A review on bio-butyric acid production and its optimization.
Butyric acid is treated as one of the renewable green fuels of tomorrow due to its high energy content and it can reduce health and environmental issues including emission of greenhouse gases global warming and climate change. The production of bio- butyric acid by microbial fermentation is not cost-effective and economically competitive due to its production at a relatively low concentration yield and rate. In order to enhance the economics of the fermentation method the butyrate production should increase using economical substrate favorable pretreatment techniques multicultural stains and fermentation conditions. In order for further investigation and improvement in butyric acid production batch repeated batch fed batch andcontinuous type butyric acid fermentation of biomass have been discussed in this review. Copyright 2014 Friends Science Publishers
Keywords: Butyric acid; Clostridium; Fermentation; Renewable biomass
Energy has been regarded as one of the main elements of human life social civilization and techno-socio-economic progress. However the advances in energy technologies have brought various revolutions throughout the world. Present global energy supply-consumption chain is obviously unsustainable from the technical environmental economic and social points of view due to limited availability of fossil fuels and inevitably be depleted (Barnes and Floor 1996). The emissions of greenhouse gases and their consequences including global warming acid rainclimate change and other environmental issues force us to think about alternative fuels although conventional fossil fuels is the world's vital energy resource (Jha and Jha2010). The application and development of bio-fuels canreduce the consumption of fossil fuels and alleviate energy crisis to some extent. The recent advances in the fields of biotechnology and microbial fermentation technologies have resulted in a renewed attention in bio-butyric acid production from low cost renewable biomass (Zhang et al.2009; Dwidar et al. 2012).A four-carbon short chain (CH3CH2CH2COOH) bio- butyric acid and its derivatives have numerous potential applications in chemical textile plastic food beverage dairy and pharmaceutical industries (Zigova and Sturdik2000). They are extensively used as solvent diluentsdrugs plasticizer perfumes fiber additive and rawmaterials (Zhang et al. 2009). Bio-butyric acid is regardedas a prospective chemical building-block to makechemicals. It is also regarded as a promising specialty chemical as it can be converted to bio-butanol. But itscommercial production is dominated by chemical synthesis. However industrial production of butyric acid mainly depends upon crude oil due to comparatively lower production cost and large scale supply consumers prefer butyric acid of natural origin especially for foodstuffadditives or pharmaceutical products (Zigova and Sturdik2000). With decreasing availability of crude oil growingdemand for natural products and rising concerns overenvironment microbial fermentation technology for butyric acid production from renewable biomass has been paying attention of many researchers because bio-butyric acid could be one of the most promising sustainable bio-fuels to meet the desires of green energy supply for replacing fossilfuels. In addition presence of profuse lignocellulose biomass as low-value agricultural commodities or obligation of apt disposal of bio-wastes to pass up pollution tribulations have been creating favorable business climate for butyric acid fermentation.Although butyric acid is a significant bio-fuel todevelop sustainable green society its production through fermentation of biomass has been regarded as very complex and hard to control. The microbial butyric acid fermentation is affected with several process dilemmas including self-inhibitory effect of end products increasing inhibition due to pretreatment slow rate of straindevelopment (Zigova' and Sturdik 2000). The productionstrains for butyric acid fermentation also produce othertypes of acids which are very difficult to separate. Higher substrate cost degeneration of the butyric acid-producing strains limited productivity lower concentration lower yield and higher products recovery cost are main factors which limit the fermentation routes as well. Limited studies on production of bio-butyric acid by anaerobic fermentation have been reported in association to bio- refinery perspectives including selection and growth of strains and physiological examination for greater yield productivity and selectivity. A higher demand in contrast to lower production through the microbial fermentation routs gives rise to the necessity of addressing and solving the problems related to the butyric acid fermentation process. In order to improve butyric acid fermentation and reduce overall production cost extensive efforts are needed for strains improvement metabolic pathways bioreactor design feed stocks effect of inhibitors fermentation process control parameters and optimization techniques.
