Journal Pre-proof Microemulsion extraction of biobutanol from surfactant based-extractive fermentation broth Preety S. Gedam, Atulkumar N. Raut, Pradip B. Dhamole
PII:
S0255-2701(19)30500-8
DOI:
https://doi.org/10.1016/j.cep.2019.107691
Reference:
CEP 107691
To appear in:
Chemical Engineering and Processing - Process Intensification
Received Date:
26 April 2019
Revised Date:
23 September 2019
Accepted Date:
17 October 2019
Please cite this article as: Gedam PS, Raut AN, Dhamole PB, Microemulsion extraction of biobutanol from surfactant based-extractive fermentation broth, Chemical Engineering and Processing - Process Intensification (2019), doi: https://doi.org/10.1016/j.cep.2019.107691
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Microemulsion extraction of biobutanol from surfactant based-extractive fermentation broth
Preety S. Gedam, Atulkumar N. Raut, Pradip B. Dhamole*
Department of Chemical engineering, Visvesvaraya National Institute of Technology, South
ro
of
Ambazari Road, Nagpur, MS, 440010, India
Corresponding author: Pradip B. Dhamole, Department of Chemical Engineering, Visvesvaraya
-p
National Institute of Technology, South Ambazari Road, Nagpur, 440010, M.S. India
re
Tel. No. 91-712-280-1788;
[email protected],
[email protected]
Jo
ur na
lP
Graphical abstract
Highlights
Two different approaches were carried out and compared for butanol extraction 1
Single stage approach showed 4 times more extraction than two stage approach
One step (i.e. coacervation) is avoided in the microemulsion extraction process
Microemulsion based approach is better than reported extraction methods.
Abstract The present work investigates the microemulsion based butanol extraction from fermentation
of
broth. Extractive butanol fermentation was carried out using a non-ionic surfactant L62.Pentane,
ro
hexane and heptane (i.e. n-C5, n-C6 and n-C7) alkanes were used for the extraction of butanol from fermentation broth. Conditions for Winsor microemulsion with n-C5, n-C6 and n-C7 alkanes
-p
were established. Hexane and heptane formed Winsor-III microemulsion. Hexane based microemulsion resulted into organic phase (Om) containing more butanol (62% w/w) than
re
pentane (12.4% w/w) and heptane (57.3% w/w) based ones. Bi-continuous phase (i.e. the
lP
surfactant phase or BC) had negligible amount of butanol. Surfactant was retained in BC and water (Wm) phases. Thus, hexane was found to be a better microemulsion extractant than the other two alkanes. Studies with two step approach i.e. coacervation followed by back-extraction
ur na
resulted in just 13% (w/w) butanol. Thus, the single stage approach with hexane not only avoided a step but also yielded about 4 times more extracted butanol than the two-stage
Jo
approach.
Keywords: Surfactant; extraction; butanol, alkanes; Winsor microemulsion
1. Introduction
Recent apprehensions about depleting crude oil reserves, impact of fossil fuels on environment, and national security threats have prompted increased interest in development of alternative fuels
2
[1]. Biobutanol derived from sustainable renewable resources has emerged as a promising renewable fuel [2-5]. Butanol has higher energy density and is less hygroscopic than ethanol, thus, making it easy to transport [2-3, 6-7]. Butanol can be produced by bacterial acetonebutanol-ethanol (ABE) fermentation with bacteria species of genus Clostridium. Product inhibition is a major issue with butanol production which limits the butanol titer to 20-22 g/L [8]. A variety of methods have been accounted for butanol separation for relieving microbes from
of
butanol toxicity. These methods include adsorption [9-11], pervaporation [12-14], perstraction
ro
[15], solvent extraction [16-17] and gas striping [18-19]. All of these methods target only butanol separation (as and when it is produced) and do not increase the butanol titer. Increased butanol
-p
titer will reduce the separation cost significantly. Dhamole et al (2012, 2015) for the first time
re
reported the extractive butanol fermentation with non-ionic surfactant resulting in enhanced titer. L62, an ambiphilic tri-block (polyethylene oxide – polypropylene oxide- polyethylene oxide i.e.
lP
PEO-PPO-PEO) copolymer improved butanol production by 50% than the control (i.e. without any surfactant) [20-21]. It was proposed that butanol is entrapped into the micelles leading to
ur na
reduced butanol toxicity. Using cloud point extraction (CPE), with the same surfactant, butanol was concentrated into surfactant rich phase (SRP also known as coacervate phase). However, only a third of butanol was extracted into SRP from fermentation broth. Back-extraction of solute from SRP is a key problem in surfactant based aqueous two-phase
Jo
separation (ATPS). Earlier work on back-extraction of butanol used two different methods i.e. evaporation of butanol from coacervate phase [20] and hexane based Winsor III microemulsion. In the former approach butanol was evaporated between 120-130°C resulting into condensate containing butanol and water. Overall extraction efficiency was just 35% and needed an additional step of purification of butanol. In the latter method, Winsor III microemulsion was
3
used to back-extract butanol. Addition of hexane formed three phases; organic phase (Om), bicontinuous phase (BC) and water phase (Wm) [22]. Organic phase (Om) and water phase (Wm), respectively contained 38% and 61% of butanol that was initially extracted into SRP. Om phase was further processed for butanol separation. Thus, effectively, an overall separation of 12-14% of butanol was achieved from the fermentation broth. The second method used two steps; coacervation followed by Winsor microemulsion and the overall yield was just 13%. To
of
summarize, both methods used resulted in an overall extraction of 12-35% of butanol.
