INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY

ISSN Print: 1560–8530; ISSN Online: 1814–9596

13–1259/2014/16–5–1019–1024
http://www.fspublishers.org

Review Article

A Review on Bio-butyric Acid Production and its Optimization

Ajay Kumar Jha1,2, Jianzheng Li1*, Yixing Yuan1, Nawraj Baral1 and Binling Ai1
1
State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering,
Harbin Institute of Technology, Harbin 150090, P. R. China
2
Kathmandu Engineering College, Tribhuvan University, Kathmandu, Nepal
*For correspondence: ljz6677@163.com

Abstract
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 and
continuous type butyric acid fermentation of biomass have been discussed in this review. © 2014 Friends Science Publishers

Keywords: Butyric acid; Clostridium; Fermentation; Renewable biomass

Introduction commercial production is dominated by chemical synthesis.

However industrial production of butyric acid mainly
Energy has been regarded as one of the main elements of depends upon crude oil due to comparatively lower
human life, social civilization and techno-socio-economic production cost and large scale supply, consumers prefer
progress. However, the advances in energy technologies butyric acid of natural origin, especially for foodstuff
have brought various revolutions throughout the world. additives or pharmaceutical products (Zigová and Šturdík,
Present global energy supply-consumption chain is 2000). With decreasing availability of crude oil, growing
obviously unsustainable from the technical, environmental, demand for natural products and rising concerns over
economic and social points of view due to limited environment, microbial fermentation technology for butyric
availability of fossil fuels and inevitably be depleted (Barnes acid production from renewable biomass has been paying
and Floor, 1996). The emissions of greenhouse gases and attention of many researchers because bio-butyric acid
their consequences including global warming, acid rain, could be one of the most promising sustainable bio-fuels to
climate change and other environmental issues force us to meet the desires of green energy supply for replacing fossil
think about alternative fuels, although conventional fossil fuels. In addition, presence of profuse lignocellulose
fuels is the world's vital energy resource (Jha and Jha, biomass as low-value agricultural commodities or
2010). The application and development of bio-fuels can obligation of apt disposal of bio-wastes to pass up pollution
reduce the consumption of fossil fuels and alleviate energy tribulations have been creating favorable business climate
crisis to some extent. The recent advances in the fields of for butyric acid fermentation.
biotechnology and microbial fermentation technologies Although butyric acid is a significant bio-fuel to
have resulted in a renewed attention in bio-butyric acid develop sustainable green society, its production through
production from low cost renewable biomass (Zhang et al., fermentation of biomass has been regarded as very
2009; Dwidar et al., 2012). complex and hard to control. The microbial butyric acid
A four-carbon short chain (CH3CH2CH2COOH) bio- fermentation is affected with several process dilemmas
butyric acid and its derivatives have numerous potential including self-inhibitory effect of end products, increasing
applications in chemical, textile, plastic, food, beverage, inhibition due to pretreatment, slow rate of strain
dairy and pharmaceutical industries (Zigová and Šturdík, development (Zigova´ and Šturdík, 2000). The production
2000). They are extensively used as solvent, diluents, strains for butyric acid fermentation also produce other
drugs, plasticizer, perfumes, fiber, additive and raw types of acids, which are very difficult to separate. Higher
materials (Zhang et al., 2009). Bio-butyric acid is regarded substrate cost, degeneration of the butyric acid-producing
as a prospective chemical building-block to make strains, limited productivity, lower concentration, lower
chemicals. It is also regarded as a promising specialty yield and higher products recovery cost are main factors,
chemical as it can be converted to bio-butanol. But its which limit the fermentation routes as well. Limited

