Co-culture of Zymomonas mobilis and Scheffersomyces stipitis immobilized in polymeric membranes for fermentation of glucose and xylose to ethanol

Biochemical Engineering Journal 145 (2019) 145–152

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Regular article

Co-culture of Zymomonas mobilis and Scheffersomyces stipitis immobilized in polymeric membranes for fermentation of glucose and xylose to ethanol Duong Thi Thuy Nguyena, Prashant Praveenb, Kai-Chee Loha, a b

T

⁎

Department of Chemical & Biomolecular Engineering, National University of Singapore, Singapore Clean Technologies, Scion, Rotorua, New Zealand

H I GH L IG H T S

hollow fiber membrane bioreactor (SHFMB) was developed. • Submerged of immobilized and suspended Z. mobilis and S. stipitis was conducted. • Co-culture alleviated catabolite repression, substrate and product inhibition. • SHFMB glucose and xylose fermentation was observed in the SHFMB. • Concomitant • Cell immobilization in SHFMB was stable and performance improved with time.

A R T I C LE I N FO

A B S T R A C T

Keywords: Bioethanol Catabolite repression Cell immobilization Fermentation Hollow fiber membranes Xylose

Submerged hollow fiber membrane bioreactor (SHFMB) was fabricated and operated to co-culture Zymomonas mobilis and Scheffersomyces stipitis for fermentation of glucose and xylose mixture. Suspended Z. mobilis and S. stipitis in co-culture exhibited poor xylose fermentation due to substrate/product inhibition and catabolite repression. Immobilization in polymeric membranes was effective in alleviating these inhibitions in the SHFMB, which resulted in concomitant fermentation of glucose and xylose at initial glucose and xylose concentrations of 20–80 g L−1 and 10–40 g L−1, respectively. Selective aeration in SHFMB resulted in 100% glucose and > 70% xylose fermentation within 48 h, producing 36.7 g L−1 ethanol from 90 g L−1 sugar mixture. Partial immobilization in the membranes allowed Z. mobilis to diffuse in suspension under benign condition, whereas S. stipitis remained inside membrane pores throughout the operation. Xylose fermentation in the SHFMB could be enhanced further by increasing the number of S. stipitis immobilized membranes. Cell immobilization in the SHFMB was stable during 12 consecutive batch runs over 24 days, and exhibited improved xylose fermentation due to gradual increase in S. stipitis accumulation within the membranes. These results indicate that SHFMB can be promising for co-culture of Z. mobilis and S. stipitis in fermentation of mixed sugars from lignocellulosic hydrolysate.

1. Introduction Lignocellulosic biomass is a readily available, low-cost, sustainable feedstock for the production of a wide variety of commodities, including biofuels [1]. Lignocellulose is comprised of cellulose, hemicellulose and lignin. Both cellulose and hemicellulose are carbohydrate polymers, and these can be hydrolyzed into pentose and hexose sugars, which are generally represented as glucose and xylose [2]. Although hemicellulose constitutes nearly one-third of lignocellulosic biomass, research on lignocellulosic biomass has been focused primarily on

utilization of cellulosic fractions, which yields glucose upon hydrolysis. Utilization of hemicellulose fraction of lignocellulose can be an important factor in boosting productivity and yield during microbial fermentation [3,4]. The primary challenge in fermentation of hydrolysate from both cellulose and hemicellulose is the inability of commonly used fermentative bacteria and yeast, such as Saccharomyces cerevisiae and Zymomonas mobilis, in fermenting both glucose and xylose into ethanol [5]. In the absence of such microorganisms, two strategies are generally adopted in co-fermentation of these sugars. The first strategy is based

Abbreviations: SHFMB, submerged hollow fiber membrane bioreactor; HFM, hollow fiber membranes; OD, optical density; DO, dissolved oxygen ⁎ Corresponding author at: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117585, Singapore. E-mail address: [email protected] (K.-C. Loh). https://doi.org/10.1016/j.bej.2019.02.019 Received 19 September 2018; Received in revised form 16 January 2019; Accepted 24 February 2019 Available online 25 February 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.

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2.2. Co-culture using suspended cells