Appropriate microorganisms selection is the base of a successful fermentation process. Numerous bacterial strains which are suitable to produce bio-butyric acid are mainly isolated from waste water excess sludge soil contaminated dairy and food products meats and animal digestive systems. Altogether more than ten butyrate-producing bacterial strains belonged to the genera Clostridium Butyrvibrio Butyribacterium Eubacterium FusobacteriumMegasphera and Sarcina are reported (Zigova and Sturdik2000). They are Gram positive chemoorganotrophicstrictly anaerobic and spore-forming bacteria. The strains ofgenera Clostridium have been extensively used and studied microorganisms due to their high productivities and relatively higher stability followed by Butyrivibrio and Butyribacterium. Among them C. butyricum C. beijerinckii C. acetobutylicum C. tyobutyricum C. populeti and C. thermobutyricum are superior strains.Favorable culture temperature ranges from 30-37C for C. butyricum C. populeti and C. tyrobutyricum while 55C is considered as optimal culture temperature for C.thermobutyrium. Although an extensive range of carbon source is able to be utilized by clostridia glucose is common substrate for bio-butyrate production. Clostridium bacteria can utilize different types of sugars including hexoses several pentoses oligo- and polysaccharides for bio-butyric acid production while C. butyricum is able to utilize glycerol pentose hexose molasses lignocellulose cheese-whey permeat and potato starch as carbon sources. However C. tyrobutyricum can only make use of glucose xylose and fructose (Matijasic et al. 2007). Baroi et al. (2013) presented C. tyrobutyricum has the ability to transfer both pentose and hexose sugars but the xylose uptaking speed is lower than that of glucose. C. thermobutyricum which is isolated from horse dung can mainly use monomeric sugars (glucose fructose maltose xylose and ribose but not arabinose galactose and mannose) dimeric (cellobiose) oligomeric and polymeric sugars.Metabolic Pathway and Inhibition in Bo-butyricAcid FermentationExtensive studies have illustrated metabolic pathways and regulations for fermentation of glucose to produce bio- butyric acid using clostridia (Zhang et al. 2009; Zigova andSturdik 2000; Ramey and Yang 2004). Glucosefermentation by C. butyricum (Eq. 1) and C. tyrobutyricum(Eq. 2) follows the stoichiometric equations below (Zhanget al. 2009):Glucose0.8 Butyrate + 0.4 Acetate + 2.4 H2 + 2 CO2(Eq. 1)Glucose0.85 Butyrate + 0.1 Acetate + 0.2 Lactate+1.9 H2 + 1.8 CO2 (Eq. 2)The metabolic route of glucose fermentation is presented in Fig. 1. Butyric acid is produced from glucose during acetogenesis stage whereas in solventogenesis phase butyrate is converted into butanol (Ramey and Yang 2004). High ATP concentration and minimal NADH:NAD ratio should be maintained in order to prevent solventogenesis(Zigova and Sturdik 2000). Glucose is metabolized to pyruvate by means of EmbdenMeyerhofParnas and generates ATP and NADH. Afterwards acetyl-CoAacetoacetyl-CoA and butyryl-CoA are formed from pyruvate as key intermediates in the main branch (Jones and Woods 1986). Butyric acid can be produced consequently in case of presence of high levels of enzymes that are concerned with the pathway of butyryl-CoA to butyrate. During the conversion of acetyl-CoA into butyryl-CoA thiolase crotonase 3-hydroxybutyryl-CoA dehydrogenase and butyryl-CoA dehydrogenase are played vital roles as key enzymes. The bio-butyrate-producing clostridia notonly produce bio-butyric acid but also several possible by- products including acetate H2 CO2 lactate and other products. The conversion of acetyl-CoA is firstly occurred into acetyl phosphate which is converted into acetate. Similarly butyryl-CoA is firstly converted into butyryl phosphate and butyrate is produced from butyryl phosphate. The acetyl-CoA is catalyzed by phosphotransacetylase (PTA) whereas phosphotransbutyrylase (PTB) catlalyzes butyryl-CoA. Correspondingly acetate kinase (AK) and butyrate kinase (BK) catalyze acetyl-phosphates and butyryl- phosphates for the production of acetate and butyrate respectively.In fact the metabolic pathway of a microorganismduring anaerobic fermentation is affected by several factors. In case of bio-butyrate-producing clostridia mainly glucose concentration pH hydrogen partial pressure acetate and butyrate are able to influence the growth rate final products concentration and distribution of the products (Kong et al.2006; Jo et al. 2008; Rodriguez et al. 2006). It is necessity to build up an appropriate and healthy process parameters that yields low amount of acetic acid (greater selectivity) has a superior yield and a greater productivity of bio- butyric acid from lignocellulosic renewable biomass.