ro
Microemulsion is a promising method for separation of solutes and recovery of surfactant. Due to its unique properties, Winsor III microemulsion has several existent and potentially valuable
-p
applications including mixing of immiscible polymers, enhanced oil recovery, coatings, multi-
re
phase reaction media and templating media for nanomaterials [23-25]. Other applications in biotechnology includes bio-nanomaterial synthesis, drug delivery system, hosting of multiphasic
lP
bio-catalytic reaction and extraction of proteins [26-27]. This work reports a microemulsion based approach, wherein, in a single stage butanol will be extracted from the fermentation broth
ur na
without coacervation. Thus, the objective of the present work is to improve the overall extraction of butanol and also to reduce the number of step/s in the extraction process. Different alkanes were studied for microemulsion formation and screened for further studies. Phase separating conditions for different Winsor microemulsion were established for surfactant L62-butanol-
Jo
simulated fermentation broth with different alkanes. These phase separating conditions were used to extract butanol using microemulsion. Initial experiments were carried out using a model system (simulated fermentation broth) followed by real fermentation broth. Two different approaches are reported in the present work (i) two-stage approach i.e. coacervation followed by Winsor microemulsion extraction and (ii) single stage approach wherein microemulsion
4
extraction of butanol is carried directly from the fermentation broth. The performance of both approaches was compared in terms of butanol extraction efficiency.
2. Materials and methods 2.1 Chemicals Non-ionic surfactant pluronic L62 which has a molecular weight of 2500 g/mol and 20% EO
of
content, was used in the present study. Its molecular formula is represented as EO6PO34EO6 and
ro
has hydrophile-lipophile balance (HLB) value of 7.0. The structure of L62 is reported elsewhere [22]. L62 was provided by BASF (as a gift sample) and used with no further purification.
-p
Butanol (analytical grade with >98% purity) was obtained from Hi-media, India. Non-polar
2.2 Bacteria culture and medium
re
solvents pentane, hexane and heptane with purity >98% were brought from Loba-Chemie, India.
lP
Clostridium acetobutylicum ATCC No. 824 (NCIM No. 2337) re-identified as Clostridium sporogenes was purchased from National Chemical Laboratory (NCL), Pune, India. Lyophilized
ur na
culture was revived using a heat shock at 80°C for 10-15 min in a cooked meat medium (CMM). Further, it was incubated at 37°C for 24-48 h. The revived cells were then inoculated into freshly prepared CMM and incubated at 37°C for 24 h. CMM with following composition was used as a seed culture media: Beef extract, 98 g/L; peptone, 20 g/L; dextrose, 2g/L; NaCl, 5g/L; cysteine
Jo
HCl, 0.5g/L and resazurin dye 1mg/L. Anaerobic environment was maintained by passing nitrogen gas through fermentation medium for 10-15 min. The medium was autoclaved at 121°C before inoculation. 10% of the actively grown cells were then used for fermentation. 2.3 Fermentation with surfactant
5
Actively grown cells of C. sporogenes (10% v/v) were inoculated into the fermentation medium containing 6% (v/v) L62. All the experiments were carried out in triplicates in serum bottles (130 mL) with 50 mL of working volume. Cheng’s media used for fermentation had following composition: Glucose, 60 g/L; ammonium sulphate, 2g/L; dipotassium hydrogen phosphate, 2 g/L; calcium carbonate, 3 g/L; magnesium sulfate, 0.5 g/L; ferrous sulfate, 0.516 g/L; cysteine HCl, 1 g/L; yeast extract, 5.133 g/L and resazurin dye 1 mg/L. After preparation, media was
of
bubbled through nitrogen for 10 min to remove oxygen and autoclaved at 121°C before
ro
inoculation. Fermentation was carried out at 37°C in orbital shaking incubator. 2.4 Cloud point extraction
-p
Cloud point extraction of simulated/fermentation system was carried out to get surfactant rich
re
phase. Simulated system containing aqueous solution of 6% (v/v) L62 and 4% (v/v) butanol was prepared. This solution (250 mL) was incubated in a conical flask (500 mL) at 45 oC.
lP
Temperature was decided based on earlier reported studies [28]. After phase separation, both SRP and dilute phase were collected separately and volume of both phases was recorded.
ur na
Butanol concentration was determined using gas chromatography. Dry weight method was employed to verify surfactant concentration in each phase. 2.5. Phase separating conditions/phase behavior for Winsor microemulsion Phase separating conditions for Winsor microemulsion were established for both surfactant rich
Jo
phase as well as simulated fermentation broth. SRP was obtained by incubating a simulated fermentation broth above the cloud point temperature. Simulated fermentation broth consisted of butanol (4%), L62 (6%) and ABE in the ratio of 3:6:1. SRP / simulated fermentation broth was added with different alkanes (1:1 volume ratio). All the experiments were conducted in a 15 mL graduated test tube. The solution was then incubated at different temperatures (4-60oC) in
6
circulating water bath (Polyscience digital temperature controller, USA, MX07R-20-A-12E). Thermo-equilibrium was attained by heating samples for 30 min at different temperatures. Change in the phase behavior was determined by rapid change in turbidity of aqueous surfactant solution. Microemulsion formation conditions were noted. These conditions were used in backextraction and extraction of butanol, respectively, from SRP and simulated fermentation broth, in
of
the subsequent studies. 2.6 Back extraction of butanol from SRP using alkanes
ro
SRP obtained from CPE of an aqueous solution containing 6% (v/v) L62 and 4% (v/v) butanol was separated and used for back-extraction of butanol using Winsor microemulsion [22]. 5 mL
-p
of SRP was taken in separate test tubes and mixed completely with highly non polar solvents
re
pentane, hexane and heptane. These solutions were then incubated in a circulating water bath for 30 min in the temperature range of 4-8°C for pentane and 45-50°C for hexane and heptane. This
lP
led to Winsor microemulsion. Samples were collected from each phase and the volume of each phase was recorded. Solvents were analyzed using gas chromatography and surfactant
ur na
concentration was verified using dry weight method. 2.7 Microemulsion extraction of butanol from fermentation broth with alkanes Simulated fermentation broth (5 mL) was mixed with highly non-polar alkanes in 1:1 volume ratio. This mixture was incubated in a circulating water bath at different temperatures (4-8 oC for
Jo
pentane and 45-50oC for hexane and heptane). Alkane with which highest butanol extraction and surfactant recover was obtained, then used with real fermentation broth. In case of real fermentation broth, cells were removed (using centrifugation and vacuum filtration) prior to microemulsion extraction. This broth and alkane were taken in the ratio 1:1 and procedure described earlier in section 2.6 was repeated.
