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Biofuels and Biomass
Reduction wastewater discharge towards the secondgeneration acetone-butanol-ethanol production: broth recycling by fermentation-pervaporation hybrid process Changwei Zhang, Siyu Pang, Meng Lv, Xiangyu Wang, Changsheng Su, Weixiang Gao, Huidong Chen, Di Cai, Peiyong Qin, and Tianwei Tan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b03526 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019
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Energy & Fuels
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Reduction wastewater discharge towards the second-generation acetone-butanol-
2
ethanol production: broth recycling by fermentation-pervaporation hybrid process
3 4
Changwei Zhang a,b, Siyu Pang b, Meng Lv b, Xiangyu Wang b, Changsheng Su b,
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Weixiang Gao b, Huidong Chen c,d , Di Cai a,b,*, Peiyong Qin a,b, Tianwei Tan a,b
6 7
* Corresponding author
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Address: No.15, East Road of the North 3rd Ring, Chaoyang District, Beijing,
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100029.
10
E-mail:
[email protected] 11 12
a
13
Technology, Beijing 100029, PR China
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b
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Technology, Beijing 100029, PR China
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c
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Beijing 100029, PR China
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d
19
Technology, Beijing 100029, PR China
National Energy R&D Center for Biorefinery, Beijing University of Chemical
College of Life Science and Technology, Beijing University of Chemical
College of Chemical Engineering, Beijing University of Chemical Technology,
Center for Process Simulation & Optimization, Beijing University of Chemical
20 21 22
Abstract 1
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In order to reduce the butanol fermentation wastewater (BFW) discharge and to
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address the difficulties in BFW treatment, a novel fermentation-pervaporation hybrid
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process was performed. After each batch of acetone-butanol-ethanol (ABE)
5
fermentation, ABE in the fermentation broth were ex situ separated by pervaporation.
6
The retentate was used as the buffer and the solution for the following batches of
7
enzymatic hydrolysis and fermentation, respectively. Results showed that there were
8
little negative influences of the phenols and acid inhibitors on the fermentation
9
performances using the recycled BFW. Benefits from the process for BFW discharge
10
reduction was obvious. After 4 cycles of BFW, ~86 % of the BFW was saved. Over
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94 % of ABE with concentrations of 42.5-50.2 g/L was recovered by pervaporation.
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Therefore, the novel process can effectively eliminate the disadvantage of large BFW
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discharge in the ABE fermentation processes.
14 15
Key words:
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Acetone-butanol-ethanol;
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Fermentation;
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Pervaporation;
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Corn stover;
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Wastewater
21 22
1. Introduction 2
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Energy & Fuels
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Biobutanol (n-butanol) from renewable biomass by acetone-butanol-ethanol
3
(ABE) fermentation has attached much attention to its significant potential applied as
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alternative biofuel. However,Bio-butanol has a poor competitiveness in comparison
5
with its petrochemical equivalent.1 Typically, there are many obstacles limited the
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commercialization of the second-generation ABE plants. One of the main bottlenecks
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is the poor inhibitor tolerances of the Clostridium sp., the ABE producing strains.2 As
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consequences, low titer biobutanol was generated in the fermentation broth. At the
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same time, costly detoxification process for the lignocellulosic hydrolysate treatment
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should be applied prior to inoculation.3
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The low titer biobutanol production also resulted in energy-intensive downstream
12
separation processes.2 In the past decades, alternative separation techniques have been
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developed for energy-efficient butanol recovery.4,5 Among different types of
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separation techniques, pervaporation, the membranes based method, showed great
15
potential due to its high efficiency, energy-saving, no harm to the culture, mitigating
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butanol inhibition, and improving the solvents productivity.6-8 More importantly,
17
pervaporation also showed the potential to recover toxic furan, phenol and organic
18
acids from the lignocellulosic hydrolysate or the fermentation broth without sugars
19
loss.9-13
20
Accompany with the low titer ABE production, an additional concern is the
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production of significant amounts of butanol fermentative wastewaters (BFW) with
22
high COD.14 Efforts have been taken to solve the problems. For example, one of the 3
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potential methods is cascading the ABE and oleaginous microorganism’s
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fermentations for microbial lipids production by organic acids and residual sugars.15-16
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The energy requirement in the cascading process was estimated to have decreased by
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at least 24 MJ/kg when further coupled the fermentation process with in situ product
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recovery (ISPR) systems.17 Another potential strategy was conversion the
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carbohydrates in BFW into bacterial cellulose, which also partially decrease the COD
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concentration.18
8 9
Unfortunately, although COD in the BFW could be significant decrease by hybrid an additional fermentation process after ABE fermentation, the total amount of the
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BFW was still large, which called for further intensive anaerobic treatment. Actually,
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the problem of large BFW discharge has long been ignored in previous researches.
