Determination of N. cameroonii growth on Enset fiber
To evaluate the growth of N. cameroonii on Enset fiber, and to analyze the fermentation products, the fungi were grown initially on 0.25 g Enset fiber as sole carbon source. Before fermentation, the bottles were filled with 8.06 mmol CO2 gas at atmospheric pressure (≈1.00 bar). As shown earlier by Theodorou M. et al. [22], pressure increase can be used as an indicator of growth and metabolite production, therefore, the bottle pressure was measured regularly. Figure 1A shows the amount of gas produced by the anaerobic fermentation of N. cameroonii along with the absolute pressure built up. Within 24 hours, an absolute pressure of 1.2 bar was developed and 0.97 mmol of CO2 were produced. As fermentation progressed, the gas composition in the bottle headspace changed to hydrogen and CO2, and a maximum pressure of 1.39 bar was generated on the fourth day. After 5 days, 0.51 mmol of hydrogen was produced with no significant changes in hydrogen quantity in the following days. However, there was a slight decrease in the amount of CO2 gas as well as the pressure. In the control experiments with 0.25 g wheat straw as substrate, the gas profiles were similar and the fungi developed a maximum pressure of 1.25 bar and 0.39 mmol hydrogen, which was lower than with Enset fiber (Fig. 1B).
The amount of soluble products released during growth of N. cameroonii on 0.25 g of Enset fiber is shown in Fig. 2A. The cultures produced 0.37 mmol acetate and 0.41 mmol formate from Enset fiber within two days, however, lactate and succinate were not detected. After 5 days, the production was increased to 1.16 mmol acetate and 1.26 mmol formate. In addition, lactate and succinate were detected in small amounts, 0.16 mmol and 0.08 mmol, respectively. During the media preparation, the hemin solution containing ethanol was added to the media; but after 5 days of fermentation, the amount of ethanol increased to 0.8 mmol, indicating that ethanol was produced during the process. At 7 days, all amounts of metabolites remained constant except for formate, which increased to 1.34 mmol. For both substrates, the maximum total metabolite production rate was observed on the third day, but with Enset fiber a higher production rate than with wheat straw was calculated, 0.73 mmol/day compared to 0.49 mmol/day for wheat straw. At the end of the fermentation process, it was observed that the anaerobic fungi produced a total of 3.94 mmol of metabolites from Enset fiber, which was considerably higher than the 2.86 mmol of metabolites produced from wheat straw (Fig. 2A&B). When comparing the percentage of each metabolite, acetate, hydrogen, and succinate were all produced from Enset fiber in the same proportions as from wheat straw, 29, 13, and 1.9% (mmol/mmol), respectively (Fig. 3C). However, there was a significant difference in the percentage of formate (34%(mmol/mmol)) as well as lactate (4%(mmol/mmol)), and it was higher with Enset fiber than with wheat straw.
In addition, to investigate the influence of substrate loading of Enset fiber on the growth of N. cameroonii, different substrate loadings of 0.5, 1, 3, 5 and 7%(w/v) were tested. Figure 3 shows the pressure generated at the end of fermentation during N. cameroonii growth at different substrate loading of Enset fiber. The highest pressure of 1.72 bar was recorded with a substrate loading of 1%(w/v), but the pressure decreased with increasing substrate loading. Statistical analysis with p < 0.05 showed a significant difference between different substrate loadings of Enset fiber on pressure development by N. cameroonii (see Additional file 1: Table S1). Moreover, the metabolite results shown in Fig. 4A indicate that all fermentation products increased when substrate loading was increased from 0.5 to 1% (w/v). Acetate, formate, and hydrogen increased to 1.89 mmol, 1.82 mmol and 0.88 mmol, respectively. There was also an increase in lactate and succinate to 0.46 mmol and 0.11 mmol, respectively, but no significant difference in ethanol production was observed. At 3% (w/v) substrate loading, acetate and succinate increased significantly to 2.44 mmol and 0.24 mmol, respectively, but formate and lactate were the same. However, hydrogen production dropped to 0.7 mmol. All metabolites decreased at both 5 and 7% (w/v) substrate loadings, except acetate which increased to 3.18 mmol. Figure 4B illustrates the percentage of each metabolite produced during N. cameroonii growth. There was a similarity between the acetate and formate percentages of 30% (mmol/mmol) when the substrate loading was low, however, the acetate percentages increased to 53% (mmol/mmol) when substrate loading was high. Additionally, as substrate loading increased, the hydrogen percentage tended to decrease from 14 to 8% (mmol/mmol) as well.
