Hydrogen is an important source of energy and is considered as the future energy carrier post-petroleum era. Nowadays hydrogen production through various methods is being explored and developed to minimize the production costs. Biological hydrogen production has remained an attractive option, highly economical despite low yields. The mixed-culture systems use undefined microbial consortia unlike pure-cultures that use defined microbial species for hydrogen production. This review summarizes mixed-culture system pretreatments such as heat, chemical (acid, alkali), microwave, ultrasound, aeration, and electric current, amongst others, and their combinations to improve the hydrogen yields. The literature representation of pretreatments in mixed-culture systems is as follows: 45–50% heat-treatment, 15–20% chemical, 5–10% microwave, 10–15% combined and 10–15% other treatment. In comparison to pure-culture mixed-culture offers several advantages, such as technical feasibility, minimum inoculum steps, minimum media supplements, ease of operation, and the fact it works on a wide spectrum of low-cost easily available organic wastes for valorization in hydrogen production. In comparison to pure-culture, mixed-culture can eliminate media sterilization (4 h), incubation step (18–36 h), media supplements cost ($4–6 for bioconversion of 1 kg crude glycerol (CG)) and around 10–15 Millijoule (MJ) of energy can be decreased for the single run.

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.