To cite this paper: Jha, A.K., J. Li, Y. Yuan, N. Baral and B. Ai, 2014. A review on bio-butyric acid production and its optimization. Int. J. Agric. Biol., 16:
1019‒1024
 Jha et al. / Int. J. Agric. Biol., Vol. 16, No. 5, 2014

studies on production of bio-butyric acid by anaerobic Metabolic Pathway and Inhibition in Bo-butyric
fermentation have been reported in association to bio- Acid Fermentation
refinery perspectives including selection and growth of
strains, and physiological examination for greater yield, Extensive studies have illustrated metabolic pathways and
productivity and selectivity. A higher demand in contrast regulations for fermentation of glucose to produce bio-
to lower production through the microbial fermentation butyric acid using clostridia (Zhang et al., 2009; Zigová and
routs gives rise to the necessity of addressing and solving Šturdík, 2000; Ramey and Yang, 2004). Glucose
the problems related to the butyric acid fermentation fermentation by C. butyricum (Eq. 1) and C. tyrobutyricum
process. In order to improve butyric acid fermentation and (Eq. 2) follows the stoichiometric equations below (Zhang
reduce overall production cost, extensive efforts are et al., 2009):
needed for strains improvement, metabolic pathways,
bioreactor design, feed stocks, effect of inhibitors, Glucose→0.8 Butyrate + 0.4 Acetate + 2.4 H2 + 2 CO2
fermentation process control parameters and optimization (Eq. 1)
techniques. Glucose→0.85 Butyrate + 0.1 Acetate + 0.2 Lactate+
1.9 H2 + 1.8 CO2 (Eq. 2)
Microbial Strains The metabolic route of glucose fermentation is
presented in Fig. 1. Butyric acid is produced from glucose
Appropriate microorganisms selection is the base of a during acetogenesis stage whereas in solventogenesis phase,
successful fermentation process. Numerous bacterial strains, butyrate is converted into butanol (Ramey and Yang, 2004).
which are suitable to produce bio-butyric acid, are mainly High ATP concentration and minimal NADH:NAD ratio
isolated from waste water, excess sludge, soil, contaminated should be maintained in order to prevent solventogenesis
dairy and food products, meats, and animal digestive (Zigová and Šturdík, 2000). Glucose is metabolized to
systems. Altogether, more than ten butyrate-producing pyruvate by means of Embden–Meyerhof–Parnas and
bacterial strains, belonged to the genera Clostridium, generates ATP and NADH. Afterwards, acetyl-CoA,
Butyrvibrio, Butyribacterium, Eubacterium, Fusobacterium acetoacetyl-CoA and butyryl-CoA are formed from
Megasphera and Sarcina, are reported (Zigová and Šturdík, pyruvate as key intermediates in the main branch (Jones and
2000). They are Gram positive, chemoorganotrophic, Woods, 1986). Butyric acid can be produced consequently
strictly anaerobic, and spore-forming bacteria. The strains of in case of presence of high levels of enzymes that are
genera Clostridium have been extensively used and studied concerned with the pathway of butyryl-CoA to butyrate.
microorganisms due to their high productivities and During the conversion of acetyl-CoA into butyryl-CoA,
relatively higher stability, followed by Butyrivibrio and thiolase, crotonase, 3-hydroxybutyryl-CoA dehydrogenase
Butyribacterium. Among them, C. butyricum, C. and butyryl-CoA dehydrogenase are played vital roles as
beijerinckii, C. acetobutylicum, C. tyobutyricum, C. key enzymes. The bio-butyrate-producing clostridia not
populeti and C. thermobutyricum are superior strains. only produce bio-butyric acid but also several possible by-
Favorable culture temperature ranges from 30-37°C for C. products, including acetate, H2, CO2, lactate and other
butyricum, C. populeti and C. tyrobutyricum, while 55°C products. The conversion of acetyl-CoA is firstly occurred
is considered as optimal culture temperature for C. into acetyl phosphate, which is converted into acetate.
thermobutyrium. Although an extensive range of carbon Similarly, butyryl-CoA is firstly converted into butyryl
source is able to be utilized by clostridia, glucose is phosphate and butyrate is produced from butyryl phosphate.
common substrate for bio-butyrate production. The acetyl-CoA is catalyzed by phosphotransacetylase
Clostridium bacteria can utilize different types of sugars (PTA) whereas phosphotransbutyrylase (PTB) catlalyzes
including hexoses, several pentoses, oligo- and butyryl-CoA. Correspondingly, acetate kinase (AK) and
polysaccharides for bio-butyric acid production while C. butyrate kinase (BK) catalyze acetyl-phosphates and
butyricum is able to utilize glycerol, pentose, hexose, butyryl- phosphates for the production of acetate and
molasses, lignocellulose, cheese-whey permeat, and potato butyrate, respectively.
starch as carbon sources. However, C. tyrobutyricum can In fact, the metabolic pathway of a microorganism
only make use of glucose, xylose, and fructose during anaerobic fermentation is affected by several factors.
(Matijasic et al., 2007). Baroi et al. (2013) presented C. In case of bio-butyrate-producing clostridia, mainly glucose
tyrobutyricum has the ability to transfer both pentose and concentration, pH, hydrogen partial pressure, acetate, and
hexose sugars but the xylose uptaking speed is lower than butyrate are able to influence the growth rate, final products
that of glucose. C. thermobutyricum, which is isolated from concentration and distribution of the products (Kong et al.,
horse dung, can mainly use monomeric sugars (glucose, 2006; Jo et al., 2008; Rodriguez et al., 2006). It is necessity
fructose, maltose, xylose, and ribose, but not arabinose, to build up an appropriate and healthy process parameters
galactose, and mannose), dimeric (cellobiose), oligomeric that yields low amount of acetic acid (greater selectivity),
and polymeric sugars. has a superior yield and a greater productivity of bio-
butyric acid from lignocellulosic renewable biomass.