on genetic engineering of model organisms to incorporate pathways for xylose utilization. Several microbial strains, such as Escherichia coli, Klebsiella oxytoca, Z. mobilis and S. cerevisiae have been genetically engineered to express both glucose and xylose fermentative pathways with promising results [6–8]. However, the recombinant microorganisms have not found industrial application, due to poor stability and susceptibility to stress associated with changes in pH or temperature, and low tolerance to lignin-derived inhibitors or ethanol [9]. Another approach for simultaneous glucose and xylose fermentation is based on co-culture, wherein microbes utilizing glucose are cultivated along with different group of microbes utilizing xylose in the same medium [10–12]. The key to establish a successful co-culture is the compatibility between the microorganisms, such that they are able to co-exist under identical fermentation conditions of pH, temperature and aeration. Some of the successful co-culture systems reported in the literature include: E. coli with S. cerevisiae; Z. mobilis with Candida tropicalis, and; S. cerevisiae with Pachysolen tannophilis [3,13–15]. Although these co-culture system have exhibited excellent results, vulnerability of xylose-fermenting strains to catabolite repression from glucose is a major challenge [16]. Besides, co-culture systems usually operate at low mixed sugars concentrations (< 40 g L−1 of glucose and 10–15 gL−1 of xylose) due to high sensitivity of xylose-utilizing microorganisms to glucose, ethanol and lignocellulose-derived inhibitors. Furthermore, some xylose-utilizing microorganisms, such as S. stipitis, require microaeration for xylose fermentation, which may compromise anaerobic fermentation of glucose [17]. Although most co-culture studies on bioethanol fermentation have been conducted using suspended cells, several studies have also demonstrated the use of immobilized co-culture system in boosting bioethanol yield and productivity [16,18,19]. Cell immobilization has been effective in protecting microorganisms from various inhibitory compounds, including sugars, ethanol and lignocellulose-derived inhibitors, and some reports have indicated that cell immobilization may also alleviate catabolite repression of xylose-utilizing microorganisms [15,20]. Among various substrates available for cell entrapment, polymeric hollow fiber membranes (HFM) provide some unique advantages. The HFMs offer partial immobilization, which allows bacteria to freely diffuse in or out of the support pores, whereas membrane materials exhibit excellent stability. Besides, tubular HFMs can also simplify oxygen transfer to xylose-utilizing microorganisms [21,22]. In this research, co-culture of Z. mobilis and S. stipitis was investigated for simultaneous glucose and xylose fermentation under different modes. Baseline experiments were conducted to investigate the productivity of co-cultures using suspended cells. A submerged hollow fiber membrane bioreactor (SHFMB) was developed for coculture of Z. mobilis and S. stipitis, immobilized separately within macroporous HFMs. The SHFMB was operated under different operating conditions to assess its performance and stability.

Baseline co-culture experiments were conducted with suspended cells of Z. mobilis and S. stipitis in 500 mL Erlenmeyer flask. The experiments were conducted in three modes at glucose and xylose concentrations of 20–80 g L−1 and 10–40 g L−1, respectively. First, both the microorganisms were added together at same concentrations in the beginning. In the second approach, the experiments were conducted in two stages: Z. mobilis was added first into the culture medium, and S. stipitis was added after 24 h, when glucose had been completely fermented. In the third approach, the two stage approach of glucose fermentation by Z. mobilis, followed by xylose fermentation by S. stipitis was followed. However, prior to S. stipitis inoculation, the medium was centrifuged and the supernatant filtered with 0.45 μm filter to completely remove Z. mobilis from the suspension. Samples were collected periodically to measure cell, ethanol, glucose and xylose concentrations. Each of these experiments were conducted for 70–72 h. 2.3. SHFMB design and operation 2.3.1. Membrane synthesis and cell immobilization Polysulfone HFMs of 450 μm inner diameter and 225 μm thickness were prepared through wet-spinning. Detailed procedure and conditions to fabricate HFMs are available elsewhere [21]. The HFMs were cut into small pieces of 6 cm length. The small pieces of 60 HFMs were bundled together in a pack by applying epoxy resins at both ends of the HFMs. The HFM bundles were washed with 1 M NaOH, followed by 70% ethanol, and rinsed with sterilized water before cell immobilization. 2.3.2. Cell immobilization 150 mL of cell culture medium (separately for Z. mobilis at OD600 of 3 and P stipitis at OD600 of 6) in late-exponential growth phase was centrifuged at 8000 rpm for 10 min at 4 °C (Eppendorf 5810R, Germany). The pellet was resuspended into 150 mL sterile ultrapure water in an Erlenmeyer flask and the HFM bundles were immersed in the culture for cell immobilization. The flask was incubated in a shaking water bath (GFL 1092, Germany) at 150 rpm and 30 °C for 6 h. At the end, cell suspension was drained out and the HFMs were rinsed with sterile ultrapure water. Two sets of immobilized HFM bundles were prepared using pure cultures of Z. mobilis and S. stipitis for use in the SHFMB. 2.3.3. SHFMB setup Fig. 1 shows the schematic diagram of the submerged SHFMB. The SHFMB comprised of a 1 L reagent bottle of 500 mL effective volume. HFM bundles immobilized separately with Z. mobilis and S. stipitis were

2. Materials and methods 2.1. Cell culture All chemicals used in this study were of analytical grade, purchased from Sigma- Aldrich (St Louis, USA) or Merck (Darmstadt, Germany). Zymomonas mobilis ATCC 31821 and Scheffersomyces stipitis ATCC 58376 (previously Pichia stipitis) were used throughout this study. The cells were grown in rich medium (20 g L−1 glucose or 10 g L−1 xylose; 10 g L−1 yeast extract; 2 g L−1 KH2PO4) in 250 mL Erlenmeyer flask with 100–150 mL effective volume. The flasks were incubated on a shaking water bath (GFL 1092, Germany) at 30 °C and 150 rpm. Prior to inoculation, cells were induced by transferring stock culture from agar slant to liquid medium. Activated cells in late exponential growth phase were used as inoculum for all experiments. All media, pipette tips, and screw-cap Erlenmeyer flasks were autoclaved at 121 °C for 20 min before use.