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Severe environment consequences would occur if we still keep a blind eye on this
13
problem. For instance, ABE concentration in fermentation broth was about 1.5 %
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(w/v), while alcohol titer in conventional ethanol plant was about 13 % (w/v).
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Therefore, for given amount of ABE production, the BFW discharge was almost 10
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times higher than that from the ethanol plant. To decrease the overall amount of BFW,
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one of the effective ways is using concentrated enzymatic hydrolysate based on fed-
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batch fermentation-ISPR integration process.19 Drawbacks, however, was the high
19
energy demand in hydrolysate concentration after fibers pretreatment and
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saccharification. Besides, phenolic inhibitors were also cumulated in the concentrated
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hydrolysate, which showed severe inhibition to the ABE fermentation.19
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Another strategy is recycling the ABE fermentation broth after distillation and 4
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subsequent adsorption.14 Based on that process, inhibitors such as organic acids and
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phenol compounds were effectively removed. Nevertheless, the process was complex
3
and the ABE separation unit was also energy-intensive. Besides, partial of the carbon
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sources including sugars and acids (acetic acid and butyric acid) were lost after
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absorption and detoxification.9,20,21 In addition, the regeneration of the micro-
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/mesoporous activated carbon was difficult, while the pH of the BFW was adjusted by
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environmental unfriendly mineral acids.
8 9
Hence, it urges to develop alternative BFW recycling processes for ABE production. Unfortunately, to our best knowledge, only a limited number of researches
10
were focused on this point. Besides, there were not any reports using the recycled
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BFW for the following cycles of lignocellulosic biomass saccharification and
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fermentation. In this study, to overcome previous remaining drawbacks and to re-use
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BTW in subsequent saccharification and fermentation, ABE fermentation-
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pervaporation hybrid process was established using corn stover as raw material. After
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batch fermentation using the enzymatic hydrolysate of corn stover, BFW in the
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retentate of pervaporation was reused in other cycles of enzymatic hydrolysis and the
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subsequent fermentation for ABE production.
18 19
2. Materials and methods
20 21
2.1 Raw material and the pervaporation membrane
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Corn stover (CS) was collected in a local farm of Qinhuangdao, Hebei province,
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China. The main compositions of the CS were: 35.4±3.2 % of glucan, 22.3±2.6 % of
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xylan, 2.1±0.3 % of arabinan and 21.2±1.8 % of acid insoluble lignin. After milled
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into 60 mesh (below 0.076 mm), the bagasse was dried out at 105 oC.
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A PDMS/PVDF membrane was prepared in our lab following the method
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described by Li et al. study,22 by mixing 3 wt% DBSA with TEOS, DTBL and PDMS
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at room temperature, followed by coating on the PVDF layer. After that, the
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membrane was dried and cured for 24 h before use. The thickness of active and
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supporting layers were 12 μm and 34μm, respectively
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2.2 Strains and medium
12 13
C. acetobutylicum ABE-P 1201 that tolerance certain amounts of organic acids
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and phenolic compounds was used for batch ABE fermentation.19 The seed medium
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contained 40 g/L of glucose, 0.1 mg/L of p-aminobenzoic acid, 0.01 mg/L of biotin,
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the mineral salts (10 mg/L of FeSO4, 10 mg/L of MnSO4, 1 g/L of KH2PO4, 1 g/L of
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K2HPO4, 0.2 g/L of MgSO4) and the nitrogen source (2.2 g/L of ammonium acetate).
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The medium was purged with nitrogen in order to generate an oxygen-free medium,
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followed by autoclaved at 121 oC for 20 min. The inoculation size was 10 % (v/v).
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The culture conditions were: 37 oC and 40 rpm.
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2.3 Experimental setup 6
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The experimental setup is shown in Fig.1. As can be seen, the CS bagasse was
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pretreated by 2 % (w/v) NaOH under a solid loading rate of 10 % (w/v). After
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maintained at 120 oC for an hour, the bagasse was separated and washed by deionized
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water until pH on the solid surface decreased to 7. After dried out, the bagasse was
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hydrolyzed by cellulase (20 FPU/g, purchased from KDN Co. Ltd, China).