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Glucose and acetate formation at pH 5.0 is associated with decreased

activities of PTB and independent lactate dehydrogenase
ADP
Acetogenesis NAD+ Solventogenesis (iLDH), and increased activities of PTA and LDH (Zhu and
Yang, 2003). In butyrate-producing strains, PTA, AK, PTB,
ATP NADH BK, iLDH, and LDH are the main enzymes relevant to
+
NADH NAD
acetate, butyrate, and lactate production. Their products
Lactate Pyruvate distribution is affected by media pH significantly. AK and
H2 BK in the direction of acyl-phosphate formation were not
CoA Fd
significantly affected by the pH between 5.0 and 7.0.
H+ However, in the acyl-phosphate-forming direction, the
CO2 FdH2 activity of PTA increased while PTB decreased with
ATP ADP NADH NAD+
NADH NAD+ increasing the pH (Zhu and Yang, 2003). It has also been
PTA H H
reported that under a low partial pressure of H2, the ratio of
acetate to butyrate increased with a decrease in hydrogen
Acetate Acetyl-P Acetyl-CoA Acetylaldehyde Ethanol
partial pressure, accompanied by an increase of ATP yield
AK
during bio-butyric acid production fermentation by C.
CoA
CoA CO2 butyricum (van Andel et al., 1985). The production of bio-
butanol from butyric acid is also affected by inadequate pH.
Acetoacetyl-CoA Acetoacetate Acetone
Soni and Jain (1997) studied the consequence of pH on bio-
butyrate uptake as a result of the transformed strain of
+
2 NADH+H clostridium acetobutylicum and found that lower pH (<4.6)
H +
unfavorably exaggerated overall metabolic activity.
2 NAD
Minimum pH 5.2 was needed for uptake of bio-butyrate at a
H + concentration of 4 g/L. They also observed that a straight
NADH NAD NADPH NADP+
connection among minimum pH prerequisite, butyrate
BK PTB H H H H
Butyrate Butyryl-P Butyryl-CoA Bulyraldehyde Butanol concentration and allied anaerobic fermentation
temperature. The end product inhibition is a challenge for
the researchers, working to enhance product concentration.
CoA Undissociated bio-butyric acid gets ahead of through the
ADP ATP
bacterial membrane and detaches within the cell. It impinges
Fig.
Fig.1: Pathways
1: Pathways for bio-butyric
for bio-butyric acid
acid fermentation fermentation
(Ramey and Yang, 2004; (Ramey
Jones and
on the transmembrane pH gradient and declines the sum of
andWoods,
Yang, 1986).2004; Jones and Woods, 1986). existing energy for biomass growth (Zigová and Šturdík,
(AK:acetate
(AK: acetate kinase,
kinase,BK: butyrate kinase, PTA:
BK: butyrate phosphotransacetylase,
kinase, PTB:
PTA: phosphotransacetylase, 2000). One of the approaches to solve this problem is to
phosphotransbutyrylase)
PTB: phosphotransbutyrylase) develop a combination of butyric acid tolerated strains while
other technique may be online separation or in situ product
Surplus carbon supplies often have an effect on osmotic removal, especially extraction and pertraction.
dehydration of microorganisms in an anaerobic The concomitant production of acetic acid as by-