7
2.8 Analysis Concentration of butanol and alkanes was determined using gas chromatography (GC) (Thermo scientific trace 1110). GC unit was equipped with flame ionization detector (FID). Fused silica capillary column, BP-20 (Forte) with 25 m length and 0.22 mm internal diameter was used. Column temperature was held at 60°C for 2 min, raised to 190°C with a ramp of 10°C/min, and again held at 190°C for 2 min. Injector temperature was kept at 210°C and detector temperature
of
was kept at 230°C. Nitrogen was used as a carrier gas (2 mL/min) [22]. Split ratio was kept at
ro
1:100.
Dry weight method was employed to determine surfactant concentration. 1 mL sample from both
-p
phases was taken in petri dishes. Initial weight of sample at 0 h was recorded and dishes were
re
kept in oven at 40-50°C to evaporate organic solvent and water. Weighing was carried out after
3. Results and discussion
lP
every 24 h till the constant final weight of samples was achieved.
ur na
3.1 Fermentation with surfactant
In earlier studies with C. pasteurianum and C. beijerinkii, it was observed presence of nonionic surfactant relieved butanol toxicity which resulted in significant increase in butanol [20, 29]. 6% L62 was observed to produce maximum butanol amongst surfactant concentrations studied
Jo
ranging from 3% to 20% (v/v) [30]. Hence, present study was carried out in presence of 6% L62. Fig. 1 shows the effect of surfactant on butanol enhancement at the end of 144 h. The butanol titer (14.9 g/L) is close to that reported in earlier studies [30].
8
3.2. Extraction of butanol using two stage approach i.e. cloud point extraction (coacervation) of butanol and back-extraction of butanol from SRP 3.2.1 Cloud point extraction of butanol Butanol was extracted from fermentation broth following a process developed earlier [22]. A simulated fermentation broth consisting 6% L62 and 4% butanol was used for extraction of butanol using cloud point extraction system. It is anticipated that a high butanol producing strain
of
(capable of producing 22 g/L butanol) would produce 33 g/L of butanol in presence of surfactant
ro
i.e. 4% on volume basis. When temperature of the system was increased above cloud point temperature of L62 (≥45°C), it resulted in two-phase separation [21]. Thus, from the simulated
-p
fermentation broth 34.9 % of butanol was extracted into SRP (Table 1). Remaining amount of
re
butanol was present in aqueous phase. The concentration of butanol in the surfactant rich phase was carried out with L62 present in simulated fermentation broth. Butanol gets entrapped into
lP
the hydrophobic micellar core which gets separated above cloud point temperature. Higher concentration of surfactants can be used to enhance butanol concentration in SRP. Extraction
ur na
efficiency of 35% was similar to that reported in earlier studies [20, 22]. 3.2.2 Establishment of microemulsion conditions for back-extraction of butanol from SRP In earlier study, butanol back extraction from SRP was successfully done employing Winsor-III microemulsion with hexane [22]. Thus, screening of alkanes was mandatory to get best results
Jo
for butanol downstream processing. Hence, in the present work three alkanes (pentane, hexane and heptane) were studied for Winsor microemulsion. The type of microemulsion is distinctly affected by different sorts of organic solvents. Polarity of an organic solvent mainly determines the type of microemulsion for a certain nonionic surfactant. Winsor microemulsion can be formed by a mixture of nonionic surfactant aqueous solution with an organic solvent by varying
9
temperatures. If an organic compound present in a cloud point system has relatively higher solubility in the organic solvent, the organic compound and the nonionic surfactant in the cloud point system should be separated successfully [31]. This principle was used to liberate butanol from SRP as well as simulated fermentation broth. Being highly non-polar solvents pentane, hexane and heptane were explored to separate butanol and surfactant using Winsor microemulsion.