Fig. 1. Schematic diagram of SHFMB setup. 146

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submerged into the liquid medium. The liquid medium was recirculated from the bioreactor using a two-channel peristaltic pump (L/S modular pump, Masterflex, USA) into the lumen side of the HFM bundle. Air at a low flow rate of 50 mL min−1 was introduced into the flow line transporting medium into the HFM bundle immobilized with S. stipitis. Samples were withdrawn periodically to measure the concentrations of glucose, xylose and ethanol in the SHFMB, as well as, OD600 in bioreactor suspension. 2.3.4. SHFMB operation During SHFMB operation, glucose concentrations were varied from 20 to 80 g L−1, whereas xylose concentration was varied from 10 to 40 g L−1, while maintaining glucose to xylose ratio of 2:1 (based on weight). The inoculum concentration of S. stipitis was varied by varying the number of HFMs in the bundle from 60 to 120 pieces of 6 cm each. Each experiment was conducted in duplicate for reproducibility. The effects of prolonged SHFMB operation on stability of immobilized cells and their fermentation performance were assessed by conducting 12 consecutive experimental runs. These consecutive runs were conducted in quadruplicate at three different sugar concentrations: 40 g L−1 glucose and 20 g L−1 xylose; 60 g L−1 glucose and 30 g L−1 xylose, and; 80 g L−1 glucose and 40 g L−1 xylose. All other operating conditions remained unchanged during these runs, and the SHFMB was operated for 47–48 h for each experiment. At the end of each experimental run, the SHFMB was washed with autoclaved ultrapure water to remove loosely attached cells adhering to the HFMs. 2.4. Analytical methods Fig. 2. Batch experiments kinetics using suspended cells: (a) Z. mobilis in 30 g L−1 glucose; (b) S. stipitis in 10 g L−1 xylose.

Cell concentration was determined by measuring optical density at 600 nm (OD600) using a UV–vis spectrophotometer (UV-1800, Shimadzu, Japan) using 1-cm path length cuvettes. It should also be noted that OD600 indicated only suspended cell concentration in the SHFMB, and it could not be used to estimate immobilized cell concentration. Since immobilized cells could not be extricated from the membranes, the exact number or viability of the microorganisms could not be estimated. Ethanol concentration was measured using gas chromatography (Clarus 600, Perkin Elmer, USA) equipped with a headspace sampler and flame ionization detector. The analysis was performed using Elite-5 capillary column (Perkin Elmer, USA) with helium as the carrier gas at a flow rate of 2 mLmin-1. Glucose and xylose concentrations were determined through HPLC using an Agilent Zorbax carbohydrate column (4.6 mm ID x 150 mm) and an RID detector. Water and acetonitrile mixture (75/25) was used as the mobile phase at a flow rate of 1.4 mL min−1. The peaks were detected using a UV detector at 280 nm. Ethanol yield was calculated by dividing the increase in ethanol concentration by decrease in total sugars concentration during the experiment.

fermentation efficiency and high bioethanol yield during glucose fermentation by Z. mobilis [23–25]. Fig. 2b shows growth kinetics of S. stipitis in 10 g L−1 xylose. The microorganisms exhibited relatively short lag phase, and biomass concentration increased with specific growth rate of 0.22 h−1. The microorganisms were able to completely assimilate xylose within 20 h and produced 4.8 g L−1 ethanol, whereas OD600 increased to about 8.0 during this period. Although growth rate of S. stipitis was slower as compared to Z. mobilis, ethanol yield of both of these microorganisms were comparable at 0.48 gg−1. The growth rate of S. stipitis obtained in this study was lower than those reported in literature, which could be due to absence of external aeration or due to relatively low initial xylose concentration [26]. Under aerobic conditions, the growth rate could be enhanced to 0.38 h−1 which resulted in improved fermentation kinetics but no significant changes in ethanol yield (results not shown). At xylose concentrations of 10–40 g L−1, there were no significant changes in cell growth rates and biomass yields (results not shown). These results are in agreement with findings describing effects of aeration and initial sugar concentrations on xylose fermentation [27,28].