fermentation process. A momentous rise in the ratio of product with butyrate production creates problems for the
butyrate to acetate is found in bio-butyric acid fermentation recovery of bio-butyric acid in downstream processing
process with limited amount of glucose and C. butyricum as (Zhang et al., 2009). For example, in the immobilized cell
working microorganism (Saint-Amans and Soucaille, 1995). anaerobic fermentation process, a greater quantity of 0.27
It is noteworthy that various pH values can influence not mol/mol of acetate was formed along with 0.95 mol/mol of
only the sharing of produced organic acids but also cell butyrate from glucose (Ramey and Yang, 2004). It is
membrane transfer behavior, and cell lysis (Zigová and obvious that reducing biomass formation and acetate
Šturdík, 2000). Relatively higher pH (e.g. >6.0) is useful for production increases butyrate yield significantly. Ramey and
cell development and biosynthesis of butyric acid, Yang (2004) reported that complete elimination of acetate
especially in the case of C. butyricum (He et al., 2005). formation could increase butyric acid yield more than 1
Furthermore, media pH also has an effect on the particular mol/mol for glucose and 0.83 mol/mol for xylose as the
growth rate, bio-butyric acid production rate and less sugars substrate for immobilized cell fermentation. Knocking out
utilization rate. For C. tyrobutyricum, various pHs are able the acetate-producing pathway can increase the butyrate
to change the distribution of the metabolic flux. At pH 6.3, production.
the highest bio-butyrate production is observed, compared
to that at pH 6.0 and 6.7 (Zhu and Yang, 2003; Jo et al., Optimization of Butyric Acid Bio-production
2008). Bio-butyrate production was lower at lower pH, with
acetate and lactate as the main acid products at pH 5.0. The The conventional bio-butyric acid production technique
metabolic shift from butyrate formation at pH 6.3 to lactate is not yet cost-effective and economically competitive

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Table 1: Some examples of butyrate-production by fermentation

Feedstocks Pretreatment Culture design Strain Cul. temp. pH Butyrate Reference

(°C) Conc. (g/L)
Cane molasses Sulfuric acid Fed batch/Immobilized fibrous C. tyrobutyricum 37 6.0 55.2 Jiang et al., 2009
bed bioreactor
Cheese whey - Batch C. beijerinckii 37 5.5 >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
Hydrochloric acid reactor thermobutyricum
Jerusalem 0.01M Sulfuric acid Fed batch/Immobilized C. tyrobutyricum 37 6.0 60.4 Huang et al.,
artichoke fibrous-bed bioreactor 2011
Sugarcane 0.1–0.5 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
hydrolysate
Glucose from Pretreated and hydrolyzed Batch C. tyrobutyricum 6.0- 7.0 71.6 Baroi et al., 2013
wheat straw
Xylose from pretreated and hydrolyzed Batch C. tyrobutyricum 6.0- 7.0 55.4 Baroi et al., 2013
wheat straw