of
Fig. 2 (a and b) shows typical phase behavior of microemulsion obtained using SRP in presence
ro
of pentane and heptane. Phase behavior of microemulsion with hexane is already reported [22]. For studies carried out in presence of pentane the solutions were incubated in the temperature
-p
range of 4°C to 25°C. From Fig. 2a, it can be seen that with SRP, from 4°C to 8°C, pentane
re
forms Winsor I microemulsion. At 10°C, slight development of Winsor III microemulsion was observed which was highly unstable as it did not attain equilibrium. From 12°C to 16°C, Winsor
lP
II microemulsion was observed whereas after 16°C Winsor IV microemulsion prevailed. Fig 2b shows phase behavior of SRP in presence of heptane. It can be seen that from 10°C to
ur na
25°C, heptane forms Winsor IV microemulsion. From 30°C to 40°C formation of Winsor I microemulsion was observed followed by Winsor III and Winsor II microemulsion at temperatures ranging from 45°C to 50°C and 55°C to 60°C, respectively. Being a thermo-separating polymer non-ionic L62 affects type of microemulsion with change in
Jo
temperature. From the results shown in Fig. 2, it is confirmed that the system containing alkanes, water and L62 fits in the universal trend of microemulsion i.e. Winsor I-III-II [32]. According to Winsor, change in phase behavior occurs due to interaction energy balance (R) between the amphiphile-oil and amphiphile-water phase [33]. Winsor III microemulsion systems exist when the affinity of the surfactant for the water is equivalent to the affinity for organic solvent,
10
producing an interface that has the minimum interfacial tension. In the present study, formation of Winsor III microemulsion was induced by the addition of alkanes (pentane, hexane and heptane). It is known that hydrophobicity of alkanes increases with increase in carbon number [34]. Also, hydrophobicity of alkanes depends on temperature and it decreases with temperature [34]. As a result, addition of alkanes reduces interfacial tension and influences curvature by its ability to penetrate and hence swell the tail group of surfactants. Due to increase in the
of
penetration of surfactant into the organic phase, curvature values reduced from positive values
ro
(Winsor I) to zero (Winsor III). Also, increasing temperature (in case of non-ionic surfactant) increases the hydrophobicity of surfactant solution and thus segregate more towards the oil–
-p
water interface, thereby reducing the surfactant film curvature and interfacial tension. At net zero
re
curvature, a Winsor III system is formed [35]. This is in accordance with the R-theory proposed by Winsor. When R<1, formation of Winsor-I takes place, when R>1 and R=1 formation of
lP
Winsor-II and Winsor-III takes place, respectively.
3.2.3 Back extraction of butanol from SRP using alkanes
ur na
Downstream process for butanol back extraction from simulated fermentation broth using alkanes (n-C5 and n-C7) is shown in scheme 1. SRP was obtained after cloud point extraction of butanol from simulated fermentation broth (containing 32.4 g/L butanol and 6% L62). 35% (w/w) of butanol was extracted into a small SRP. Butanol back extraction from SRP to organic
Jo
phase was performed using alkanes (n-C5 and n-C7). Stable formation of Winsor III was not obtained using pentane as discussed in section 3.2.1, so Winsor-I microemulsion was tested for butanol back extraction using pentane. Butanol partitioned into organic phase (30.2%) and in oil in water (O/W) microemulsion (69.8 %). L62 remained in Wm phase when pentane was used. However, when hexane and heptane was used in 1:1 ratio with SRP, stable formation of Winsor-
11
III microemulsion was observed at 45°C. Hexane was successfully used for butanol back extraction from SRP using Winsor microemulsion [22]. Performances of pentane and heptane along with hexane are included in Table 1. Winsor-III microemulsion based back extraction resulted into organic phase (Om or solvent rich phase), bi-continuous phase (BC or surfactant phase) and water phase (Wm phase). Om phase was free from surfactant and it partitioned into BC and Wm phase. Om phase obtained using pentane and heptane contained 30% and 52.8%
of
(w/w) butanol (extracted from the SRP). Thus, overall extraction of butanol from the broth was
ro
just 10% and 17% (w/w) with pentane and heptane based microemulsion. Hexane was found to
-p
extract 13% (w/w) of butanol from broth [22].
re
3.3 Microemulsion extraction of butanol from the fermentation broth Studies were carried out with C5, C6 and C7 alkanes to establish the microemulsion conditions
actual fermentation broth.
lP
with simulated broth and the extraction efficiencies were estimated for simulated as well as
broth
ur na
3.3.1 Establishment of microemulsion conditions for extraction of butanol from fermentation
Fig. 3 (a, b and c) shows phase behavior of microemulsion obtained using simulated ABE broth with pentane, hexane and heptane. From Fig. 3a and 2a, it can be seen that the phase behavior
Jo
was not significantly affected by the presence of ethanol and acetone. Figs. 3b and 3c show phase behavior of simulated fermentation with hexane and heptane respectively. With hexane Winsor IV microemulsion formed between 10°C to 25°C (Fig. 3b). Winsor I microemulsion (30°C to 40°C) was followed by Winsor III (45°C to 50°C) and Winsor II microemulsions (55°C to 60°C) (Fig. 3b). Similar phase behavior was noted with heptane (Fig. 3c). Phase behavior
12
trend in fermentation broth or in SRP is quite similar for hexane [22] as well as heptane (Fig. 2c and 3c). Thus, it can be concluded that presence of acetone and ethanol does not alter the phase behavior of microemulsion formed. 3.3.2 Microemulsion extraction of butanol from fermentation broth Downstream process for butanol and surfactant L62 separation from simulated fermentation broth (SFB) using alkanes (C5, C6 and C7) is shown in scheme 2. Simulated fermentation broth
of
having 32.4 g/L butanol, 5.4 g/L ethanol, 16.2 g/L acetone and 6% (v/v) surfactant L62 was
ro
used. Alkanes were used in 1:1 ratio with simulated fermentation broth to obtain Winsor-III microemulsion. As shown in Fig. 3 (a, b and c), pentane does not favor formation of Winsor-III
-p
microemulsion, hence study was carried out with Winsor I microemulsion for pentane; however,
re
hexane and heptane favor formation of Winsor III microemulsion at moderate temperature (45°C). Similar to the addition of alkanes to SRP, addition of alkanes to simulated fermentation