3. Results & discussion 3.1. Growth kinetics of Z. mobilis and S. stipitis

3.2. Co-culture using suspended cells Batch experiments were conducted with suspended cells of Z. mobilis and S. stipitis to investigate their growth kinetics, sugar utilization and ethanol productivity. Fig. 2a shows the growth of Z. mobilis in 30 g L−1 glucose. The microorganisms exhibited relatively short lag phase and biomass concentration increased exponentially with specific growth rate of 0.37 h−1. Glucose was completely removed within 12 h and OD600 reached a maximum of 3.82. During this period, ethanol concentration increased gradually and stabilized at 14.3 g L−1, resulting in an ethanol yield of 0.48 gg−1. No significant changes were observed in growth rate sand ethanol yields of Z. mobilis, when initial glucose concentrations were varied between 20–80 g L−1 (results not shown). These results were consistent with other findings in literature on high

In order to investigate the mutual compatibility and other challenges associated with co-culture of Z. mobilis and S. stipitis, co-culture experiments were conducted with suspended cells in three different modes. In the first mode, both Z. mobilis and S. stipitis were added simultaneously to the liquid medium, whereas the microorganisms were added sequentially in the second and the third modes. Glucose and xylose were supplied in 2:1 ratio (based on concentration), and total sugar concentration was varied from 30 to 120 g L−1. 3.2.1. Simultaneous co-culture Fig. 3 shows cell growth, sugar uptake and ethanol production 147

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At high sugar concentrations of 60–80 g L−1 glucose and 30–40 g L−1 xylose, the co-culture was deemed unsuccessful, as xylose removal was almost negligible (< 7%). Although glucose was completely metabolized during these experiments, ethanol yield decreased sharply due to non-utilization of xylose, and possibly due to substrate inhibition of Z. mobilis [25]. Ethanol yield was only 0.28 gg−1 during the experiment conducted using 80 g L−1 glucose and 40 g L−1 xylose. It was also observed that OD600 was nearly proportional to initial glucose concentration in the sugar mixture, which was indicative of poor fermentation of xylose in the mixture, and suggested that microbial growth in the medium was dominated by Z mobilis. Besides, the inability of S. stipitis to grow at higher sugar concentrations indicated that apart from catabolite repression and product inhibition, these cells were also susceptible to substrate inhibition at high sugar concentrations.

Fig. 3. Simultaneous co-culture of suspended Z. mobilis and S. stipitis in 20 g L−1 glucose and 10 g L−1 xylose.

3.2.2. Sequential Co-culture In order to alleviate the effects of catabolite repression on S. stipitis during xylose metabolism, the next set of experiments were conducted in sequential mode. These experiments were started only with Z. mobilis and S. stipitis was inoculated after 24 h, once glucose had been completely fermented. Table 1 summarizes the results obtained under sequential mode at different sugars concentrations. It can be seen that the sequential approach was better than concomitant co-culture, at low xylose concentration of 10 g L−1 and 20 g L−1, when > 75% and > 18% xylose could be fermented within 24 h, respectively. However, at higher xylose concentrations of 20–40 g L−1, xylose removal was < 10%, and no improvement in xylose fermentation was detected, as compared to previous experiments, when both Z. mobilis and S. stipitis were inoculated together. Poor performance of S. stipitis at higher sugar concentrations can be attributed to higher ethanol levels at the time of inoculation, owing to complete fermentation of glucose by Z. mobilis with ethanol yields of about 0.48 gg−1. However, it was also observed that the sequential mode did not yield dramatic improvement in performance, even at a low xylose concentration of 20 g L−1. Therefore, it is possible that there was negative synergism between the two microorganisms that retarded S. stipitis growth and prevented complete utilization of xylose. These challenges in sequential co-culture have been reported, and an effective strategy is to add the microorganisms and the respective sugars sequentially, such that glucose is added to the system only after xylose has been completely fermented [31]. However, this strategy may not be practical in fermentation of lignocellulosic hydrolysate rich in both pentose and hexose sugars.

during co-culture of Z. mobilis and S. stipitis at initial glucose and xylose concentrations of 20 g L−1 and 10 g L−1, respectively. It can be seen that sugar removal during the co-culture occurred in two stages. In the first stage (first 16 h), glucose was completely metabolized but there was a marginal change in xylose concentration. During this period, ethanol concentration and OD600 increased to 9.4 g L−1 and 2.62, respectively. These results were in agreement with biomass growth and ethanol yield observed during Z. mobilis growth on glucose (Fig. 2a), indicating dominant role of Z. mobilis in the first stage. Xylose metabolism occurred in the second stage of the co-culture. Once glucose had been completely exhausted, there was a brief lag phase, when there were no significant changes in OD600 and xylose or ethanol concentrations. Changes in these parameters were observed after 20 h, as OD600 and ethanol concentration increased gradually with concomitant decrease in xylose levels. At the end of the experiment after 70 h, OD600 and ethanol concentration had increased to 2.9 and 12.4 g L−1, whereas xylose concentration had reduced to 3.1 g L−1. Overall, glucose, xylose and total sugar removal were 100%, 68% and 89%, respectively, whereas ethanol yield was 0.45 gg−1. These results highlight the role of catabolite repression on co-utilization of glucose and xylose by the co-culture system. Despite the availability of xylose-fermenting microorganisms, glucose and xylose were metabolized sequentially, and xylose metabolism occurred only after glucose had been completely removed from the medium. Catabolite repression by glucose has been widely reported in literature [29], and it is generally considered as one of the key challenges in simultaneous fermentation of both pentose and hexose sugars in bioethanol production. This phenomena results in repression of enzymes and transporters necessary for xylose metabolism, and it may occur at glucose concentrations as low as 2 g L−1 [15]. It was also observed that even after complete glucose removal, the growth of S. stipitis was relatively slow, and not all the xylose was metabolized, despite a relatively long operating period of 70 h. Similar sequential trends were observed during fermentation of 40 g L−1 glucose and 20 g L−1 xylose using the mixed co-culture system. While glucose was completely metabolized within 20 h, yielding 16 g L−1 ethanol and OD600 of 4.9, xylose concentration remained unchanged during this period. Xylose fermentation began after 24 h, but cell growth and sugar uptake by S. stipitis were low, and only 11% xylose was consumed within 70 h. Both ethanol concentration and OD600 increased marginally during this period, and the final values were 19 g L−1 and 5.43, respectively (Table 1). Although overall ethanol yield was relatively high at 0.45 gg−1, this was due primarily to complete fermentation of glucose by Z. mobilis. Low xylose conversion during the experiment also signifies the disruptive role of ethanol, and the resulting product inhibition, on xylose metabolism by S. stipitis. The sensitivity of S. stipitis to ethanol has been reported previously, and ethanol concentrations above 30 g L−1 may be detrimental for these microorganisms [30].