due to lower concentration, lower productivity and lower fermentable carbon substrates can be obtained from
yield of the bio-butyrate. The production of byproducts such lignocellulosic biomass while the cellulosic and
as acetic acid, propionic acid and ethanol causes further hemicellulosic hydrolysates and starch can be used to
reduction in butyric acid concentration and increases the produce butyric acid production. However, these
costs for product recovery and purification. It means the lignocellulosic residues are abundant available in the
complicated and expensive isolation process also limits its developing countries, they are being used inefficiently and
commercialization. In order to increase the economics of consequently cause considerable environmental evils. The
butyrate production, various optimizing techniques may be beauty of cellulosic materials is that significant amounts of
useful. different types of sugar can be obtained through hydrolysis
process, which are useful for bio-production of butyric acid
Feed stocks and bio-butanol and consequently minimize waste
generation (Li et al., 2011).
The cost of feed stocks is regarded as a key issue for
economical production of bio-butyric acid by microbial Pretreatment
fermentation process. Considering economics of the bio-
butyric acid fermentation, it can be pointed out that The degradation of lignocelluloses is the major obstacle for
agricultural products would not be feasible substrates due to utilizing renewal and cost-effective biomass including
high cost and direct uses for human beings and other agricultural residues to produce bio-butyric acid.
animals. In such situations, agricultural residues and other Pretreatment methods are considered as one of the approach
industrial wastes are the favorable substrates based on its to solve this problem. The treatment of molasses by sulfuric
composition, availability, cost, good water retention acid increased butyric acid concentration by 32.6%, yield by
capacity and ease of pretreatment. Lignocellulosic materials 31.8% and sugar utilization 12.3%, compared to untreated
such as maize straw, rice straw, barley, wheat straw, molasses (Jiang et al., 2009). In contrast, high costs of
molasses and dairy wastes have potential to serve as low hydrolyzing cellulose into simple monomeric sugars and
cost renewable raw materials for bio-butyric acid formation of inhibition products during the hydrolysis are
fermentation, mainly in agriculture based countries. They the major limitation for pretreatment techniques. The effect
are readily available and inexpensive renewable biomass. In of different pretreatment methods including acid, base,
fact, they consist of cellulose, hemi-cellulose, lignin and thermal, thermal-acid and thermal-alkali with various
smaller quantities of pectin, protein, extractives and ash. Li pretreatment time, temperature and concentration for higher
et al. (2011) observed that corn stalk has a great potential to glucose production and low quantity of inhibition products,
immobilize C. thermobutyricum for bio-butyric acid and consequently higher butyrate production will be focus
fermentation. Similarly, Jiang et al. (2009) and Vandák et for the future researches. In addition, physical pretreatment
al. (1995) have used cane molasses for bio-butyric acid such as appropriate size for substrate, and biological
fermentation using C. butyricum and C. tyrobutyricum treatment are very significant for higher butyric acid
respectively. Baroi et al. (2013) reported the main contents production as well.
in the sugars yielded from wheat straw haves glucose (71.6
g/L) and xylose (55.4 g/L) respectively. Multicultural Strains
Hemicelluloses, the second most abundant available
polysaccharides in nature, represent around 20-35% of Inability of most of the single butyrate-producing strains to
lignocellulosic biomass (Ezeji et al., 2007). Enough grow and degrade substrate at different fermentation