Surplus carbon supplies often have an effect on osmotic dehydration of microorganisms in an anaerobic fermentation process. A momentous rise in the ratio of butyrate to acetate is found in bio-butyric acid fermentation process with limited amount of glucose and C. butyricum as working microorganism (Saint-Amans and Soucaille 1995). It is noteworthy that various pH values can influence not only the sharing of produced organic acids but also cellmembrane transfer behavior and cell lysis (Zigova and Sturdik 2000). Relatively higher pH (e.g. greater than 6.0) is useful for cell development and biosynthesis of butyric acidespecially in the case of C. butyricum (He et al. 2005). Furthermore media pH also has an effect on the particular growth rate bio-butyric acid production rate and less sugars utilization rate. For C. tyrobutyricum various pHs are able to change the distribution of the metabolic flux. At pH 6.3 the highest bio-butyrate production is observed compared to that at pH 6.0 and 6.7 (Zhu and Yang 2003; Jo et al.2008). Bio-butyrate production was lower at lower pH with acetate and lactate as the main acid products at pH 5.0. The metabolic shift from butyrate formation at pH 6.3 to lactate and acetate formation at pH 5.0 is associated with decreased activities of PTB and independent lactate dehydrogenase (iLDH) and increased activities of PTA and LDH (Zhu and Yang 2003). In butyrate-producing strains PTA AK PTB BK iLDH and LDH are the main enzymes relevant to
acetate butyrate and lactate production. Their products distribution is affected by media pH significantly. AK and BK in the direction of acyl-phosphate formation were not significantly affected by the pH between 5.0 and 7.0. However in the acyl-phosphate-forming direction the activity of PTA increased while PTB decreased withincreasing the pH (Zhu and Yang 2003). It has also beenreported that under a low partial pressure of H2 the ratio of acetate to butyrate increased with a decrease in hydrogenpartial pressure accompanied by an increase of ATP yield during bio-butyric acid production fermentation by C.butyricum (van Andel et al. 1985). The production of bio- butanol from butyric acid is also affected by inadequate pH.
Soni and Jain (1997) studied the consequence of pH on bio- butyrate uptake as a result of the transformed strain of clostridium acetobutylicum and found that lower pH (less than 4.6)unfavorably exaggerated overall metabolic activity.Minimum pH 5.2 was needed for uptake of bio-butyrate at aconcentration of 4 g/L. They also observed that a straight connection among minimum pH prerequisite butyrateconcentration and allied anaerobic fermentationtemperature. The end product inhibition is a challenge for the researchers working to enhance product concentration.Undissociated bio-butyric acid gets ahead of through the bacterial membrane and detaches within the cell. It impingeson the transmembrane pH gradient and declines the sum of existing energy for biomass growth (Zigova and Sturdik2000). One of the approaches to solve this problem is todevelop a combination of butyric acid tolerated strains whileother technique may be online separation or in situ product removal especially extraction and pertraction.The concomitant production of acetic acid as by- product with butyrate production creates problems for the recovery of bio-butyric acid in downstream processing (Zhang et al. 2009). For example in the immobilized cell anaerobic fermentation process a greater quantity of 0.27 mol/mol of acetate was formed along with 0.95 mol/mol of butyrate from glucose (Ramey and Yang 2004). It is obvious that reducing biomass formation and acetate production increases butyrate yield significantly. Ramey and Yang (2004) reported that complete elimination of acetate formation could increase butyric acid yield more than 1 mol/mol for glucose and 0.83 mol/mol for xylose as the substrate for immobilized cell fermentation. Knocking out the acetate-producing pathway can increase the butyrate production.