lP
broth forms Winsor III microemulsion and separation of butanol takes place in Om phase and Wm phase. With pentane, butanol partitioned into solvent rich phase (Om also referred as excess
ur na
oil phase) and in O/W microemulsion (Wm) as 12.4 % (w/w) and 87.7 % (w/w) respectively (Table 2). Hexane and heptane were mixed with simulated fermentation broth in volume ratio of 1:1 and equilibrated at 45°C to obtain Winsor III microemulsion which was used to extract butanol into the Om phase and Wm phase whereas L62 was extracted into the BC phase and Wm
Jo
phase. Using hexane, butanol partitioned into Om phase and Wm phase was respectively, 60.5 % (w/w) and 37.1% (w/w). Similar results were obtained when hexane was replaced by heptane as organic solvent for formation of Winsor III microemulsion. With heptane, butanol distribution in Om phase and Wm phase was respectively, 58.6 % (w/w) and 31.4 % (w/w). The detailed compositions of all the phases obtained with microemulsion extraction from simulated
13
fermentation broth are included in Table 2. It can be seen that butanol was extracted into only two phases i.e. Om and Wm; whereas bi-continuous phase was devoid of butanol. More importantly, the Om phase was free from surfactant. This clearly shows that there are no surfactant losses and one can recycle the water phase (Wm) and bi-continuous phase (BC). Thus, pentane based microemulsion resulted in 10-12% butanol extraction using both approaches (i.e. two stage approach and single stage approach) (Table 1 and Table 2). Hexane and heptane
of
showed better performance while following a single stage approach. Using simulated
ro
fermentation broth, the overall butanol extraction with hexane and heptane was 60.5% and 58.6% (w/w), respectively (Table 2). With two stage approach, the overall extraction was just
-p
13.3% and 18.2%, with hexane and heptane, respectively (Table 1). Therefore, hexane and
re
heptane were selected for butanol extraction studies from real fermentation broth. Microemulsion extraction with hexane from simulated fermentation broth was higher than with
lP
heptane. The detailed compositions of all the phases obtained with microemulsion extraction from microbial fermentation broth (i.e. actual fermentation broth) are included in Table 3. Using
ur na
hexane, butanol partitioned into Om phase and Wm phase was respectively, 62 % (w/w) and 34.7 % (w/w), whereas, using heptane, butanol partitioned into Om phase and Wm phase was 57.3 % (w/w) and 33.3 % (w/w), respectively. Thus, butanol extracted using single step approach (5762%) was almost 4 times higher (Table 3) than that of two step approach of coacervation
Jo
followed by back-extraction (Table 1). Further, the steps in the extraction process are reduced.
As compared to the different solvent extraction methods reported earlier, the extraction efficiency obtained in the present work is higher. Adhami et al., [36] achieved 45-51% butanol extraction from model system (containing 150-225 mM butanol) using soybean-derived biodiesel as an extractant. Extraction efficiency of 45% was achieved when methylated crude palm oil was
14
used as an extractant in an extractive fermentation [37]. In case of ionic liquids, the highest butanol extraction efficiency obtained was about 74% at 50°C [38]. Also, when the energy requirement is calculated for both processes i.e. two-step process and single step process, it was observed that energy requirement is two times higher in case of two step process (42.23 kJ/g of butanol recovered) than single step (17.73 kJ/g of butanol separated). Basis of 3% (v/v) L62 and 4%(v/v) butanol with 100 mL of initial volume of simulated
of
fermentation broth was considered for calculation purpose. The energy requirement was
-p
required for evaporation of hexane from the Om phase.
ro
calculated from the energy required for incubation to obtain phase separation and the energy
re
4. Conclusions
Both, single stage and two-stage approaches were used for extraction of butanol with Winsor
lP
microemulsion. Conditions for Winsor microemulsion using n-C5, n-C6 and n-C7 alkanes were established. Formation of Winsor III microemulsion was highly unstable in case of pentane.
ur na
Hexane and heptane formed Winsor III microemulsion at relatively low temperature of 45 oC. Further, single stage microemulsion extraction using hexane (62% w/w) and heptane (57% w/w) extracted four times more butanol than the two-stage approach (13-18% w/w). Thus, the single stage microemulsion approach was found to be better than the two-stage approach as it not only
Jo
reduces the number of steps but also enhances the separation efficiency.
Acknowledgement: Dr. Pradip B. Dhamole would like to thank Department of Biotechnology (Govt. of India) for funding this work (vide Sanction order No. BT/PR5886/PBD/26/304/2012 dated 26.12.2013).
15
References [1] J. Zucchetto, Trends in oil supply and demand, potential for peaking of conventional oil production, and possible mitigation options, A Summary Report of the Workshop, The National Academies Press Washington, D.C. (2006). [2] P. Dürre, Biobutanol: an attractive biofuel, Biotechnology Journal: Healthcare Nutrition Technology 2(12) (2007) 1525-1534.
of
[3] K. Michael, N. Steffi, D. Peter, The past, present, and future of biofuels–biobutanol as
ro
promising alternative, Biofuel Production-Recent Developments and Prospects, Dr. Marco Aurelio Dos Santos Bernardes (Ed.), InTech, Rijeka, Croatia (2011).
-p
[4] I.S. Maddox, The acetone-butanol-ethanol fermentation: recent progress in technology,
re
Biotechnology and Genetic Engineering Reviews 7(1) (1989) 189-220.
[5] A. Ranjan, V.S. Moholkar, Biobutanol: science, engineering, and economics, International
lP
Journal of Energy Research 3 6(3) (2012) 277-323.
[6] E.M. Green, Fermentative production of butanol—the industrial perspective, Current opinion
ur na
in biotechnology 22(3) (2011) 337-343.
[7] V. García, J. Päkkilä, H. Ojamo, E. Muurinen, R.L. Keiski, Challenges in biobutanol production: How to improve the efficiency?, Renewable and sustainable energy reviews 15(2) (2011) 964-980.
Jo
[8] G.M. Awang, G. Jones, W. Ingledew, A. Kropinski, The acetone-butanol-ethanol fermentation, CRC Critical reviews in microbiology 15(sup1) (1988) S33-S67. [9] N. Qureshi, S. Hughes, I. Maddox, M. Cotta, Energy-efficient recovery of butanol from model solutions and fermentation broth by adsorption, Bioprocess and biosystems engineering 27(4) (2005) 215-222.
