## 3 Results and Discussion
### Two-Stage Anaerobic Digestion of Wheat Straw and Algal Biomass
#### 3.2.4 Microbial Communities' Composition  
The bioconversion of lignocellulosic into hydrogen, methane and carbon dioxide was carried out with the participation of specific microbial communities. The microbial community composition was characterized and so the biodiversity in the hydrogen- and methane-producing bioreactors was determined. A metagenomics analysis of the communities performing anaerobic digestion and gas production showed that completely different species were present in the two bioreactors. The reason for this is that the acidogenic and acetogenic microbes differ fundamentally from methanogenic microbes in their physiological needs and surrounding condition requirements. Bacterial species diversities in BR-1 and BR-2 are presented and compared in Figure 8. Remarkably, the hydrogen-producing fermenter contained exclusively Clostridia (99.69%) with two predominant species in the community: _Thermocaproicibacter melisse_ (44.86%) and _Clostridium cellulosic_ (41.94%). Two other Clostridal species (each of them close to 4% of the community) were _Thermoanaerobacterium butyriciformis_ (3.90%) and _Calorimonas adulescens_ (3.77%). Less abundant Clostridal genera were _Anaerocolumina_ of the family _Lachospiraceae_ (1.70%) and _Caldanaerobius_ of the family _Thermoanaerobacteraceae_ (1.16%), followed by other minor _Clostridia_ such as _Bionaibacter_ (of the family _Tepidanaerobacteraceae_--0.18%) and _Syntrophomonas zehnderi_, _Ruminococcus champanellensis_, _Saccharofermentans acetigenes_, _Eubacterium coprostanoligenes_ and _Ruminococcus graurus_, as the last five species sharing totally 0.05% of the community. The aerobic/microaerophilic bacilli were very limited: _Caldibacillus thermoamylocorans_ (0.08%) and _Niallia endozanthoxylica_ (0.03%). Pseudomonads were about 2%, represented by _Pseudomonas aeruginosa_ (Figure 9). The presence of _Thermocaprolactone melisse_ (genus _Oscillospecaceae_) was determined, as the first bioreactor operates at 55 \({}^{\circ}\)C and the strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37-60 \({}^{\circ}\)C, with an optimum production at 50-55 \({}^{\circ}\)C and at pHs of 5.0-7.0. This is the most probable producer of caproic acid in BR-1. A very similar thermophilic chain-elongating bacterium MDTJ8 producing n-caproate from polymeric saccharides starch and hemicellulose was recently isolated from a thermophilic acidogenic anaerobic digester by [PERSON] et al., producing n-caproate from human waste (2023) [34]. However, the authors identified the bacterium at the genus level only. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**HRT,** & **Energy Carrier** & **Total Yield, dm3** & **Yield, m3/t VS** & **Lower Heating Value, kW.h/cm3** & **Total Energy for the Whole System, kW.h/t** \\ \hline
2 & Hydrogen & 16.92 & 120.85 & 2.99 & \\  & Methane & 81.44 & 581.75 & 9.94 & 2458 \\ \hline
3 & Hydrogen & 8.23 & 58.80 & 2.99 & \\  & Methane & 42.37 & 302.67 & 9.94 & 1274 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont.__Clostridium_ species are of utmost importance among other microorganisms for fermentative H\({}_{2}\) production. _Clostridium cellulosi_ is known to convert cellulose into ethanol, acetate and hydrogen at the optimal temperature of 60 \({}^{\circ}\)C [35]. Considering the role of _Theromanaerobacterium butyrateiformis_, this species was reported to produce hydrogen from lignocellulosic sugars [36], similar to another newly isolated _Theromanaerobacterium sp._ strain MJ1. High hydrogen yields have been obtained by MJ1 using acid-pretreated  
Figure 8: Bacterial diversity in BR-1 (hydrogen-producing bioreactor) and BR-2 (methanogenic bioreactor) analysed via metagenomics. The share of the main classes as a percentage of the whole bacterial community is presented. Figure 9: Bacterial diversity in BR-1 analysed by metagenomics. The species are listed in order of decreasing shares of the bacterial community in percent. sugarcane bagasse hydrolysateate [37].