Syntrophic metabolism of a co-culture containing Clostridium cellulolyticum and Rhodopseudomonas palustris for hydrogen production

Abstract

Several studies have explored combining fermentative and purple bacteria to increase hydrogen yields from carbohydrates, but the metabolic interaction between these organisms is poorly understood. In an artificial co-culture containing Clostridium cellulolyticum and Rhodopseudomonas palustris with cellulose as the sole carbon source, we examined cell growth kinetics, cellulose consumption, H₂ production, and carbon transfer from C. cellulolyticum to R. palustris. When cultured alone, C. cellulolyticum degraded only 73% of the supplied cellulose. However, in co-culture C. cellulolyticum degraded 100% of the total cellulose added (5.5 g/L) and at twice the rate of C. cellulolyticum monocultures. Concurrently, the total H₂ production by the co-culture was 1.6-times higher than that by the C. cellulolyticum monoculture. Co-culturing also resulted in a 2-fold increase in the growth rate of C. cellulolyticum and a 2.6-fold increase in final cell density. The major metabolites present in the co-culture medium include lactate, acetate and ethanol, with acetate serving as the primary metabolite transferring carbon from C. cellulolyticum to R. palustris. Our results suggest that the stimulation of bacterial growth and cellulose consumption under the co-culture conditions is likely caused by R. palustris' removal of inhibitory metabolic byproducts (i.e., pyruvate) generated during cellulose metabolism by C. cellulolyticum.

Introduction

Cellulolytic microorganisms, such as cellulolytic clostridia, can break down cellulosic materials and ferment the sugars to hydrogen gas (H₂), a promising transportation fuel [1], [2], [3]. As such, using cellulolytic clostridia may serve an economically relevant role in H₂ production in industrial digestion processes, particularly in anaerobic digesters fed with municipal waste or where agricultural raw materials containing a high percentage of cellulosic materials are used as feedstock [4], [5]. However, fermentative H₂ yields are low because electrons are lost in the obligate excretion of organic acids and alcohols, indicating a relatively inefficient pathway of carbon flow by cellulolytic clostridia [1], [6], [7], [8]. A solution to this obstacle could be co-culturing of cellulolytic clostridia with photosynthetic purple bacteria that can consume fermentation products and produce H₂ via nitrogenase during nitrogen fixation [9], [10], [11]. Most anoxygenic photosynthetic purple bacteria cannot consume sugars but thrive and produce H₂ when growing on another microbe's fermentation waste products. Thus, purple bacteria are a natural choice co-culturing with cellulolytic clostridia to increase H₂ production from carbohydrates.

Previous work combining these bacterial metabolisms has focused on a two-stage process. In the first stage, fermentative bacteria are used to ferment carbohydrates to H₂ and soluble products. The fermentative effluent is then transferred to a photobioreactor containing purple bacteria where the organic acids are further converted to H₂ in the second stage [10], [11], [12]. However, efforts have also been made to form a consolidated bioprocess in which the same two groups of microbes, fermentative and purple bacteria, are co-cultured [13], [14], [15], [16], [17], [18]. While modest H₂ production has been reported, all but one of these studies used simple sugar feedstocks (such as glucose) rather than polymers such as cellulose. The one exception showed that purple bacteria could be used to produce H₂ from cellulose by co-culturing with a cellulose-degrading bacterium, but in this case the cellulose-degrading bacterium itself did not produce H₂ [19]. These studies showed that co-culturing can improve H₂ production from sugars. However, there is still much to be understood about the key metabolic interactions and population dynamics in such co-cultures that are critical for improvement of syntrophic efficiency and H₂ productivity [20]. Continued improvements in our ability to track metabolite fluxes will provide a more comprehensive picture of intermediary metabolism and the factors that control the flux of organic matter that result in H₂ production.

To obtain a better understanding of syntrophic metabolism, we examined the kinetics of cellular growth and carbon transfer in an artificial syntrophic co-culture containing Clostridium cellulolyticum H10 and purple bacterium Rhodopseudomonas palustris CGA676 [21], [22], [23] with the goal of increasing H₂ yields from carbohydrates. Constitutive nitrogenase activity in CGA676 allows for H₂ to be produced, even in media containing ammonia, which would normally inhibit H₂ production by nitrogenase [23]. In this co-culture system, cellulose is the sole carbon and energy source for C. cellulolyticum. R. palustris utilizes the fermentation products secreted by C. cellulolyticum, and H₂ is produced by both organisms. Because both organisms have been fully sequenced, their co-culture is an ideal way to begin to understand the genomic underpinnings of syntrophic interactions. Both genomic information and an understanding of the physiology of the co-culture could eventually lead to a robust metabolic model of the consortium, an important first step toward a systems biology understanding of microbial communities. We found that the presence of R. palustris in the co-culture greatly stimulated cellulose degradation and H₂ production, which likely resulted from accelerated metabolism of C. cellulolyticum and consumption of acetate and pyruvate by R. palustris.