16
[10] T.J. Levario, M. Dai, W. Yuan, B.D. Vogt, D.R. Nielsen, Rapid adsorption of alcohol biofuels by high surface area mesoporous carbons, Microporous and Mesoporous Materials 148(1) (2012) 107-114. [11] A. Oudshoorn, L.A. Van Der Wielen, A.J. Straathof, Assessment of options for selective 1butanol recovery from aqueous solution, Industrial & Engineering Chemistry Research 48(15) (2009) 7325-7336.
of
[12] N. Qureshi, H.P. Blaschek, Production of acetone butanol ethanol (ABE) by a
pervaporation, Biotechnology progress 15(4) (1999) 594-602.
ro
hyper‐ producing mutant strain of Clostridium beijerinckii BA101 and recovery by
-p
[13] A. Rozicka, J. Niemistö, R.L. Keiski, W. Kujawski, Apparent and intrinsic properties of
re
commercial PDMS based membranes in pervaporative removal of acetone, butanol and ethanol from binary aqueous mixtures, Journal of Membrane Science 453 (2014) 108-118.
lP
[14] S.-Y. Li, R. Srivastava, R.S. Parnas, Separation of 1-butanol by pervaporation using a novel tri-layer PDMS composite membrane, Journal of Membrane Science 363(1-2) (2010) 287-294.
ur na
[15] N. Qureshi, I. Maddox, Reduction in butanol inhibition by perstraction: utilization of concentrated lactose/whey permeate by Clostridium acetobutylicum to enhance butanol fermentation economics, Food and Bioproducts Processing 83(1) (2005) 43-52. [16] P.J. Evans, H.Y. Wang, Enhancement of butanol formation by Clostridium acetobutylicum
Jo
in the presence of decanol-oleyl alcohol mixed extractants, Applied and environmental microbiology 54(7) (1988) 1662-1667. [17] K.A. Taconi, K.P. Venkataramanan, D.T. Johnson, Growth and solvent production by Clostridium pasteurianum ATCC® 6013™ utilizing biodiesel‐ derived crude glycerol as the sole
17
carbon source, Environmental Progress & Sustainable Energy: An Official Publication of the American Institute of Chemical Engineers 28(1) (2009) 100-110. [18] N. Qureshi, H. Blaschek, Recovery of butanol from fermentation broth by gas stripping, Renewable Energy 22(4) (2001) 557-564. [19] T.C. Ezeji, P.M. Karcher, N. Qureshi, H.P. Blaschek, Improving performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation,
of
Bioprocess and biosystems engineering 27(3) (2005) 207-214.
ro
[20] P.B. Dhamole, Z. Wang, Y. Liu, B. Wang, H. Feng, Extractive fermentation with non-ionic surfactants to enhance butanol production, biomass and bioenergy 40 (2012) 112-119.
-p
[21] P.B. Dhamole, R.G. Mane, H. Feng, Screening of non-ionic surfactant for enhancing
re
biobutanol production, Applied biochemistry and biotechnology 177(6) (2015) 1272-1281. [22] A.N. Raut, P.S. Gedam, P.B. Dhamole, Back-extraction of butanol from coacervate phase
lP
using Winsor III microemulsion, Process Biochemistry 70 (2018) 160-167. [23] R. Latsuzbaia, E. Negro, G. Koper, Bicontinuous microemulsions for high yield, wet
ur na
synthesis of ultrafine nanoparticles: a general approach, Faraday discussions 181 (2015) 37-48. [24] S. Vargas-Ruiz, O. Soltwedel, S. Micciulla, R. Sreij, A. Feoktystov, R. von Klitzing, T. Hellweg, S. Wellert, Sugar surfactant based microemulsions at solid surfaces: influence of the oil type and surface polarity, Langmuir 32(45) (2016) 11928-11938.
Jo
[25] R.J. Hickey, T.M. Gillard, M.T. Irwin, T.P. Lodge, F.S. Bates, Structure, viscoelasticity, and interfacial dynamics of a model polymeric bicontinuous microemulsion, Soft Matter 12(1) (2016) 53-66.
[26] M. Tagavifar, S. Jang, L. Chang, K. Mohanty, G. Pope, Controlling the composition, phase volume, and viscosity of microemulsions with cosolvent, Fuel 211 (2018) 214-222.
18
[27] D.G. Hayes, R. Ye, R.N. Dunlap, M.J. Cuneo, S.V. Pingali, H.M. O’Neill, V.S. Urban, Protein extraction into the bicontinuous microemulsion phase of a Water/SDS/pentanol/dodecane winsor-III system: Effect on nanostructure and protein conformation, Colloids and Surfaces B: Biointerfaces 160 (2017) 144-153. [28] A.N. Raut, P.S. Gedam, P.B. Dhamole, Determination of phase transition temperatures of PEO-PPO-PEO block copolymer L62 in presence of fermentation media components, Fluid
of
Phase Equilibria 460 (2018) 126-134.
ro
[29] K. Singh, P.S. Gedam, A.N. Raut, P.B. Dhamole, P. Dhakephalkar, D.R. Ranade, Enhanced n-butanol production by Clostridium beijerinckii MCMB 581 in presence of selected surfactant,
-p
3 Biotech 7(3) (2017) 161.
re
[30] P.S. Gedam, A.N. Raut, P.B. Dhamole, Effect of Operating Conditions and Immobilization on Butanol Enhancement in an Extractive Fermentation Using Non-ionic Surfactant, Applied
lP
biochemistry and biotechnology (2018) 1-13.
