is the fermentative
conversion of organic substrate to biohydrogen
, it is a complex process manifested by diverse group of bacteria
by a series of biochemical
reactions involving three steps similar to anaerobic conversion
. Dark fermentation differs from photofermentation
because it proceeds without the presence of light
Fermentative/hydrolytic microorganisms hydrolyze complex organic polymers to monomers which further converted to a mixture of lower molecular weight organic acids and alcohols by obligatory H2 producing acidogenic bacteria.
Utilization of wastewater as a potential substrate for biohydrogen production has been drawing considerable interest in recent years especially in dark fermentation process. Industrial wastewater as fermentative substrate for H2 production addresses most of the criteria required for substrate selection viz., availability, cost and biodegradability (Angenent, et al., 2004; Kapdan and Kargi, 2006). Chemical wastewater (Venkata Mohan, et al., 2007a,b), cattle wastewater (Tang, et al., 2008), diary process wastewater (Venkata Mohan, et al. 2007c), starch hydrolysate wastewater (Chen, et al., 2008) and designed synthetic wastewater (Venkata Mohan, et al., 2007a,2008b) have been reported to produce biohydrogen apart from wastewater treatment from dark fermentation process using selectively enriched mixed culture under acidophilic conditions. Various wastewaters viz., paper mill wastewater (Idania, et al., 2005), starch effluent (Zhang, et al., 2003), food processing wastewater (Shin et al., 2004, van Ginkel, et al., 2005), domestic wastewater (Shin, et al., 2004, 2008e), rice winery wastewater (Yu et al., 2002), distillery and molasses based wastewater (Ren, et al., 2007, Venkata Mohan, et al., 2008a), wheat straw wastes (Fan, et al., 2006) and palm oil mill wastewater (Vijayaraghavan and Ahmed, 2006) were also studied as fermentable substrates for H2 production along with wastewater treatment. Using wastewater as a fermentable substrate facilitates both wastewater treatment apart from H2 production. The efficiency of dark fermentative H2 production process was found to depend on the pre-treatment of the mixed consortia used as biocatalyst, operating pH, organic loading rate apart from wastewater characteristics (Venkata Mohan, et al., 2007d,2008c,d, Vijaya Bhaskar, et al., 2008d).
Employing mixed culture is extremely important and well-suited to the non-sterile, ever-changing, complex environment of wastewater treatment (Angenent, et al., 2004, Das, 2008). Typical anaerobic mixed cultures can not produce H2 as it is rapidly consumed by the methane-producing bacteria (Sparling, et al., 1997). Successful biological H2 production requires inhibition of H2 consuming microorganisms, such as methanogens and pre-treatment of parent culture is one of the strategies used for selecting the requisite microflora. The physiological differences between H2 producing bacteria (also referred to as acidogenic bacteria) and H2 consuming bacteria (methanogenic bacteria) form the fundamental basis behind the development of various methods used for the preparation of H2 producing seeds (Zhu and Beland, 2006). When parent inoculum was exposed to extreme environments such as high temperature, extreme acidity and alkalinity, spore forming H2 producing bacteria such as Clostridium survived, but methanogens had no such capability. Pre-treatment helps to accelerate the hydrolysis step, thus, reducing the impact of rate limiting step and augment the anaerobic digestion to enhance the H2 generation (Kim, et al., 2003, Venkata Mohan, et al. 2007d, 2008c). Several pre-treatment procedures viz., heat-shock, chemical, acid, alkaline, oxygen-shock, load-shock, infrared, freezing, etc., were employed on a variety of mixed cultures (Sparling, et al., 1997; Logan et al., 2002; Ferchichi et al., 2005; Kim, et al., 2003, Valdez-Vazquez, et al., 2006; Kraemer and Bagley, 2007; Venkata Mohan et al., 2007c,d,2008a,e) for selective enrichment of acidogenic H2 producing inoculum. pH also plays a critical role in governing the metabolic pathways of the organism where the activity of acidogenic group of bacteria is considered to be crucial (Fan, et al., 2006). Optimum pH range for the methanogenic bacteria is reported to be between 6.0 and 7.5, while acidogenic bacteria functions well below 6 pH (van Ginkel, et al., 2005). The pH range of 5.5-6.0 is considered to be ideal to avoid both methanogenesis and solventogenesis (Fan, et al., 2006, Venkata Mohan, et al., 2007d,2008c,d) which is the key for effective H2 generation.
In spite of advantages, the main challenge observed with fermentative H2 production process is relatively low energy conversion efficiency from the organic source. Typical H2 yields range from 1 to 2 mol of H2/mol of glucose, which results in 80-90% of the initial COD remaining in the wastewater in the form of various volatile organic acids (VFAs) and solvents, such as acetic, propionic, butyric acids and ethanol (Logan, 2004). Even under optimal conditions about 60-70% of the original organic matter remains in solution (Das and Veziroglu, 2001, Venkata Mohan et al., 2007a,2008f). Bioaugmentation with selectively enriched acidogenic consortia to enhance H2 production was also reported (Venkata Mohan, et al., 2007b). Generation and accumulation of soluble acid metabolites causes sharp drop in the system pH and inhibit the H2 production process. Usage of unutilized carbon sources present in acidogenic process for additional biogas production sustains the practical applicability of the process. One way to utilize/recover the remaining organic matter in a useable form are to produce additional H2 by terminal integration of photo-fermentative process H2 production (Venkata Mohan, et al., 2008e) and methane by integrating acidogenic process to terminal methanogenic process (Venkata Mohan, et al., 2008b).
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