Caproate production from Enset fiber in one-pot two-step fermentation using N. cameroonii and C. kluyveri
The purpose of this study was to produce caproate directly from Enset fiber without any pretreatment or addition of external enzymes. N. cameroonii was cultivated on Enset fiber and then fermented with C. kluyveri to produce caproate in an one-pot two-step fermentation. Figure 5AB&C shows the steps of one-pot two-step fermentation process, the results of two-step fermentation, and microscopic images of N. cameroonii and C. kluyveri growth before and after the two-step fermentation using Enset fiber as sole carbon source. In the first step fermentation, N. cameroonii grew on 0.25 g Enset fiber for 7 days and produced 1.36 mmol acetate, 0.69 mmol hydrogen, and 1.11 mmol of ethanol. Within 18 hours of starting the second fermentation, C. kluyveri produced 0.21 mmol caproate and 0.33 mmol butyrate. After 42 hours, a maximum of 0.3 mmol caproate and 0.46 mmol butyrate were produced and thereafter there was no significant difference in production. In addition, the hydrogen production increased to 0.95 mmol and ethanol was completely consumed within 27 hours. However, the remaining 0.76 mmol of acetate and other metabolites from anaerobic fungi were not consumed at the end of fermentation. As a control experiment, 2.63 mmol acetate and 1.1 mmol ethanol were used as a substrate for the C. kluyveri fermentation, and after 42 hours 0.27 mmol caproate, 0.68 mmol butyrate and 0.34 mmol hydrogen were produced (Fig. 6). Similarly to the two-step fermentation, all ethanol was consumed at the end of the fermentation, but 1.57 mmol of acetate remained unconsumed.
In addition, separate experiments were conducted in which ethanol was added during C. kluyveri inoculation in the two-step fermentation to maximize acetate consumption and study the effect of ethanol. Figure 7 shows the results of two-step fermentation using Enset fiber and with the addition of 2.18 mmol ethanol. After second fermentation, there was no difference in the production of metabolites during the first 27 hours of the experiment compared to previous experiments. However, the fermentation products after 42 hours were significantly different from the previous experiment, more caproate was found than butyrate, 1.13 mmol and 0.5 mmol, respectively, and hydrogen production increased to 1.35 mmol. It was also found that all ethanol was consumed within 42 hours and only 0.32 mmol acetate was not consumed.
Carbon balance
Carbon balance estimation was performed for N. cameroonii growth on Enset fiber, and for one-pot two-step fermentation using N. cameroonii and C. kluyveri (Table 1). The results showed that 86.3% of the carbon was recovered as metabolites by N. cameroonii growth using Enset fiber as a carbon source. The remaining carbon in the Enset fiber might not be degraded by anaerobic fungi. In the two-step fermentation, C. kluyveri achieved 94.1% carbon recovered as metabolites after the fungi were grown on Enset fiber, therefore it is likely that the missing carbon is bacterial cell biomass.
Table 1
Carbon balance for N. cameroonii growth on Enset fiber, and two-step fermentation using N. cameroonii and C. kluyveri on Enset fiber without additional carbon source
Compound | Carbon per compound (mmol) for N. cameroonii growth | Carbon per compound (mmol) for two-step fermentation |
| Substrates | Substrates |
Enset fiber | 8.58 | - |
Acetate | - | 2.72 |
Ethanol | 1.28* | 2.22 |
Succinate | - | 0.44 |
Lactate | - | 0.27 |
Formate | - | 1.14 |
CO2 | 8.06 | 9.19 |
| Products | Products |
Acetate | 2.22 | 1.69 |
Ethanol | 1.43 | 0 |
Succinate | 0.32 | 0.24 |
Lactate | 0.49 | 0.25 |
Formate | 1.34 | 1.11 |
CO2 | 9.66 | 8.32 |
Caproate | - | 1.65 |
Butyrate | - | 1.78 |
Carbon recovery (%) | 86.3 | 94.1 |
* The amount of ethanol came from the hemin solution in the medium |