3.2.3. Sequential co-culture with cell removal In order to verify the hypothesis that the presence of Z. mobilis was not favorable for xylose metabolism by S. stipitis, next set of sequential co-culture experiments were conducted with cell removal. In these experiments, Z. mobilis was first inoculated in the medium to ferment glucose. Once glucose had been completely fermented (after 24 h), the medium was filtered to remove the cells, and S. stipitis was inoculated in the filtered medium for xylose fermentation. It can be seen from Table 1 that the removal of Z. mobilis from the medium had a favorable impact on xylose fermentation. At lowest total sugars concentration of 30 g L−1 (including 10 g L−1 xylose), > 90% xylose could be fermented within 24 h, whereas > 27% xylose could be fermented at a higher total sugar concentration of 60 g L−1 (including 20 g L−1 xylose). However, the effects of cell removal at higher sugar concentrations were insignificant, which signified the adverse role of product inhibition in preventing xylose fermentation at high sugars concentrations (> 60 g L−1). These results also implied that additional measures were needed to protect S. stipitis from inhibitory effects of ethanol at higher sugar concentrations. Based on the results obtained during co-culture of suspended Z. mobilis and S. stipitis, it could be inferred that the co-culture system was 148

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Table 1 Co-fermentation of glucose and xylose using suspended cells. Expt. mode

Simul-taneous

Seq-uential

Seq-uential with cell removal

Initial conc.

Removal efficiency

Ethanol

OD600

Glucose (gL−1)

Xylose (gL−1)

Total (%)

Glucose (%)

Xylose (%)

Conc. (gL−1)

Yield (gg−1)

Prod. (gL−1 h-1)

20

10

89.4 ± 1.3

68 ± 3.8

0.17 ± 0.006

3.01 ± 0.16

20

70.2 ± 0.6

0.45 ± 0.022

0.27 ± 0.013

5.43 ± 0.35

60

30

69.0 ± 0.3

0.43 ± 0.024

0.38 ± 0.021

6.78 ± 0.49

80

40

0.27 ± 0.015

0.31 ± 0.018

9.13 ± 0.65

20

10

67.5 ± 0 91.5 ± 1.4

0.45 ± 0.022

0.18 ± 0.009

4.34 ± 0.17

40

20

72.7 ± 0.3

0.44 ± 0.024

0.27 ± 0.015

5.05 ± 0.31

60

30

69.5 ± 0.1

0.43 ± 0.022

0.39 ± 0.019

6.97 ± 0.24

80

40

0.36 ± 0.011

0.41 ± 0.012

9.18 ± 0.89

20

10

66.9 ± 0 96.4 ± 1.9

0.46 ± 0.016

0.19 ± 0.007

–

40

20

75.5 ± 0.8

0.46 ± 0.023

0.30 ± 0.015

–

60

30

71.6 ± 0.3

0.45 ± 0.024

0.41 ± 0.022

–

80

40

67.6 ±0

12.1 ± 0.4 18.8 ± 0.9 26.6 ± 1.5 21.6 ± 1.3 12.5 ± 0.6 19.2 ± 1.1 27.0 ± 1.4 28.8 ± 0.8 13.3 ± 0.5 21 ± 1.1 28.9 ± 1.6 28.0 ± 1.8