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H3PO4/KH2PO4 buffer (0.01M) for enzymatic hydrolysis was conducted so that
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provides suitable phosphate sources and potassium sources to the subsequent ABE
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fermentation.23 For the first cycle of fermentation, H3PO4/KH2PO4 buffer was mixed
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well with 6 % (w/v) of the pretreated bagasse. The enzymatic hydrolysis conditions
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were: 50 oC, pH 4.8, 200 rpm and 72 h. After separating the hydrolysate, pH of the
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liquid was adjusted to 7 by 20 % (w/v) of ammonium hydroxide.
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For ABE fermentation, similar conditions were performed using the enzymatic
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hydrolysate as substrate. pH in bioreactor kept at ~4.5 by ammonium hydroxide/KOH
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aqueous mixture (10 %/5 %, w/v, ammonium hydroxide and KOH were dissolved
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together in aqueous solution, the solution was not only used as pH adjustor, but also
17
used as supplemented potassium and nitrogen sources for the following ABE
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fermentation). The working volume of the 1st cycle of fermentation was 1 L. After 72
19
h of batch fermentation, cells were separated by microfiltration (0.22 μm, Tianjin
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Motianmo Co. Ltd., China). The remaining cells were reused as the seed in the
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following cycles and were stored in the seed medium, in order to ensure fresh
22
inoculums conducted for each cycle. The cells free fermentation broth was passed 7
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through the pervaporation module similar with the method described in Cai et al.9 The
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area of pervaporation membrane was about 47 cm2. For each batches of
3
pervaporation, 0.25 L fermentation broth was added into a conical flask, and was
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passed through the membrane module at a speed of 0.2 L/min (~25 oC). The pressure
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on the permeate side of pervaporation membrane was kept below 200 Pa. The
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separated ABE was collected in a cold trap in liquid nitrogen. The feed pressure was
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maintained at 0.1 Mpa.
8 9
After pervaporation separation, the retentate was collected, namely BFW. After adjusted pH to 4.8 by KOH, the BFW was used as the buffer for the following batches
10
of enzymatic hydrolysis and ABE fermentation. In the current work, the BFW, the
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ABE free fermentation broth, was recycled for 4 batches.
12 13
2.4 Assay
14 15
Sugars in the enzymatic hydrolysate of CS, fermentation broth and the retentate
16
of pervaporation were determined by high performance liquid chromatography
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(HPLC, Shimadzu LC-10A, Japan) following the method of Cai et al. study.24 An
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Aminex HPX-87P column (Biorad) and a differential refraction index detector were
19
used. ABE and the organic acids concentration in the fermentation broth and the
20
permeate were determined by a gas chromatography (Shimadzu GC-2010, Japan). A
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hydrogen flame detector and a Porapack Q column were applied.25 Total phenolic
22
compounds were analyzed by the Folin-Ciocalteu method.26 8
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Energy & Fuels
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Aspen plus V 8.0 was used to simulate the distillation process for ABE
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separation after ex situ pervaporation. Similar to our previous studies,4,27 NRTL
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property model and Sequential Modular Approach were applied. The product purities
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were: >92 wt % of ethanol, >99.9 wt% of butanol, and >99.7 wt. % of acetone in
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order to meet the national standard of China (GB/T 6026-1998).
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3. Results and discussion
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3.1 Enzymatic hydrolysis using the recycled ABE fermentation broth
10 11
The ABE free fermentation broth was not only used as the aqueous solution for
12
the following batches of fermentation, but also used as the buffer for enzymatic
13
hydrolysis (see Fig.1). Acids by-products including acetic acid and butyric acid was
14
reacted with KOH before the subsequent enzymatic hydrolysis steps. As was shown in
15
Fig.2a, except for the first batch of hydrolysis, before being reused in the following
16
batches of hydrolysis, 10-12 g/L of residual sugars remained in retentate. Therefore,
17
the glucan and xylan recovery rates were little decrease after the 2nd cycle (Fig.2b).
18
Specifically, 88.7 % of glucan and 63.6 % of xylan were recovered as reducing sugars
19
after the 1st cycle, and the glucan and xylan recovery rates were 81.2 % and 58.6 %,
20
respectively, after the 4th cycle of enzymatic hydrolysis.
