## 3 Results and Discussion
### Two-Stage Anaerobic Digestion of Wheat Straw and Algal Biomass
#### 3.2.2 Volatile Fatty Acid Formation  
Cellulose and hemicellulose are degraded and fermented by anaerobic microbes to produce volatile fatty acids, the main intermediates, which are transferred to the second bioreactor. VFAs are generally produced through anaerobic digestion. In fermentative hydrogen production, metabolic end products are known to affect the hydrogen yield. Figure 6 represents VFA formation in BR-1 and Figure 7--their further utilization during methane formation. In BR-1, the main VFAs present were acetate and butyrate acids, with propionate and caproate acids present in lower quantities. The influence of the HRT on VFA production was not strong. In BR-2, the quantity of propionate exceeded that of acetate and the other VFAs registered. At HRT = 7.5, a whole spectrum of VFAs was detected. At HRT = 10, only acetate and propionate were found in small quantities, which is probably due to the better utilization of the VFAs. This result is in relation to the highest methane yield at the same HRT. The VFA content could be altered according to the type of substrate. In the present study, the organic loading was greater. With these high loads and the presence of easily digestible organic matter (algal biomass), it is possible to obtain only the basic fatty acids in BR-1. In BR-2, the loads from the residual organic matter from the hydrogenic BR-1 were also high, and the used retention time allows for a more complete utilization of different classes of biochemical compounds. Hence, a greater variety of fatty acids was obtained. During the consecutive stages of this biochemical process, e.g., hydrolysis, acidogenesis, acetogenesis and methanogenesis, complex organic substrates are converted into various short-chain volatile fatty acids such as acetic, propionate, lactic and butyric acid by acidogenic bacteria in the hydrolysis and acidogenesis stages [32]. In the later stages, acetogenic microbes oxidize VFAs into acetate, hydrogen and carbon dioxide, which are the main substrates for methane production by methanogens. The anaerobic processes in the two-stage energy carrier production rely on differences in the activities of the participating acidogens and methanogens that reside in the two bioreactors and in their different physiological needs, bearing in mind the high physiological specialization and growth rates. In the first stage in BR-1, acidogens transform the substrate to obtain H2, CO2, volatile fatty acids, lactic acid and alcohols at optimal pHs of 5-6. In the second stage in BR-2, the remaining volatile fatty acids, lactic acid, and alcohols in the first stage are converted to CH4 and CO2 by methanogens at optimal pHs of 7-8. The two-stage system permits a higher organic loading at lower retention times. However, the retention time should be sufficient for the microorganisms to have enough time to degrade the substrate [33]. For the operation of two bioreactors in a coupled system, we have assumed that the synchronization of the flows of waste raw material (substrate) in BR-1 and BR-2 is important. When implementing this process, it is good to synchronize the volumes of the bioreactors, so that both processes can take place without inhibition. In this study, the chosen ratio between the working volumes of the two reactors was 1:5. This is why the HRT of the second bioreactor is always a multiple of the HRT of the first reactor. The organic loading at all HRTs of BR-1 remains the same. Therefore, in Table 1, reflecting the total energy that could be obtained from the combustion of the produced hydrogen and methane, only the HRT of the first reactor is indicated in the first column.