Mesophilic biohydrogen production from buffalo slurry co-fermented with cheese whey and crude glycerol: Optimization using Mixture Design

Biohydrogen production from buffalo slurry (BS) co-fermented with cheese whey (CW) and crude glycerol (CG) was investigated using a suitable microbial community (F210) as inoculum. Mixture Design was used to individuate the optimal composition (%) of the three substrate components and to investigate the effect of the mixing ratio on Bio-H₂ yields. Maximum H₂ yield estimated through the model was around 117 mL H₂/g VS_added, while the maximum experimentally detected was 111.6±21.8 mL H₂/g VS_added, obtained for a mixing ratio of substrate composition of 66% BS and 33% CW (R²=0.962; p-value=0.0001). CW was clearly the most suitable substrate (with a relative contribution higher than 46%), but led to a rapid drop in pH from 6.5 to 4, while BS showed high buffering capacity by maintaining the pH above 6. The results demonstrate the usefulness of the mixture design for finding the optimal substrate composition using BS as co-fermentation substrate to obtain high H₂ production yields. Moreover the response surface shows the possibility of mixing the substrates in different ways, while maintaining H₂ production within an optimum range: 105-117 mL H₂/g VS_added. This might offer a considerable advantage in the effective management of systems or processes in which the substrates availability may change over the time.