21
The inhibition of the recycled fermentation broth was likely due to the lignin
22
debris or the phenolic compounds.28-30 There was 0.6 g/L of total phenolic compounds 9
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in the ABE absence broth after the 1st cycle of pervaporation (see Fig.4a). In addition,
2
similar saccharification results obtained after the 2nd cycle was influenced by lignin
3
toxicity and the remaining cellulase from the front-cycles of fermentation broth.
4
We also speculated that the organic acids from fermentation broth have negative
5
influence on the enzymatic hydrolysis, as was indicated in Chylenski et al. study.31
6
Luckily, the increasing acids by-products concentration in BTW (as the buffer)
7
showed no obvious influence on sugars production (Fig.S1a to Fig.S1c). To further
8
determine the influence of acetic acid and butyric acid in the recycled fermentation
9
broth on cellulase activity, proof experiments were carried out using different types of
10
buffer include acetic acid/ KOH and butyric acid/ KOH. At the same time, the
11
conventional citric acid/ sodium citrate and the H3PO4/KH2PO4 buffers were also
12
performed as the control groups. Although it was evidenced in literature that acetic
13
acid could be the inhibitor to cellulase,32 results obtained in Fig.S1d indicated that the
14
glucose and xylose concentration remained similar no matter which buffer used.
15
Therefore, the by-products acids showed no influence on enzymatic hydrolysis. The
16
effect of residual sugars concentration on the enzymatic hydrolysis process was also
17
evaluated, which showed no impact on sugars production. We finally speculated that
18
phenols and lignin debris in the recycled broth may the key influencers to the short
19
decrease of the saccharification performance. Phenols and lignin debris would be
20
irreversible bonding with cellulase so as to hinder the adhesion of cellulase to the
21
surface of cellulose.29,30
22 10
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3.2 Batch fermentation using the recycled ABE fermentation broth
2 3
It is vital to evaluate the differences of fermentation performances using the
4
recycled BFW, in order to test the feasibility of recycle BFW for ABE production.
5
Batch fermentation was carried out using the recycled BTW. As was shown in Fig.3a,
6
in comparison with the 10.1 g/L of total ABE in the end of the 1st cycle of
7
fermentation, ABE concentration in the following batches decreased, even though
8
there were only slight differences in the initial sugars concentration at the beginning
9
of each cycle. Therefore, the recycled BFW showed negative impact on ABE
10
fermentation. Correspondingly, as it also indicated in Fig.2a, residual sugars
11
concentration after each cycles were slight increased. The fermentation period was
12
also increased with the increase of the broth recycling (Fig.3a and Fig.3b). In
13
consequence, accompany with the decrease of ABE concentrations in the end of each
14
batches of fermentation, ABE yield and productivity were also gradually decreased
15
with the increase batches of fermentation based on the BFW (Fig.4).
16
Acid by-products concentrations, however, were increased after the 1st cycle of
17
BFW. There were 5.3 g/L of acetic acid and 1.6 g/L of butyric acid in the BFW after
18
the 2nd cycle. In contrast, the acetic acid and butyric acid concentrations in the end of
19
the 1st cycle were only 3.1 g/L and 0.8 g/L, respectively. After the 2nd cycle, the acids
20
concentrations were slightly increased. For each cycles of ABE fermentation, acids
21
concentrations were always increased significantly before reaching the logarithmic
22
phase. Then, a short decreased of acid concentrations were occurred until the end of 11
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fermentation. This phenomenon could be explained by two distinct steps in the
2
metabolic of C.acetobutylicum. It has long been recognized that ABE fermentation
3
was a biphasic system.33,34 The first phase was the acidogenic phase. In this phase,
4
acids producing pathways were active, acetic acid and butyric acid were produced
5
during the exponential growth phase.35 The second phase was the solventogeneis
6
phase, of which the solventogenic genes were expression.36 During the solventogeneis
7
phase, acid by-products were likely to be reused as additional carbon source to
8
produce alcohols by the effect of acetoacetyl-CoA acetate/butyrate-CoA-transferase
9
catalysis.37,38 As results, acids concentrations were decreased slightly after the phase
10
transitions (see Fig. 3c). Similar trends were also obtained in our previous works.9,39
11
Nevertheless, acids concentrations were not significantly changed in comparison with
12
the broth before inoculation after the 2nd cycle. It might be caused by the relatively
13
high acids concentration in BFW after three cycles. Acids in the former broth were
14
subsequently consumed in the following batches of fermentation.40
15
However, literatures showed that the ABE concentrations were almost unchanged
16
but the butanol ratio was improved if adding suitable amount of acetic acid and
17
butyric acid in the initial medium.41 Organic acids are related to the ATP regeneration
18
system that was derived by several enzymes such as acetate kinase.42 Another few
19
works found that the ABE production only increased slightly under low acetic and
20
butyric acids concentration environments.43,44 On the contrary, superfluous organic
21
acids also showed inhibition to the metabolism of Clostridia.40,45 This might be one of
22
the reasons for the poor performances of ABE fermentation when increasing the broth 12
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recycle times. To test the influence of acid by-products concentrations on batch fermentation
3
performances, confirmatory experiments were also carried out by adding acids in the
4
CS hydrolysate before inoculation. Generally, butyric acid showed severer inhibition
5
to the ABE production compared with the effect of acetic acids (Fig.S2a and Fig.S2b).
