven genera identified with proportion of more than 0.5%, which was higher than at pH 4.0 (Fig. 2b). Lactobacillus and Bifidobacterium, represented the largest proportion of genera (62.1%). Other genera identified included Acetobacter, Olsenella, and Prevotella, with proportions of 5.8%, 2.7%, and 2.4%, respectively. Therefore, pH had a large influence on the microbial community structure in CSTR reactor. Lactobacillus and Bifidobacterium were favored both at pH 4.0 and 5.0, but microbial community increased at pH 5.0.
3.2 UASB experiments
3.2.1 Fermentative products
Different amounts of NaHCO3 were added to adjust the pH of influent between 6.5 and 7.5. Due to the production of VFAs, the effluent pH of UASB reactor decreased and eventually maintained between 3.2 and 4.0 (Fig. 3). During the experiments, HRT was gradually decreased to 4 h and the concentration of glucose in influent increased from 5 g/L (stage II: 4~19 days) to 7 g/L (stage III: 20~40 days). And thus OLR of UASB reactor was gradually increased from 12 g/(L.d) to 42 g/(L.d). The concentration of fermentative products in effluent of UASB reactor was presented in Fig.4a. At stage II, the average concentration of tCODp was 3.5 gCOD/L. And at stage III, the concentration of tCODp firstly obviously increased and then gradually deceased with the decrease of HRT and increase of OLR. And the average concentration of fermentative products was 2.7 gCOD/L at HRT of 4h and OLR of 42 g/(L.d), and thus the yield rate of total fermentative products was 16.4 gCOD/(L.d), which was much higher than 3.7 gCOD/(L.d) at the CSTR reactor.
At day 28~40, the ratio of ethanol, lactate and acetate was dominant up to more than 90% (Fig. 4b), which was similar with at the CSTR reactor. However, propionate and butyrate were also produced in UASB reactor besides ethanol, lactate and acetate, which was different from the distribution of fermentative products in CSTR reactor. In addition, hydrogen gas took large proportion of 43.7% and methane was also found with small proportion of 1.5% among the biogas produced. The pH in the UASB reactor was gradually decreased from 6.5~7.5 to 3.2~4.0, while the pH in the CSTR reactor was always kept at 4.0. Therefore, the different variation of pH in CSTR and UASB might lead to the large difference in fermentative products.
3.2.2 Acidogenic granular sludge
At day 20, obvious granular sludge appeared at bottom of the UASB reactor. The acidogenic granular sludge was white irregular spheres (Fig.5 a, b). At day 40, the concentration of sludge in UASB was 2.35 gVSS/l and particle size distribution was shown in Table 3. The granular sludge with diameter of more than 0.5 mm in this study took proportion of 43.3%. Hulshoff Pol et al. [33] firstly proposed selection pressure theory and the granulation process was believed to be the continuous selection of sludge particles under the sum of hydraulic loading rate and the gas loading rate. During the experiments, the surface velocity was gradually increased from 0.25 m3/(m2.h) to 0.625 m3/(m2.h) leading to a gradually increased selection pressure. As shown in Fig.3, each time when surface velocity increased, the particle COD increased correspondingly and then decreased, which meat that the increase of surface velocity realized the selection of heavier sludge particles retaining in the UASB reactor.
A close examination of the surfaces of 4 granules revealed the presence of a large diversity of microbial morphotypes. These included long rods, cocci, chain cocci, small rods and thin filaments (Fig. 5b,c). During these different morphologic microorganisms, a kind of rod-shaped microorganism (0.5-1μm×1-3μm) shown in Fig.5d was predominant.
3.2.3 Microbial community
The microbial community structure of acidogenic granular sludge was presented in Fig. 6. Same with in CSTR reactor, Lactobacillus was still with the largest proportion of 64.29%, however, there were thirteen different kinds of genes with proportion more than 0.1%. Clostridium took proportion of 14.52%, followed by Prevotella (7.79%), Bifidobacterium (1.99%), Acetobacter (1.73%), Enterobacter (1.49%), Dialister (1.33%), Ethanoligenes (1.19), Pectinatus (1.15%). Bifidobacterium as a kind of lactic acid bacteria could produce 1.5 molecules of acetate and one molecule of lactic acid per molecule of glucose[34]. Clostridium, Enterobacter and Ethanoligenens have been widely considered as the biohydrogen-producing anaerobes [35]. Clostridium species usually are the dominant H2-producers in acetate/butyrate fermentation while Ethanoligenens species usually are the dominant H2-producers in acetate/ethanol fermentation [13, 32]. Kim et al. [36] found H2, ethanol and acetate were produced by Enterobacter sp T4384 at an initial pH range of 4.5~9.5. Prevotella were reported to be able to grow at pH as low as 5.0-5.5 and could utilize glucose to produce organic acids such as formic acid, acetate, fumaric acid succinic acid [37]. Dialister are obligate anaerobic and nonfermentative organisms [38], While Pectinatus could degrade organic substrate to acetate, propionate and H2S[39]. The acidogenic granular sludge in this study was mixed-cultured and the different microorganisms were likely with different fermentative routes.
The fermentative products were more complexity in UASB than CSTR, which might result from the complexity of the microbial community in UASB reactor. Lactic acid bacteria (Lactobacillus and Bifidobacterium), biohydrogen-producing bacteria (Clostridium, Enterobacter , Ethanoligenens etc.) and other acids production bacteria (Prevotella, Pectinatus, etc.) coexisted in the acidogenic granular sludge, which resulted in the coexistence of different fermentative routes and different fermentative products.