Optimization of Butyric Acid Bio-production
The conventional bio-butyric acid production technique is not yet cost-effective and economically competitive
Table 1: Some examples of butyrate-production by fermentation
Feedstocks###Pretreatment###Culture design###Strain###Cul. temp. pH###Butyrate###Reference
Cane molasses###Sulfuric acid###Fed batch/Immobilized fibrous###C. tyrobutyricum###37###6.0###55.2###Jiang et al. 2009
Cheese whey###-###Batch###C. beijerinckii###37###5.5###greater than 12###Alam et al. 1988
Corn stalk###5-8 mm size 1% (v/v) Immobilized###continuous###C.###55###6.0###15.82###Li et al. 2011
Jerusalem###0.01M Sulfuric acid###Fed###batch/Immobilized###C. tyrobutyricum###37###6.0###60.4###Huang et al.
Sugarcane###0.10.5 M HCl and enzymatic Fed batch culture/Immobilized###C. tyrobutyricum###37/###200 6.0###20.9###Wei et al. 2013
bagasse###hydrolysis with cellulases fibrous bed bioreactor###rpm
Glucose###from Pretreated and hydrolyzed###Batch###C. tyrobutyricum###6.0- 7.0###71.6###Baroi et al. 2013
Xylose###from pretreated and hydrolyzed###Batch###C. tyrobutyricum###6.0- 7.0###55.4###Baroi et al. 2013
due to lower concentration lower productivity and lower yield of the bio-butyrate. The production of byproducts such as acetic acid propionic acid and ethanol causes further reduction in butyric acid concentration and increases the costs for product recovery and purification. It means the complicated and expensive isolation process also limits its commercialization. In order to increase the economics of butyrate production various optimizing techniques may be useful.
The cost of feed stocks is regarded as a key issue for economical production of bio-butyric acid by microbial fermentation process. Considering economics of the bio- butyric acid fermentation it can be pointed out that agricultural products would not be feasible substrates due to high cost and direct uses for human beings and other animals. In such situations agricultural residues and other industrial wastes are the favorable substrates based on itscomposition availability cost good water retention capacity and ease of pretreatment. Lignocellulosic materials such as maize straw rice straw barley wheat strawmolasses and dairy wastes have potential to serve as low cost renewable raw materials for bio-butyric acid fermentation mainly in agriculture based countries. They are readily available and inexpensive renewable biomass. In fact they consist of cellulose hemi-cellulose lignin and smaller quantities of pectin protein extractives and ash. Li et al. (2011) observed that corn stalk has a great potential to immobilize C. thermobutyricum for bio-butyric acid fermentation. Similarly Jiang et al. (2009) and Vandak et al. (1995) have used cane molasses for bio-butyric acid fermentation using C. butyricum and C. tyrobutyricum respectively. Baroi et al. (2013) reported the main contents in the sugars yielded from wheat straw haves glucose (71.6 g/L) and xylose (55.4 g/L) respectively.Hemicelluloses the second most abundant available polysaccharides in nature represent around 20-35% of lignocellulosic biomass (Ezeji et al. 2007). Enoughfermentable carbon substrates can be obtained from lignocellulosic biomass while the cellulosic and hemicellulosic hydrolysates and starch can be used to produce butyric acid production. However these lignocellulosic residues are abundant available in the developing countries they are being used inefficiently andconsequently cause considerable environmental evils. The beauty of cellulosic materials is that significant amounts of different types of sugar can be obtained through hydrolysisprocess which are useful for bio-production of butyric acid and bio-butanol and consequently minimize waste generation (Li et al. 2011).
The degradation of lignocelluloses is the major obstacle for utilizing renewal and cost-effective biomass including agricultural residues to produce bio-butyric acid. Pretreatment methods are considered as one of the approach to solve this problem. The treatment of molasses by sulfuric acid increased butyric acid concentration by 32.6% yield by31.8% and sugar utilization 12.3% compared to untreated molasses (Jiang et al. 2009). In contrast high costs of hydrolyzing cellulose into simple monomeric sugars and formation of inhibition products during the hydrolysis are the major limitation for pretreatment techniques. The effect of different pretreatment methods including acid base thermal thermal-acid and thermal-alkali with various pretreatment time temperature and concentration for higher glucose production and low quantity of inhibition products and consequently higher butyrate production will be focus for the future researches. In addition physical pretreatment such as appropriate size for substrate and biological treatment are very significant for higher butyric acid production as well.