6
A synergic effect of acetic acid and butyric acid was proved because higher ratios of
7
butanol was obtained (see Fig.S2c), which was also obtained in previous studies.46
8
However, acetone, butanol and ethanol concentrations were only 1 g/L, 0.3 g/L and
9
0.7 g/L in end of fermentation using the CS hydrolysate that contain 10 g/L acetic acid
10
and 4 g/L of butyric acid. In this case, ‘acid crush’ was occurred (Acids by-products
11
were no longer reassimilated in the solventogeneis phase, which was caused by the
12
high undissociated acids concentrations in broth).47 Fortunately, for the ABE
13
fermentation using the recycled BFW, acids in the recycled BFW no longer ledto the
14
‘acid crush’ by pH control. Only a small decrease of ABE production was obtained in
15
Fig.3.
16
Fermentation performances of the 4th cycled BFW and the synthetic CS
17
hydrolysate, under similar initial acids concentrations, were further compared
18
( Fig.S2d). ABE production from the 4th cycled BFW group was lower than that of the
19
synthetic CS hydrolysate, indicating other toxic compounds in the recycled BFW also
20
influenced on the fermentation performances. We speculated thatphenols existing in
21
BFW, mainly generated from the pretreatment of lignocellulosic materials, are another
22
group of inhibitors. A various works have proved phenols inhibition to the Clostridia 13
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strains.48,49 Cells growth of C. beijerinckii was decreased by 64-75 % when the
2
phenols concentration was 1 g/L in substrate.43 Our previous study also indicated the
3
phenolic compounds had severe inhibition to the metabolism of C. acetobutylicum
4
ABE-P 1201.19 Here, to minimize the influence of phenols inhibition to the cycled
5
BFW, alkaline pretreatment was performed, which also suggested have little phenol
6
inhibitors in enzymatic hydrolysate.23,50
7
Fig.4a shows the total phenols concentration remained in the BFW after
8
fermentation and pervaporation. The detoxified effects of pervaporation separation to
9
the volatility furan by-products and phenols contained in the lignocellulosic
10
hydrolysate were well proved.9,10 However, the removal of phenols in BFW by
11
pervaporation was not obvious in the current work. One of the reasons for this
12
phenomenon is the low titer phenols contain in BFW. For another reason, lignin debris
13
and low-volatility phenols can hardly permeate through the active layer of
14
pervaporation membrane. In the cold trap, no phenols and furans were detected. As a
15
result, accompany with the increase of BFW cycling times, total phenolic compounds
16
concentration in broth was increased from 0.6 g/L after the 1st cycle to 2.2 g/L after
17
the 4th cycle. Moreover, no additional nutrients were added in the BFW during the
18
broth recycling process except for the N, P, and K. Thus, the lack of metal ions and
19
other nutrients might be another factor for the decrease of the fermentation
20
performances using the recycled BFW. In brief, accompany with the reduction of
21
BFW discharge by recycling the fermentation broth, the fermentation performances
22
was negatively influenced by the inhibitors in BFW. Fortunately, the inhibition effect 14
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of BFW on ABE fermentation was acceptable when compared with the greater benefit
2
from wastewater management.
