Anaerobic digestion using mixed-culture with broader choice of pretreatments for hydrogen (H₂) production was investigated. Pretreatment of wastewater sludge by five methods, such as heat, acid, base, microwave and chloroform was conducted using crude glycerol (CG) as substrate. Results for heat treatment (100 °C for 15 min) showed the highest H₂ production across the pretreatment methods with 15.18 ± 0.26 mmol/L of medium at 30 °C in absence of complex media and nutrient solution. The heat-pretreated inoculum eliminated H₂ consuming bacteria and produced twice as much as H₂ as compared to other pretreatment methods. The fermentation conditions, such as CG concentration (1.23 to 24 g/L), percentage of inoculum size (InS) (1.23% to 24% v/v) along with initial pH (2.98 to 8.02) was tested using central composite design (CCD) with H₂ production as response parameter. The maximum H₂ production of 29.43 ± 0.71 mmol/L obtained at optimum conditions of 20 g/L CG, 20% InS and pH 7. Symbiotic correlation of pH over CG and InS had a significant (p-value: 0.0011) contribution to H₂ production. The mixed-culture possessed better natural acclimatization activity for degrading CG, at substrate inhibition concentration and provided efficient inoculum conditions in comparison to mono- and co-culture systems. The heat pretreatment step used across mixed-culture system is simple, cheap and industrially applicable in comparison to mono-/co-culture systems for H₂ production.
Keywords: 1,3-propanediol; crude glycerol; hydrogen; mixed-culture; secondary wastewater sludge.