0.45 ± 0.015

40

100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0

0.34 ± 0.020

0.40 ± 0.024

–

10.6 ± 0.3 7.1 ± 0.3 2.4 ± 0.1 74.6 ± 4.1 18.2 ± 0.8 8.5 ± 0.3 0.67 ±0 89.2 ± 5.6 26.6 ± 2.3 14.9 ± 0.8 2.8 ± 0.1

xylose were metabolized simultaneously. Glucose was completely consumed within 24 h due to higher growth and uptake rate of Z. mobilis, which was consistent with the results obtained using suspended cells. However, more than 30% xylose was also metabolized during the first 24 h, concomitantly with glucose. Xylose metabolism continued after glucose had been completely fermented, and > 45% xylose had been fermented at the end of experiment after 47 h. Co-fermentation of both glucose and xylose resulted in total production of 33.7 g L−1 ethanol, and an ethanol yield of 0.44 gg−1. Interestingly, high levels of suspended cells were observed in the SHFMB, and OD600 increased gradually from 0.1 in the beginning, to 8.3 at the end of the experiment. Since biomass accumulation occurred mostly in the first 24 h, it was inferred that Z. mobilis had diffused out of the HFM pores, and they were growing preferentially in suspension, due to non-inhibitory ethanol concentration. In contrast, ethanol concentrations of 25–30 g L−1 were of moderate toxicity to S. stipitis [30], which could have forced these cells to remain inside the HFM pores for better stress management. These results are supported by the stabilization of OD600 in suspension at the end of 24 h, indicating little or no diffusion of S. stipitis from the HFM pores. Compared to the results obtained in co-culture using suspended cells (Table 1), SHFMB performance in xylose removal was excellent. Maximum xylose uptake by S. stipitis in suspended co-culture under identical conditions was < 15%, despite a long operating period of 70 h. Besides, xylose uptake by S. stipitis in suspended co-culture occurred only when glucose had been completely metabolized. In contrast, glucose and xylose fermentation occurred simultaneously in the SHFMB, and > 45% xylose was fermented within 47 h. These results highlighted the efficacy of immobilized-cell based SHFMB in alleviating the inhibitory effects of sugars and ethanol on S. stipitis, as well as, in preventing the adverse effects of catabolite repression on xylose fermentation. Although the advantages of immobilized cell system have been amply demonstrated in lignocellulosic biomass fermentation [32,33], the uniqueness of SHFMB was found in partial immobilization of microorganisms in macro-porous HFMs. Since a large amount of microorganisms, especially Z. mobilis, diffused out of the HFM pores and stayed in suspension, the SHFMB resembled co-culture system of suspended Z. mobilis and immobilized S. stipitis. Under these conditions, it

susceptible to substrate and product inhibition, catabolite repression and negative synergism between the two microorganisms. Although these limitations had minimal effects on glucose fermentation, xylose metabolism was adversely affected, as S. stipitis was more susceptible to these inhibitory effects. These results are in agreement with other findings reported in literature highlighting the challenges in co-culture of S. stipitis with commonly used glucose fermenting microorganisms, including Z. mobilis and S. cerevisiae [4,17]. 3.3. SHFMB operation Prior to operation, microorganisms were immobilized separately in HFMs. SHFMB operation was started with 60 fibers immobilized with Z. mobilis and S. stipitis each. Subsequently, several experimental runs were conducted to investigate the effects of aeration, inoculum ratio and concentrations of the sugars. Finally, the SHFMB was operated to assess its stability under long-term operation. 3.3.1. Co-culture using immobilized cells Fig. 4 shows the results obtained during SHFMB operation at a relatively high sugar levels of 60 g L−1 glucose and 30 g L−1 xylose, in the absence of any external aeration. It can be seen that both glucose and

Fig. 4. SHFMB operation at 60 g L−1 glucose and 30 g L−1 xylose. 149

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productivity and OD600 increased proportionately with sugar concentration. At higher total sugar concentrations of 80–120 g L−1, > 70% xylose could be fermented within 48 h, although ethanol yield decreased significantly during SHFMB operation at the highest total sugar concentration of 120 g L−1. The maximum theoretical yield of ethanol on glucose is 0.51 gg−1 [35]. However, due to cell growth and maintenance requirements, ethanol yield is limited to 90–95% of theoretical yield under favourable condition, or lower under adverse conditions. In the SHFMB, high ethanol yield of 0.44-0.48 gg−1 was obtained at lower sugars concentrations, indicating favourable conditions for cell growth and metabolism. On the other hand, the decrease in ethanol yield at higher sugars concentration could be a result of substrate or product inhibition of microorganisms, especially S. stipitis [9,36]. Although relatively low suspended cell concentration (OD600 of 7.5) in the SHFMB at total sugar concentration of 120 g L-1 indicated that these conditions could have been inhibitory for Z. mobilis as well. These results also highlighted the flexibility offered by HFM in cell immobilization, as microorganisms could choose to grow in suspension or in membrane pores depending on the growth conditions [25].