3 4
3.3 Pervaporation for ABE separation from the recycled BFW
5 6
Batch pervaporation was carried out using the recycled BFW and the
7
PDMS/PVDF membrane. Fig.6 shows the kinetics of pervaporation separation of
8
ABE in fermentation broth. Accompany with the decrease of ABE concentration in
9
feed, ABE concentration in permeate was also decreased.8,9 In all of the tested groups,
10
compared with butanol and ethanol, acetone concentration in retentate was dropped
11
sharply with lengthen the operation time. It was due to the high solubility of acetone
12
in the membrane.51 In contrast, the poor permeation of ethanol was caused by the
13
bonding and aggregation of ethanol to water molecular.52 It showed the pervaporation
14
efficiency was decreased significantly by the fouling effect of components in the
15
fermentation broth.53,54 Fortunately, the results showed in Fig.6 indicated that the ABE
16
separation performance was not hugely dropped after overall 4 batches of operation.
17
The cross-flow under high speed of the recycled BFW in the pervaporation process
18
might be a reason for the stable ABE separation performances.55 Detail of the
19
pervaporation performances, including the separation factor of solvents, and ABE and
20
water fluxes, are also presented in Fig.S3 and Fig.S4, respectively.
21
Finally, approximately 94.1 %, 98.1%, 95.5 %, and 97.9 % of ABE was recycled
22
by pervaporation from the 1st, 2nd, 3rd and 4th cycled of BFW, respectively. The overall 15
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1
permeate ABE concentrations were 42.5 g/L, 50.2 g/L, 44.3 g/L and 44.7 g/L,
2
respectively. The permeate ABE in the pervapration condensate can be easily purified
3
by downstream distillations.56 The high titer ABE condensate also benefited to
4
decrease the energy requirement of ABE separation process.4,5 To evaluate the energy
5
requirement for ABE production based on ex situ pervaporation-distillation, static
6
process simulation was conducted using Aspen plus V8.0. Based on the distillation
7
series established in our previous research,57 heating and cooling energy requirement
8
were then calculated (Fig.S5, Table S1). The conventional distillation processes that
9
hybrid with ABE fermentation unit was also simulated as the control. Similar with our
10
previous estimation results that was based on in situ pevaporation-distillation,4,27 the
11
overall downstream ABE separation process based on ex situ pervaporation-
12
distillation was also more energy-saving in comparison with the conventional
13
distillation. 25.61 MJ/kg (of butanol) heat was required for ABE separation
14
(excluding the evaporation energy in pervaporation unit), which was only 33.46 % of
15
the control group based on conventional distillation (Fig.S6). Therefore, the novel
16
BFW recycle process presented in this study is not only of low wastewater discharge,
17
but also energy-effective.
18 19
3.4 Comparison of the BFW recycling times on ABE production.
20 21
As was indicated in the results above, the more the recycle times of BFW was,
22
the higher the inhibition of BFW to the ABE fermentation would be. However, the 16
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more the recycle times of BFW was, the little the overall wastewater would discharg.
2
Therefore, ABE production was partially sacrificed in order to gain the benefits from
3
the decreasing effluent discharge. Key parameters of the current work were
4
summarized in Table 1. Although the ABE fermentation was gradually inhibited by
5
the toxic compounds cumulated in the BFW, the discharge of BFW was also
6
significantly reduced. Because of the reusable of the BFW as the buffer and
7
fermentation media, after 4 cycles of fermentation, only ~560 mL of BFW was
8
obtained, which was only ~14 % of the conventional batch fermentation process that
9
not recycle the BFW and discharge the BFW directly. Therefore, the environmental
10
benefits of the current broth recycling process were significant. With lower BFW
11
discharge, the energy-intensive downstream product recovery and water treatment
12
processes were further improved.58
13
In addition, the current work avoided using citrate buffer. The citrate strengths
14
inhibited the bacteria growth, which was likely due to the permeability changing of
15
membrane, reducing the cells internal pH, and chelating trace elements in
16
solution.59,60 Instead, the acid by-products in BFW were recycled as buffer and
17
medium in the following batches of CS saccharification and fermentation. Although
18
the metabolism of C.acetobutylicum was inhibited by superfluous acids in medium,
19
the acetic acid/butyric acid based buffer showed little influence on the enzymatic
20
hydrolysis performances.61 The acids concentration after the 3rd and the 4th cycles of
21
fermentation were decreased, indicating that acids remained in the hydrolysate could
22
be partially reused as the carbon source for fermentative ABE production (Table 1). 17
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1
Compared with our previous work based on fermentation-gas stripping coupled
2
process,19 results obtained in current work were also attractive because the BFW
3
recycling process not only has batter stability and construability,it also avoidedthe
4
energy-intensive hydrolysate concentration process. Attributed to the gradually
5
decrease of ABE concentration that remained in the retantate in batch fermentation,
6
the permeate ABE concentration was not as high as the previous works that integrated
7
the fermentation unit and pervaporation unit for in situ ABE recovery.62-64
8
Nevertheless, the efficiency of pervaporation separation was not decreased. Actually,
9
pervaporation was also highly effective when feeding the lower concentration
10
ABE.65,66 In this study, a permeate ABE concentration of 42.5-50.2 g/L was obtained
11
(Table 1). As was estimated, energy requirement of the downstream purification
12
process was lower than 1/3 of the butanol combustion heat when feeding the 4 wt. %
13
butanol aqueous into distillation columns.56, 66 When further purifying the permeated
14
ABE mixture by distillation, the theoretical energy requirement for dehydrated
15
butanol production was 25.61 MJ/kg based on process simulation.
