Application of molecular techniques on heterotrophic hydrogen production research

Abstract

This paper reviews the application of molecular techniques in heterotrophic hydrogen production studies. Commonly used molecular techniques are introduced briefly first, including cloning-sequencing after polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP), fluorescence in situ hybridization (FISH) and quantitative real-time PCR. Application of the molecular techniques in heterotrophic hydrogen production studies are discussed in details, focusing on identification of new isolates for hydrogen production, characterization of microbial compositions in bioreactors, monitoring microbial diversity variation, visualization of microbial distribution in hydrogen-producing granular sludge, and quantification of various microbial populations. Some significant findings in recent hydrogen production studies with the application of molecular techniques are discussed, followed by a research outlook of the heterotrophic biohydrogen field.

Introduction

Energy and environment are essential for sustainable development of the global prosperity. Currently, over 80% of energy supply is dependent on fossil fuels, which cause the deterioration of environment and rapid exhaustion of natural energy sources (Guo et al., 2010). This has led to the search for alternative energy sources, among which hydrogen has attracted much attention recently. As a clean energy, producing only water after combustion, hydrogen may become an alternative to fossil fuels in the future. It also has a high energy yield of 122 kJ/g, which is about 2.75 times that of fossil fuels (Kim et al., 2006a).

At present, hydrogen is commercially produced by either thermocatalytic reformation of hydrocarbons or electrolysis of water, both of which are highly energy consuming and unsustainable processes (Das and Veziroglu, 2008). Heterotrophic biological production of hydrogen has, however, attracted research interests due to its potential ability of degrading organic pollutants which serve as carbon and energy sources for the microbes during harvesting hydrogen (Li and Fang, 2009). Heterotrophic hydrogen production is often classified into two categories depending on whether light is required, i.e. dark fermentation and photo fermentation (Levin et al., 2004). Dark fermentation converts organic pollutants into hydrogen by dark fermentative bacteria in the absence of light, producing organic acids, mainly acetate and butyrate, and alcohols as by-products. Photo fermentation is potentially able to convert acids and alcohols, which are the by-products of dark fermentation, into hydrogen by photosynthetic bacteria using light as energy source. Dark fermentation has a high production rate of hydrogen, but with low hydrogen yield, converting no more than 40% of the chemical energy in the organic pollutants into hydrogen (Li and Fang, 2007). In comparison, photo fermentation produces little organic residues, resulting in higher hydrogen yield, but has much lower hydrogen production rate than dark fermentation (Lee et al., 2010).

Various factors have been studied for heterotrophic hydrogen production including source of inoculation, feeding substrates, reactor design and operating conditions such as pH, temperature and hydraulic retention time (HRT) etc. With the development of molecular techniques, identification and quantification of microorganism communities involved in hydrogen production become more convenient, effective and accurate. The nucleic acid based techniques have been widely used in heterotrophic hydrogen production studies in the past decade, which contributed much to identification of the new isolated hydrogen-producing bacteria, exploration of the metabolic functions and interactions of different species in hydrogen production system, and investigation on the effects of operational factors on microbial communities. The application of molecular techniques will thus help to optimize the operational conditions of the bioreactors, improve the reactor stability, and increase the hydrogen production rate and yield.

This article aims to review the application of molecular techniques in heterotrophic hydrogen production studies. Commonly used molecular techniques are introduced briefly first, including cloning-sequencing after polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993), terminal-restriction fragment length polymorphism (T-RFLP) (Liu et al., 1997), fluorescence in situ hybridization (FISH) (Wagner et al., 2003) and quantitative real-time PCR (qRT-PCR) (Zhang and Fang, 2006). Applications of the above molecular techniques in analysis of microbial communities in hydrogen production bioreactors are discussed in details, focusing on identification of new isolates for hydrogen production, characterization of microbial compositions in bioreactors, monitoring microbial diversity variation, visualization of microbial distribution in hydrogen-producing granular sludge, and quantification of various microbial populations. Four tables were compiled for heterotrophic hydrogen production data scattered in literature in terms of aforementioned respective applications except visualization of microbial distribution in granular sludge, which had very limited reports. Data summarized in tables include fermentation type (dark or photo-fermentation), seed sludge (or isolation source for Table 1), reactor design (batch or type of continuous reactor), culture volume (l), substrate, maximum volumetric hydrogen production rates (l-H₂/l/h), hydrogen yield (mol H₂/mol carbohydrates consumed for dark fermentation and compared to stoichiometry (%)), cell density (g-VSS/l), molecular techniques used and microbial analysis results.