Fig. 5. Effects of micro-aeration on SHFMB performance at 60 g L−1 glucose and 30 g L−1 xylose.

was unlikely that mass transfer resistance could have been a rate-limiting factor in glucose fermentation [25]. During xylose fermentation by S. stipitis, several studies have highlighted the importance of aerobic or microaerobic conditions [28,34]. Therefore, experiments were conducted to assess the effects of microaerobic conditions on SHFMB performance. Fig. 5 shows a comparison of SHFMB operation with and without air circulation. It can be seen that microaeration was favorable for xylose fermentation and xylose uptake increased from 45%, in the absence of external aeration, to > 70% under microaerobic conditions. The increase in xylose uptake, and consequent production of ethanol, resulted in higher total sugars removal of > 90%, higher ethanol concentration of 37 g L−1 and higher ethanol productivity of 0.78 g L−1 h−1 during 48 h operation. Although rate of glucose uptake by Z. mobilis decreased slightly under microaerobic conditions, glucose was completely fermented within 30 h. Besides, the microaerobic conditions did not have any significant effects on overall ethanol yield, ethanol productivity or suspended cell concentrations in the SHFMB. It is difficult to supply oxygen to a co-culture system comprising of anaerobic microorganisms, such as Z. mobilis, and aerobic microorganisms, such as S. stipitis, due to the adverse effects of aeration on glucose fermentation. Therefore, rate of aeration, mixing and dissolved oxygen (DO) concentration are critical to maintain a balance in coculture, so that both the microorganisms can function well to maximize ethanol productivity [12]. Under these considerations, the HFM-based SHFMB provided unique advantages in co-culture microaeration, as air was supplied directly to the HFMs immobilized with S. stipitis. Although DO levels increased (< 1.5 mg L−1) due to aeration, immobilization of Z. mobilis in a separate set of HFMs and presence of membrane diffusion barrier would have reduced their exposure to DO. Consequently, there was only a small decrease in glucose uptake rate by Z. mobilis under microaerobic conditions, whereas their accumulation in suspension remained unaffected. Since glucose uptake was not the limiting factor on ethanol productivity in the SHFMB, the overall impact of microaeration on co-fermentation was positive. Therefore, all subsequent experiments in the SHFMB were conducted under microaerobic condition. Under microaerobic conditions, the SHFMB was operated under different initial total sugar concentrations, varying from 30 to 120 g L−1. At a fixed glucose to xylose ratio of 2:1, glucose concentrations were varied from 20 to 80 g L−1, whereas xylose concentrations were varied from 10 to 40 g L−1. SHFMB performance under these conditions have been summarized in Table 2. Under low total sugar concentrations of 30 g L−1 including (10 g L−1 xylose) and 60 g L−1 including (20 g L−1 xylose), both glucose and xylose were completely fermented within 48 h of operation. While ethanol yield remained stable at 0.44-0.48 gg−1 under these conditions, ethanol

3.3.2. Effects of increased S. stipitis inoculum Although SHFMB showed excellent performance in xylose fermentation, the rate of xylose uptake by S. stipitis was low, as compared to glucose uptake rate of Z. mobilis. In order to enhance xylose uptake rate in the SHFMB, the experimental setup was modified by incorporating additional HFMs immobilized with S. stipitis. Thus, SHFMB operation was conducted using 60 HFMs immobilized with Z. mobilis and 120 HFMs immobilized with S. stipitis. Fig. 6 shows SHFMB operation under increased number of S. stipitis immobilized HFMs during fermentation of 60 g L−1 glucose and 30 g L−1 xylose. As expected, the change in operating conditions had no influence on glucose uptake rate or fermentation. Glucose was completely fermented within 25 h, and OD600 increased to 7.8 during this period. On the other hand, xylose uptake in the SHFMB improved significantly. About 50% xylose was fermented simultaneously with glucose in the first 25 h, whereas xylose removal at the end of experiment (after 47 h) was > 88%. The final ethanol concentration in the SHFMB was 38 g L−1, which translated to an ethanol yield of 0.44 gg−1, and an ethanol productivity of 0.81 g L−1 h−1. There was increase in OD600 towards the end, and it stabilized at 8.7 at the end of operation. Clearly, increasing S. stipitis immobilized HFMs in the SHFMB was favorable for enhancing xylose uptake and overall performance of the SHFMB. These results are important, as these indicate 25% increase in xylose uptake rate and 13% increase in ethanol production in the SHFMB, when it was operated with higher number of S. stipitis immobilized HFMs. This increase in xylose uptake rate and the high ethanol yield (86% of theoretical yield) could be attributed to several factors. The most important factor was the increase in S. stipitis inoculum with increasing number of S. stipitis immobilized HFMs. The increase in inoculum not only had direct influence on sugar uptake rate, but a higher cells to sugars ratio could have improved the tolerance of the microorganisms to inhibitory substrates and products during fermentation [15]. Increasing the number of HFMs could also enhance sugars uptake rate by increasing the effective mass transfer area for xylose diffusion into the membranes pores [37]. Furthermore, increasing the number of S. stipitis immobilized HFMs was advantageous for oxygen mass transfer to immobilized S. stipitis in the SHFMB, and it could have contributed to the performance enhancement [22,34]. 3.3.3. Stability Immobilized cell bioreactors have been widely used in bioprocesses, especially in alleviating substrate and product inhibitions [21,25,38,39]. One of the most important factors determining applicability of these bioreactors is the stability of the immobilization support. In order to assess the stability of cell immobilization in the 150

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Table 2 Effects of initial sugar levels on fermentation in SHFMB. Initial conc.

