## 3 Results and Discussion
### Metabolite Analysis  
Isolated _Enterobacter_ sp. KBH 6958 was then used as inoculum for biohydrogen production using four different fermentation media. Dark anaerobic biohydrogen fermentation is usually accompanied by the production of organic acids [27; 28]. The type of organic acids accumulated is highly dependent on biohydrogen-producing strains and their biochemical pathways. Two major biohydrogen-producing biochemical pathways are pyruvate:ferredoxin oxidoreductase (PFOR) pathway and the pyruvate formate lyase (PFL)  
Figure 2: Phylogenetic dendrogram constructed using the neighbor-joining method based on 16s rRNA sequences indicating the position of isolate KBH 6958 among closely related members of the genus _Enterobacter_. The numbers at the nodes indicate the levels of bootstrap support percentages of 1000 replicates. pathway. The major acids accumulated through the PFOR pathway are acetic acid and/or butyric acid, whereas formic acid is produced through PFL pathway [9]. While almost all biohydrogen-producing _Clostridia_ accumulate acetic and/or butyric acid as end products, the literature reports on final organic acids accumulated in _Enterobacter_ were not unanimous. Researchers has reported that formate-hydrogen lyase enzymes found in _Enteric_ bacteria catalyzed the biohydrogen evolution through PFL pathway [9]. [PERSON] et al. reported that biohydrogen fermentation using _Enterobacter cloacae_ KBH3 isolated from termites' gut accumulated both formic and lactic acid as the final metabolites [8]. There are an equal number of published reports citing _Enteric_ bacteria, such as _Enterobacter cloacae_ IIT-BT 08 and _Enterobacter aerogenes_, that produce butyric acid, acetic acid and ethanol as their main fermentative products, which has similar traits as _Clostridia_[10; 31; 32]. In order to understand the metabolic pathway of this isolate, _Enterobacter sp_. KBH 6958, the fermented medium was analyzed qualitatively using GCMS. The final metabolites detected in all substrate media are summarized in Table 2.  
At the end of fermentability test, the highest biohydrogen potential of 50.6 mmol/L was observed when AFEX-pretreated OPEFB hydrolysate was used, whereas, the lowest biohydrogen potential of 35.3 mmol/L was observed with DAA molasses. Lower biohydrogen potential from DAA OPEFB molasses was expected due to the furfural content, which was not found in other fermentation media. Furthermore, the sugars in DAA molasses were mostly xylose-based oligosaccharides, whereas other media were rich in monosaccharides (glucose). Alternatively, AFEX-pretreated hydrolysate contained almost no inhibitors coupled with other nutrients originating from OPEFB structures. Figure 3: FESEM image of isolate KBH 6958 at (**a**) magnification of 2000\(\times\); (**b**) magnification of 5000\(\times\) showing nano-interlinkages among the bacteria; and (**c**) magnification of 10,000\(\times\) showing individual bacterium. Through the GCMS results, it was clearly demonstrated that isolate _Enterobacter_ sp. KBH 6958 produced biohydrogen through PFOR pathway. This conclusion was made because neither formic acid nor lactic acid was detected in any of the four different fermentation media, which is in agreement with previous studies [33,34]. Acetic acid, butyric acid and ethanol were detected in all the fermented samples. Moreover, propionic acid was observed in both RCM and DAA OPEFB molasses medium indicating higher fermentation stress, possibly due to the higher biohydrogen and NADH evolution rates [35]. In these two media, isolate _Enterobacter_ sp. KBH 6958 produced 2, 3-butanediol. The production of 2, 3-butanediol resulted in lower biohydrogen potential, as demonstrated by [PERSON] et al. [31]. Acetamide was discovered in the AFEX-fermented OPEFB hydrolysate. The residual ammonia in the AFEX-treated OPEFB may react with acetic acid to form acetamide. Similarly, the presence of furan compounds in DAA molasses as a result of sugar degradation during the OPEFB pretreatment resulted in the detection of 2-furan methanol compounds.