16
In fact, another obstacle of the ISPR based on fermentation-pervaporation
17
integration processes is the inconstant working volume of the bioreactor. It is caused
18
by the high titer ABE output and lower sugars supplement rate. To achieve long-term
19
operation, adjusting the working volume of bioreactor constantly, two-streams of ABE
20
aqueous that output from fermentation broth and the permeate condensate of
21
pervaporation membrane were separately fed into the beer column of distillation.67-69
22
On the contrary, the distillation process feeding the ex situ ABE solvent was simpler, 18
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which was also energy-saving. Moreover, another benefit of the batch pervaporation separation process is the
3
high ABE recovery rate. Above 94 % of ABE after each batches of fermentation was
4
successfully recovered on the permeate side of membrane (Table 1). In contrast, there
5
were still high concentrations of ABE remained in the bioreactor in the ISPR
6
process.19 Besides, in comparison with the inevitable problem of membrane in long-
7
term of ISPR processes,54 the intermittent batch pervaporation showed superiority in
8
bio-fouling elimination. Microfiltration can be operated before each batches of
9
pervaporation started.70,71 Overall, using the novel hybrid process for ABE production
10
and separation, the advantages of BFW discharge reduction far outweigh the
11
disadvantages of the decrease of fermentation performance. The process was
12
generally energy-saving and environmentally-friendly.
13 14
4. Conclusions
15 16
A novel BFW recycle method for ABE fermentation was developed in the
17
current work. The recycled BFW after pervaporation can not only be used as the
18
buffer for saccharification, it also can be used as the solution for ABE fermentation.
19
Based on this method, after 4 cycles of fermentation, about 86 % of BFW discharge
20
can be reduced with partially decreased of ABE yield and productivity. Above 4 %
21
(w/v) of ABE was recovered on the permeate side of pervaporation, which can greatly
22
reduce the downstream energy requirement. 19
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1 2 3 4
Acknowledgements
5 6
This work was supported in part by the National Nature Science Foundation of
7
China (Grant Nos. 21676014, and 21706008), Beijing Natural Science Foundation
8
(2172041), China Postdoctoral Science Foundation (Grant Nos.2017M610037;
9
2018T110036) and the Fundamental Research Funds for the Central Universities
10
(Grant No. ZY1832).
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butanol-ethanol production from sweet sorghum bagasse. Biotechnol. Biofuels
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cells in an integrated fermentation-pervaporation process. Process Biochem. 2015,
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of citrate buffer in acetone-butanol-ethanol (ABE) fermentation using corn stover
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and high-efficient product recovery by vapor stripping-vapor permeation (VSVP)
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coupled process. Bioresour. Technol. 2014, 169, 251-257.
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composite poly (dimethyl siloxane) membrane for butanol recovery from model
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(67) Van Hecke, W.; Vandezande, P.; Claes, S.; Vangeel, S.; Beckers, H.; Diels, L.; De
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Wever, H. Integrated bipprocess for long-term continuous cultivation of
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Clostridium acetobutylicum coupled to pervaporation with PDMS composite
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membranes. Bioresour. Technol. 2012, 111, 368-377.
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pervaporation: experimental results and conceptual plant design. J. Ind. Microbiol.
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Figure captions
4 5
Fig.1 Flow chart of the BFW recycling process. PDMS/PVDF membrane was used
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for ABE separation. The fermentation broth that without ABE product was
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recycled.