[31] Z. Wang, J.-H. Xu, R. Liang, H. Qi, A downstream process with microemulsion extraction
ur na
for microbial transformation in cloud point system, Biochemical Engineering Journal 41(1) (2008) 24-29.
[32] R. Liang, Z. Wang, J.-H. Xu, W. Li, H. Qi, Novel polyethylene glycol induced cloud point system for extraction and back-extraction of organic compounds, Separation and Purification
Jo
Technology 66(2) (2009) 248-256.
[33] P. Winsor, Hydrotropy, solubilisation and related emulsification processes, Transactions of the Faraday Society 44 (1948) 376-398. [34] C. Marche, C. Ferronato, J. Jose, Solubilities of n-Alkanes (C6 to C8) in Water from 30 C to 180 C, Journal of Chemical & Engineering Data 48(4) (2003) 967-971.
19
[35] E. Szekeres, E.J. Acosta, J.F. Faller, Microemulsions of triglyceride-based oils: the effect of co-oil and salinity on phase diagrams, J. Cosmet. Sci 55 (2006) 309-325. [36] L. Adhami., B. Griggs, P. Homebrook, K. Taconi, Liquid-Liquid extraction of butanol from dilute aqueous solutions using soybean derived biodiesel, J. Am.Oil Chem. Soc. (2009), 11231128
by liquid-liquid extraction, Process Biochem., (2010), 1890-1903.
of
[37] S. H. Ha, N.L. Mai, Y-M, Koo, Butanol recovery from aqueous solution into ionic liquids
ro
[38] A. Ishizaki, S. Michiwaki, E. Crabbe, G. Kobayashi, K. Sonomoto, S. Yoshino, Extractive acetone-butanol-ethanol fermentation using methylated crude palm oil as extractant in bath
-p
culture of clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564), J. Biosci. Bioeng.,
Jo
ur na
lP
re
(1999), 352-256.
20
Figure legends Figure 1: Butanol fermentation in absence (control) and presence of surfactant 6% (v/v) L62 at 37°C using 10% (v/v) inoculum. Empty square with dotted line shows butanol concentration and empty triangle with dotted line shows glucose concentration in absence of surfactant L62. Filled square with solid line shows butanol concentration and filled triangle with solid line shows glucose concentration in presence of surfactant 6% (v/v) L62.
of
Figure 2: Phase behavior of Winsor microemulsion using SRP and alkanes (double stage
ro
approach: cloud point extraction followed by microemulsion based extraction); (a) In case of pentane Winsor I microemulsion was obtained in the temperature range of 4-8°C, and (b) In case
-p
of heptane Winsor III microemulsion was obtained in the temperature range of 45-50°C.
re
Surfactant rich to alkanes ratio was 1:1, time=30 min. In both cases, with increase in temperature type of microemulsion changed from Winsor I to III and III to II.
lP
Figure 3: Phase behavior of Winsor microemulsion using simulated ABE broth and alkanes (single stage approach: direct microemulsion based extraction); (a) In case of pentane, Winsor I
ur na
microemulsion was obtained in the temperature range of 4-10°C (b) In case of hexane Winsor III microemulsion was obtained in the temperature range of 45-50°C and (c) In case of heptane Winsor III microemulsion was obtained in the temperature range of 45-50°C. Simulated ABE broth to alkanes ratio 1:1, time=30 min
Jo
Scheme 1: Extraction of butanol and recovery of surfactant using two stage approach: Stage I Cloud point extraction of butanol into SRP (i.e. coacervation) and Stage II – Microemulsion based back extraction of butanol from SRP using alkanes. Cloud point extraction was carried out at 45°C to achieve coacervate and aqueous phases. Butanol was extracted into coacervate phase. Alkanes were added in the volume of 1:1 to the coacervate phase and heated at 45°C for hexane
21
and heptane to get Winsor III microemulsion and 4°C for pentane to get Winsor I microemulsion. Scheme 2: Single stage approach- Downstream process of butanol from simulated ABE fermentation broth with alkanes. Volume ratio of simulated broth to alkane was 1:1. In case of hexane and heptane the solution was heated at 45°C to get Winsor III microemulsion and in case
Jo
ur na
lP
re
-p
ro
of
of pentane solution was cooled to 4°C to get Winsor I microemulsion.
