Bioproduction of butanol from biomass: from genes to bioreactors

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Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield. Advances in integrated fermentation and in situ product removal processes have resulted in a dramatic reduction of process streams, reduced butanol toxicity to the fermenting microorganisms, improved substrate utilization, and overall improved bioreactor performance.

Introduction

Anaerobic bacteria such as the solventogenic clostridia are capable of converting a wide range of carbon sources (e.g. glucose, galactose, cellobiose, mannose, xylose and arabinose) to fuels and chemicals such as butanol, acetone, and ethanol [1••]. However, butanol toxicity to the fermenting microorganisms limits its concentration in the fermentation broth, resulting in low butanol yields and a high cost for butanol recovery from the dilute solutions. During the past decade, the application of molecular techniques to the solventogenic clostridia — combined with recent advances in fermentation techniques — have resulted in the development of a hyper-butanol-producing strain and an integrated acetone-butanol-ethanol (ABE) fermentation system for the simultaneous production and removal of butanol from the fermentation broth [2].

Efforts have been made to understand the mechanisms of sugar transport, regulation of butanol production, butanol tolerance, utilization of lignocellulosic biomass hydrolysates, and cell inhibition by lignocellulosic degradation products, with the aim of improving butanol productivity, titer and yield. Genetic manipulation of the microorganism depends on the development of effective gene transfer and associated systems. Genetic tools for transformation of the solventogenic clostridia have been developed and transformation of this genus with suitable genes coding for active hydrolytic enzymes would increase not only the utilization of a variety of carbon sources but also the efficiency. This review will focus on current advances in biobutanol production with reference to biomass utilization, strain improvement, bioreactor design and operations.

Section snippets

Regulation of carbohydrate utilization in solventogenic clostridia

The solventogenic clostridia have received much attention in recent years, because of their ability to produce industrially relevant chemicals such as butanol and 1,3-propanediol. The clostridia secrete numerous enzymes that facilitate the breakdown of polymeric carbohydrates into monomers (Figure 1). The secreted carbohydrate-degrading enzymes include, but are not limited to, α-amylase, α-glucosidase, β-amylase, β-glucosidase, glucoamylase, pullulanase, and amylopullulanase. The various

Butanol fermentation by solventogenic clostridia

The fermentation profile of a microorganism is ultimately determined by its genetic makeup, which in turn controls the expression of relevant enzymes. An important advantage of the solventogenic clostridia is the variety of fermentation products (acetone, butanol, ethanol, acetic, butyric, lactic acids, etc.) that can be synthesized by this group of microorganisms. However, the loss of available carbon as a result of the formation of unwanted products is an undesirable property of the

Genetic strain improvement

The biobutanol fermentation suffers from several limitations (e.g. low titer, yield and productivity) and improvements in the performance of the solventogenic clostridia are necessary to move biobutanol fermentation research to a competitive commercial position. Several approaches have been employed to improve the performance of solventogenic clostridia with the aim of generating strains that can be used in industrial biobutanol production. Recombinant DNA technology, in addition to traditional

Advanced fermentation techniques and novel downstream processing

Traditionally, batch fermentations were commonly used for butanol production because suitable technologies to address the product toxicity problems associated with ABE fermentation were not available. During the 1940s and 1950s, biobutanol production on an industrial scale (Terre Haute, IN and Peoria, IL) was carried out using large batch fermentors ranging in capacity from 200 000 to 800 000 L. The industrial process used 8–10% corn mash, which was cooked for 90 min at 130–133 °C. Sugar cane

Immobilized and cell recycle continuous bioreactors

In a biobutanol batch process, reactor productivity is limited to less than 0.50 g/L/h for a number of reasons, including low cell concentration, down time, and product inhibition (reviewed in [19]). In a batch reactor a cell concentration of <4 g/L is normally achieved. The cell concentration inside the bioreactor can be increased by one of two techniques, namely ‘immobilization’ or ‘cell recycle’. In a study to explore different cell supports (e.g. clay brick) for C. beijerinckii cells, Qureshi

Gas stripping

Gas stripping is a technique that can be applied for in situ butanol recovery during the ABE fermentation [19, 21, 22]. The production of ABE is associated with the generation of gases (CO2 and H2). In an attempt to make the process of ABE recovery from the fermentation broth simpler and more economical, these fermentation gases are used to recover butanol during simultaneous fermentation and in situ recovery by gas stripping [19, 21, 22]. The gases are bubbled through the fermentation broth

Liquid–liquid extraction

The removal of butanol or ABE from fermentation broth by liquid–liquid extraction is considered an important technique. Usually, a water-insoluble organic extractant is mixed with the fermentation broth. Butanol is more soluble in the organic (extractant) phase than in the aqueous (fermentation broth) phase; therefore, butanol selectively concentrates in the organic phase. As the extractant and fermentation broth are immiscible, the extractant can easily be separated from the fermentation broth

Perstraction

Several problems are associated with liquid–liquid extraction, such as toxicity to the cells, the formation of an emulsion, loss of extraction solvent, and the accumulation of microbial cells at the extractant and fermentation broth interphase. To solve these concerns, a new technique called ‘perstraction’ was developed [19]. In a perstractive separation, the fermentation broth and the extractant are separated by a membrane. The membrane contactor provides surface area where the two immiscible

Pervaporation

Pervaporation is a technique that allows the selective removal of volatile compounds from model solution/fermentation broth using a membrane. The membrane is placed in contact with the fermentation broth and the volatile or organic component selectively diffuses through the membrane as a vapour. The compound is then recovered by condensation. In this process, a phase change occurs from liquid to vapor. As it is a selective removal process, the desired component requires a heat of vaporization

Economics of the ABE fermentation

In recent years several economic studies have been performed on the production of butanol from corn (dry corn and wet corn milling) whey permeate, and molasses [19, 21]. In these studies it was determined that the distillative recovery of biobutanol from the fermentation broth is not economical when compared with butanol derived from the current petrochemical route. Nevertheless, studies employing C. beijerinckii BA101, C. acetobutylicum P260, hydrolyzed DDGS and wheat straw suggest that

Conclusions

The use of lignocellosic biomass as a substrate for biobutanol fermentation has great potential. Much progress has been made with the use of DDGS (TC Ezeji, HP Blaschek, unpublished), wheat straw [18••], and corn fiber xylan [27••]. Nevertheless, to use substrates such as corn fiber hydrolysate and to meet the economic challenges associated with this fermentation, new strains that are both capable of utilizing mixtures of lignocellulosic-derived sugars and resistant to microbial inhibitors

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by grants from the United States Department of Agriculture (USDA) [231 AG2006-35504-17419] and the Illinois Council on Food and Agricultural Research (CFAR) [IDA CF 06DS-01-03].

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