Removal efficiency

Ethanol

OD600

Glucose (gL−1)

Xylose (gL−1)

Total (%)

Glucose (%)

Xylose (%)

Conc. (gL−1)

Yield (gg−1)

Prod. (gL−1 h-1)

20

10

40

20

60

30

80

40

100 ± 0 100 ± 0 90 ± 1.8 91 ± 1.1

100 ± 0 100 ± 0 100 ± 0 100 ± 0

100 ± 0 100 ± 0 71.1 ± 5.5 72.5 ± 3.2

14.3 ± 0.6 26.7 ± 1.5 36.7 ± 1.3 41.9 ± 3.2

0.48 ± 0.020 0.44 ± 0.025 0.45 ± 0.016 0.37 ± 0.028

0.40 ± 0.017 0.78 ± 0.044 0.78 ± 0.028 0.87 ± 0.066

3.4 ± 0.22 6.2 ± 0.46 7.9 ± 0.53 7.5 ± 0.71

These results showed that SHFMB performance did not deteriorate over time. In contrast, the improvement in xylose uptake indicated robustness and efficacy of the immobilized microorganisms. The improvement in xylose fermentation could be attributed to increase in S. stipitis accumulation in the HFMs during consecutive runs, although it could also be a result of gradual acclimatization and adaption of immobilized cells to inhibitory conditions in the SHFMB [22]. Similar results were obtained during the third set of experiments (Run 9–12), at 80 g L−1 glucose and 40 g L−1 xylose. During these runs, there were no changes in glucose removal, which remained consistently at 100%. Xylose removal was partial during these runs, although removal efficiency improved from 77% during Run 9, to 83% during Run 12. More dramatic changes were observed in ethanol concentration during these runs, which increased gradually from 42 g L−1 during Run 9 to 48 g L−1 during Run 12. These translated into ethanol yield of 0.37 gg−1 during Run 9 and 0.42 gg−1 during Run 12. This increase in ethanol yield was could not be explained based on fermentation of additional xylose in the SHFMB. It was also noted that the performance of S. stipitis during the consecutive runs was much better than those seen earlier under identical conditions (Table 2). Therefore, it could be inferred that the consecutive runs allowed microorganisms to better acclimatize to increased sugar loadings and better manage the stress due to gradual increase in S. stipitis accumulation in the HFMs with time [40]. The results obtained during the prolonged operation also highlighted the potential of polymeric HFMs to be excellent support for cell immobilization, owing to their stability, high porosity, high mass transfer area, as well as, the flexibility in allowing cells to move freely in and out of the porous walls depending on the culture environment [21,41]. Since these HFMs have been shown to be effective in mitigating the toxicity arising from lignocellulose-derived inhibitors in bioethanol fermentation [25], it could be concluded that the SHFMB can be an excellent platform to maximize ethanol production from mixed sugars in lignocellulosic hydrolysate.

Fig. 6. Effects of high S. stipitis inoculum on fermentation in SHFMB at 60 g L−1 glucose and 30 g L−1 xylose.

SHFMB, 12 consecutive runs were conducted under different sugar concentrations using 60 fibers each immobilized with Z. mobilis and S. stipitis. The HFMs were washed with sterile ultrapure water after each run to remove loosely attached cells. Fig. 7 shows glucose, xylose and ethanol profiles during the consecutive runs over 24 days of SHFMB operation. During the first four runs (Run 1–4), at initial glucose concentration of 40 g L−1 and xylose concentrations of 20 g L−1, both glucose and xylose were completely fermented during each run. Ethanol concentration at the end of these runs was stable at 26–27 g L−1, which translated into ethanol yield of 0.43-0.45 gg−1. In the next set of experiments (Run 5–8), at 60 g L−1 glucose and 30 g L−1 xylose, glucose was completely fermented during each experimental run, but xylose could not be fermented completely within 48 h. Nevertheless, xylose removal increased gradually in consecutive runs, from 83% during Run 5 to 93–94% during Run 7-8. The increase in xylose uptake rate resulted in slight increase in ethanol concentrations, from 37 g L−1 during Run 5 to 39 g L−1 during Run 8.

4. Conclusions The SHFMB was designed and operated with immobilized Z. mobilis and S. stipitis for simultaneous fermentation of glucose and xylose. HFM-based immobilization prevented catabolite repression, alleviated substrate and product inhibition, facilitated selective aeration and allowed movement of microbes in suspension under favorable conditions. While glucose was completely removed under all the conditions, xylose removal was 100% up to total sugar concentrations of 60 g L−1, and > 70% up to total sugar concentration of 120 g L-1. Both aeration and S. stipitis inoculum size were critical to improve xylose fermentation, and doubling S. stipitis inoculum improved xylose uptake to 88% and ethanol yield to 0.44 gg-1 at total sugar concentration of 90 g L-1. Cell immobilization in the SHFMB was stable and fermentation performance improved during 12 consecutive batch runs. The use of SHFMB can be promising in mitigating various operating challenges and improving ethanol production in fermentation of lignocellulosic

Fig. 7. Stability of SHFMB during 12 consecutive runs at different initial mixed sugar concentrations. 151

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