Inability of most of the single butyrate-producing strains to grow and degrade substrate at different fermentationconditions is one of the major barriers for butyrate fermentation. The identification of a combination of multicultured microbes and their metabolic pathways will accelerate fermentation process to produce higher rate of butyric acid because (i) different conditions and process parameters might be favorable for different microbes; (ii) the inhibition problems due to hydrolysis and fermentation products will be decreased by having better tolerance to butyric acid inhibition for the microbes. The depth insight for finding combinations of microbial strains will be major focus for future researches.
Reactor Design and its Operation
Batch repeated batch fed batch continuous and cell recycle anaerobic fermentation processes are widely utilized for the researches on butyric acid bio-production (Table 1). It was observed that greater butyrate concentrations could be achieved in batch culture while higher productivity might be obtained in continuous cultures (Michel-Savin et al. 1990). Repeated batch culture could eliminate the lag phase by providing adaptation period required for cells to survive in a changed environment. Adaptation of bacterial cultures and shift in metabolic pathways in fed batch fermentation could increase further butyrate concentration and consequently butyrate/acetate ratio. The productivity can be enhanced using cell recycling process or cell immobilization. Jiang et al. (2009) studied performance of a fibrous bed bioreactor (FBB) with immobilized C. tyrobutyricum under batch repeated batch and fed batch fermentation systems in order to optimize bio-butyric acid production from cane molasses. The fed-batch fermentation produced 61.9% higher butyrate concentration and reduced 50.9% acetic acid but yield and productivity were decreased by 16.4 and 46.9% compared to the batch fermentation process. Similarly Mitchell et al. (2009) found that the continuous culture system with immobilized cells has improved butyric acid productivity and consequently reduced product separation cost. The fibrous bed bioreactors due to their regeneratives and higher mass transfer capabilities can maintain the productivity over a longer period. Huang et al. (2002) reported that cells immobilized FBB with packed fibrous matrix is successful in producing most of the commercially used organic acids.
The identification of relatively optimum process parameters will solve the problems related to efficient butyrate production to some extent. It is believed that thermophilic microorganisms can produce higher fermentation products due to higher rate of hydrolysis improved mass transfer and reduced susceptibility to contamination. The pH value and acetic acid concentration have also significant effects on the total organic acids and butyric acid production productivity and yield. As overall metabolic activity of Clostridium bacterium is decreased at low pH it is considered that a pHrange from 4.5 to 7.0 is favorable for butyric acid fermentation (Zigova et al. 1999). According to Jiang et al. (2009) the optimum pH for butyric acid production and its microorganism growth is 6.0. They further explained that lowering pH to 5.0 brings a change in metabolic shift for acetate production from butyric acid production route and consequently acetate becomes the major product. Alam et al. (1988) noted that a constant pH 5.5 provides highest level of butyric acid from cheese whey using C. beijerinckii at 37C in batch culture. Similarly Li et al. (2011) observed highest butyric acid yield from corn stalk at pH 6.0 while pH 7.0 was favorable for maximal butyrate/acetate ratio in a continuous type immobilized cell reactor using C. thermobutyricum at 55C.
Separation of Butyrate
Difficulty in separating butyric acid from anaerobic fermentation broth is considered as one of the major bottlenecks for butyrate production. Solvent extraction and distillation processes are extensively applied to separate butyric acid from other byproducts mainly acetic acid. The main drawbacks of these processes are solvent toxicity in extraction and huge energy consumption in distillation which prohibit their uses in the separation of bulk chemicals from bio-production. Wu et al. (2010) reported salting out" separation method based on an aqueous two-phase system with inorganic salts such as calcium chloride for effective separation of bio-butyric acid from anaerobic fermentation broth and consequently increase butyrate/acetate ratio.
It is possible to significantly obtain butyric acid from renewable biomass by anaerobic fermentation using different strains of the genera Clostridium Butyrivibrio Butyribacterium Sarcina and others but low productivity rate and concentration are questions for its economicalproduction. Isolation of butyrate tolerate strains cost effective lignocellulosic materials optimum fermentation condition low cost culture media materials and economical hydrolysis are the key factors which could reduce challenges of butyric acid bio-production and accelerate its research and commercial production.
The authors gratefully acknowledge the National Natural Science Foundation of China (Grant No. 51178136) and the State Key Laboratory of Urban Water Resource and Environment Harbin Institute of Technology (Grant No. HCK201206) for valuable financial support.
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