22
Table Legends Table 1: Parameters for butanol separation using CPE from simulated fermentation broth and back-extraction from the SRP by Winsor Microemulsion (two stage approach) Table 2: Parameters for butanol separation from simulated ABE broth using Winsor Microemulsion (Single stage approach) Table 3: Parameters for butanol separation from real fermentation broth using Winsor III
Jo
ur na
lP
re
-p
ro
of
microemulsion (Single stage approach)
23
Tables: Table 1: Parameters for butanol separation using CPE from simulated fermentation broth and back-extraction from the SRP by Winsor Microemulsion (two stage approach)
250
BtOH Conc (g/L) 32.4
BtOH Absolute (g) 8.1
200
26.35
5.27
65.1
SRP
50
56.57
2.83
34.9
SRP Used
5
56.57
0.283
Upper Phase
5
17.2±0.8
0.086±0.004
Lower Phase
5
39.53±0.70
SRP Used
20
54.4±4.38
Upper Phase (Om)
17
24.47±5.1
0.41±0.09
38.2
Lower Phase (Wm)
10.5±0.5
57.96±9.2
0.57±0.1
56.4
Middle Phase (BC) SRP Used
56.57 32.84±1.88
0.283 0.150±0.011
-
Upper Phase (Om)
12.5±0.6 5 4.55±0.05
Lower Phase (Wm) Middle Phase (BC)
3.35±0.15 2.1±0.1
29.58±1.69 -
0.099±0.001 -
35.0 -
Volume (mL)
Initial AqP
System Used Simulated Fermentation Broth
CPE Back Extraction using Winsor I
SRP + Pentane (1:1)
SRP + Hexane (1:1)
Back Extraction using Winsor III
SRP + Heptane (1:1)
Efficiency (%)
30.2
52.8
lP
SFB + Hexane (1:1)
Jo
Separation using Winsor III
SFB + Pentane (1:1)
Separation using Winsor III
SFB + Heptane (1:1)
Conc
BtOH Absolute (g) 8.1
Efficiency (%)
SFB Used
5
32.4
0.162
Upper Phase
5
4±0.2
0.020±0.001
12.4
Lower Phase
4.5
31.59±0.2
0.142±0.001
87.7
SFB Used
5
32.4
0.162
Upper Phase
4.55±0.05
21.5±0.66
0.098±0.002
60.5
Lower Phase
3
19.92±0.67
0.06±0.002
37.0
Middle Phase
2.45±0.05
-
-
-
SFB Used
5
32.4
0.162
Upper Phase
3.9±0.1
24.385±0.045
0.095±0.002
58.6
Lower Phase
3.3±0.1
15.5±1.13
0.051±0.002
31.5
Middle Phase
2.8
-
-
-
ur na
Separation using Winsor I
BtOH (g/L) 32.4
24
Present Study
69.8
Table 2: Parameters for butanol separation from simulated ABE broth using Winsor Microemulsion (Single stage approach) Separation of butanol using Winsor Microemulsion Volume Experiment System Used Sample (mL) Simulated Fermentation Broth Initial 250
Present Study
2.54±0.02
re
Back Extraction using Winsor III
0.198±0.004
ro
Sample
-p
Experiment
of
Separation of butanol using CPE followed by Winsor Microemulsion
[22]
Present Study
Table 3: Parameters for butanol separation from real fermentation broth using Winsor III microemulsion (Single stage approach) Separation of butanol using Winsor Microemulsion Volume BtOH Conc BtOH Efficiency Operation System Used Sample (mL) (g/L) Absolute (g) (%) Microbial Fermentation Broth Initial 50 15 0.75
MFB + Heptane (1:1)
5
15
0.075
Upper Phase
4.55±0.05
10.2±0.03
0.047±0.001
62
Lower Phase
3.35±0.15
7.76±0.06
0.026±0.001
34.8
Middle Phase
2.1±0.1
-
-
-
MFB Used
5
15
0.075
Upper Phase
4.2
10.22±0.16
0.043±0.001
57.3
Lower Phase
3.3±0.1
7.61±0.55
0.025±0.001
33.3
Middle Phase
2.5±0.1
-
-
-
Jo
ur na
lP
re
-p
ro
Separation using Winsor III
MFB + Hexane (1:1)
MFB Used
of
Separation using Winsor III
25
60
18 16 14 12 10 8 6 4 2 0
40 30 20 10 0 48
72 Time (h)
96
120
144
Glucose 6% (v/v) L62
Glucose_control
Butanol 6% (v/v) L62
Butanol_control
of
24
-p
0
ro
Glucose (g/L)
50
Butanol (g/L)
Figures:
Jo
ur na
lP
re
Figure 1: Butanol fermentation in absence (control) and presence of surfactant 6% (v/v) L62 at 37°C using 10% (v/v) inoculum. Empty square with dotted line shows butanol concentration and empty triangle with dotted line shows glucose concentration in absence of surfactant L62. Filled square with solid line shows butanol concentration and filled triangle with solid line shows glucose concentration in presence of surfactant 6% (v/v) L62.
26
re
-p
ro
of
(a)
Jo
ur na
lP
(b)
Figure 2: Phase behavior of Winsor microemulsion using coacervate phase and alkanes (double stage approach: cloud point extraction followed by microemulsion based extraction); (a) In case of pentane Winsor I microemulsion was obtained in the temperature range of 4-8°C, and (b) In case of heptane Winsor III microemulsion was obtained in the temperature range of 45-50°C. Surfactant rich phase to alkanes ratio = 1:1; incubation time=30 min. In both cases, with increase in temperature type of microemulsion changed from Winsor I to III and III to II.
27
(b)
of
ro
-p
re
lP
ur na
Jo (a)
28
-p
ro
of
(c)
Jo
ur na
lP
re
Figure 3: Phase behavior of Winsor microemulsion using simulated ABE broth and alkanes (single stage approach: direct microemulsion based extraction); (a) In case of pentane, Winsor I microemulsion was obtained in the temperature range of 4-10°C (b) In case of hexane Winsor III microemulsion was obtained in the temperature range of 45-50°C and (c) In case of heptane Winsor III microemulsion was obtained in the temperature range of 45-50°C. Simulated ABE broth to alkanes ratio 1:1, time=30 min
29
of ro -p re lP ur na
Scheme 1: Extraction of butanol and recovery of surfactant using two stage approach: Stage I - Cloud point extraction of butanol into SRP (i.e. coacervation) and Stage II – Microemulsion based back extraction of butanol from SRP using alkanes. Cloud point extraction was carried out at 45°C to achieve coacervate and aqueous phases. Butanol was extracted into coacervate phase. Alkanes were added in the volume of 1:1 to the coacervate phase and
Jo
heated at 45°C for hexane and heptane to get Winsor III microemulsion and cooled to 4°C for pentane to get Winsor I microemulsion.
30
of ro -p re lP ur na
Scheme 2: Single stage approach- Downstream process of butanol from simulated ABE fermentation broth with alkanes. Volume ratio of simulated broth to alkane was 1:1. In case of hexane and heptane the solution was heated
Jo
at 45°C to get Winsor III microemulsion and in case of pentane solution was cooled to 4°C to get Winsor I microemulsion.
31