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Fig.2 Enzymatic hydrolysis of the pretreated CS bagasse using the recycled ABE
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fermentation broth after pervaporation. The loading rate of the pretreated CS
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bagasse was 6 % (w/v). The abscissa axis refers to the sum of hydrolysis time in
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different cycles. To ensuring high enzymatic efficiency, 72 h of hydrolysis time was
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performed. (a) Kinetics of sugars production in the enzymatic hydrolysis process in
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each cycles; (b) sugars recovery rates after each cycles of BFW.
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Fig.3 Time course of the batch ABE fermentation using the recycled fermentation
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broth. CS hydrolysate was used as the substrate. The abscissa axis refers to the
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sum of fermentation time in different cycles. And ABE products were separated
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by pervaporation before recycled in the next period. (a) ABE concentrations; (b)
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residual sugars concentration remained in the fermentation broth; (c) Time
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course of acid by-products during each cycles of batch fermentation. The results
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showed decrease ABE concentrations with the recycled times. Acid by-products
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were increased sharply after the 1st cycle of BFW.
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Fig.4 Effects of recycling times of the BFW on ABE (a) concentrations, (b) yields and 32
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(c) productivities. In comparison of the light decrease of ABE concentrations and
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yields with the times of cycles, dramatic change was occurred in ABE productivity.
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It might be caused by the inhibitions remained in the recycled BFW.
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Fig.5 Inhibitors accumulation during the BFW recycling process. (a) Total phenolic compounds; (b) acids-by products. Fig.6 Kinetics of the batch pervapration feeding different cycles of BFW. The abscissa
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axis refers to the sum of the operation time of pervaporation in different cycles.
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The pervaporation unit was turned off when the ABE concentration remained in
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the retentate was lower than 1.5 g/L. The retentate kept at room temperature (25
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o
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condensate of pervaporation. The volume of the pervaporation condensates after
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the 1st cycle, 2nd cycle, 3rd cycle and 4th cycle were 224 mL, 161 mL, 142 mL and
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116 mL, respectively.
C). (a) ABE concentration remained in retentate; (b) ABE concentration in the
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Fig.1
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Fig.2
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Fig.3
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Fig.4 37
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Fig.5 38
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Fig.6 39
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Energy & Fuels
Table 1 Key parameters of batch ABE fermentation and pervaporation using the recycled BFW. Parameters 1st cycle 2nd cycle 3rd cycle 4th cycle BFW volume (mL) ~1000f ~780 ~690 ~560 a d Wastewater saving rate (%) 20 ~65 ~80 ~86 ABE concentrations (g/L) 9.9 8.5 8.7 8.1 Acetone 2.1 1.6 1.9 1.8 Butanol 7.2 6.1 5.5 5.6 Ethanol 0.1 0.8 1.3 0.8 e Acids by-products (g/L) 3.9 3.0 -0.3 -0.1 e Acetic acid 3.1 2.2 -0.2 e -0.1 e Butyric acid 0.8 0.8 -0.1 e 0e Overall ABE yield b(g/g) 0.32 0.30 0.29 0.28 c Overall ABE productivity (g/L h) 0.17 0.13 0.11 0.09 Cumulative of ABE condensate (mL) ~224 ~385 ~527 ~643 ABE in condensate (g/L) 42.5 50.2 44.3 44.7 Acetone 8.1 10.1 9.1 9.3 Butanol 32.5 37.0 31.6 31.6 Ethanol 1.9 3.1 3.6 3.9 a The wastewater saving rate is the amount of BFW after each batches of pervaporation divide by the volume of broth that after the conventional batches of fermentation. Except for the recycle of BFW, the separation of water and ABE by pervaporation also contributed to the decrease of fermentation wastewater discharge. Moreover, the additional water from the seed was also considered to calculate the wastewater saving rate. b ABE yield is the ABE production of the cycled BFW towards the total reducing sugars consumed in batches of fermentation. c ABE productivity is the ABE production of the cycled BFW towards the volume of BFW and the sum of fermentation time based on the BFW cycles. d The decrease of BFW volume in the 1st cycle is mainly caused by the solvents and water separated in the pervaporation process. 41
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e
The differences of the initial BFW and the fermentation broth. Data shows that the net acids consumption is negative, indicating that the acids could be reused as additional carbon source for the metabolism of C. acetobuylicum ABE-P 1201. f The working volume in the first batch ABE fermentation using the CS hydrolysate.
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