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conditions is one of the major barriers for butyrate range from 4.5 to 7.0 is favorable for butyric acid
fermentation. The identification of a combination of fermentation (Zigová et al., 1999). According to Jiang et al.
multicultured microbes and their metabolic pathways will (2009), the optimum pH for butyric acid production and its
accelerate fermentation process to produce higher rate of microorganism growth is 6.0. They further explained that
butyric acid because (i) different conditions and process lowering pH to 5.0 brings a change in metabolic shift for
parameters might be favorable for different microbes; (ii) acetate production from butyric acid production route and
the inhibition problems due to hydrolysis and fermentation consequently acetate becomes the major product. Alam et
products will be decreased by having better tolerance to al. (1988) noted that a constant pH 5.5 provides highest
butyric acid inhibition for the microbes. The depth insight level of butyric acid from cheese whey using C. beijerinckii
for finding combinations of microbial strains will be major at 37°C in batch culture. Similarly, Li et al. (2011) observed
focus for future researches. highest butyric acid yield from corn stalk at pH 6.0 while
pH 7.0 was favorable for maximal butyrate/acetate ratio in a
Reactor Design and its Operation continuous type immobilized cell reactor using C.
thermobutyricum at 55°C.
Batch, repeated batch, fed batch, continuous and cell recycle
anaerobic fermentation processes are widely utilized for the Separation of Butyrate
researches on butyric acid bio-production (Table 1). It was Difficulty in separating butyric acid from anaerobic
observed that greater butyrate concentrations could be fermentation broth is considered as one of the major
achieved in batch culture while higher productivity might be bottlenecks for butyrate production. Solvent extraction and
obtained in continuous cultures (Michel-Savin et al., 1990). distillation processes are extensively applied to separate
Repeated batch culture could eliminate the lag phase by butyric acid from other byproducts, mainly acetic acid. The
providing adaptation period required for cells to survive in a main drawbacks of these processes are solvent toxicity in
changed environment. Adaptation of bacterial cultures and extraction and huge energy consumption in distillation,
shift in metabolic pathways in fed batch fermentation could which prohibit their uses in the separation of bulk chemicals
increase further butyrate concentration and consequently from bio-production. Wu et al. (2010) reported “salting out”
butyrate/acetate ratio. The productivity can be enhanced separation method based on an aqueous two-phase system
using cell recycling process or cell immobilization. Jiang et with inorganic salts such as calcium chloride for effective
al. (2009) studied performance of a fibrous bed bioreactor separation of bio-butyric acid from anaerobic fermentation
(FBB) with immobilized C. tyrobutyricum under batch, broth and consequently increase butyrate/acetate ratio.
repeated batch and fed batch fermentation systems in order
to optimize bio-butyric acid production from cane molasses. Conclusion
The fed-batch fermentation produced 61.9% higher butyrate
concentration and reduced 50.9% acetic acid but yield and It is possible to significantly obtain butyric acid from
productivity were decreased by 16.4 and 46.9%, compared renewable biomass by anaerobic fermentation using
to the batch fermentation process. Similarly, Mitchell et al. different strains of the genera Clostridium, Butyrivibrio,
(2009) found that the continuous culture system with Butyribacterium, Sarcina and others but low productivity,
immobilized cells has improved butyric acid productivity rate and concentration are questions for its economical
and consequently reduced product separation cost. The production. Isolation of butyrate tolerate strains, cost
fibrous bed bioreactors due to their regeneratives and higher effective lignocellulosic materials, optimum fermentation
mass transfer capabilities can maintain the productivity over condition, low cost culture media materials, and economical
a longer period. Huang et al. (2002) reported that cells hydrolysis are the key factors, which could reduce
immobilized FBB with packed fibrous matrix is successful challenges of butyric acid bio-production and accelerate its
in producing most of the commercially used organic acids. research and commercial production.

Fermentation Conditions Acknowledgements

The identification of relatively optimum process parameters The authors gratefully acknowledge the National Natural
will solve the problems related to efficient butyrate Science Foundation of China (Grant No. 51178136) and the
production to some extent. It is believed that thermophilic State Key Laboratory of Urban Water Resource and
microorganisms can produce higher fermentation products Environment, Harbin Institute of Technology (Grant No.
due to higher rate of hydrolysis, improved mass transfer and HCK201206) for valuable financial support.
reduced susceptibility to contamination. The pH value and
acetic acid concentration have also significant effects on the References
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