Zymomonas mobilis immobilization in polymeric membranes for improved resistance to lignocellulose-derived inhibitors in bioethanol fermentation

Accepted Manuscript Title: Zymomonas mobilis Immobilization in Polymeric Membranes for Improved Resistance to Lignocellulose-Derived Inhibitors in Bioethanol Fermentation Authors: Nguyen Thi Thuy Duong, Prashant Praveen, Kai-Chee Loh PII: DOI: Reference:

S1369-703X(18)30314-0 https://doi.org/10.1016/j.bej.2018.09.003 BEJ 7032

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

29-6-2018 23-8-2018 3-9-2018

Please cite this article as: Thuy Duong NT, Praveen P, Loh K-Chee, Zymomonas mobilis Immobilization in Polymeric Membranes for Improved Resistance to LignocelluloseDerived Inhibitors in Bioethanol Fermentation, Biochemical Engineering Journal (2018), https://doi.org/10.1016/j.bej.2018.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Zymomonas mobilis Immobilization in Polymeric Membranes for Improved Resistance to Lignocellulose-

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Derived Inhibitors in Bioethanol Fermentation

Department of Chemical & Biomolecular Engineering, National University of Singapore,

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Nguyen Thi Thuy Duong1, Prashant Praveen2, Kai-Chee Loh1,*

Clean Technologies, Scion, Rotorua, New Zealand

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Singapore

*Corresponding author:

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Associate Professor, Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585.

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Highlights

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Email: [email protected]; Tel.: +65 6516 2174; Fax: +65 6779 1936

Zymomonas mobilis was immobilized in macroporous hollow fiber membranes

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Immobilized cells showed excellent tolerance to lignocellulose-derived inhibitors

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Sorption of inhibitors on membranes reduced their effective concentrations

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Cells achieved 95% theoretical ethanol yield under inhibitory conditions

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Cell immobilization was stable under 20 consecutive batch runs over 10 days

Abstract Immobilized cell hollow fiber membrane bioreactor (ICHFMB) was designed and operated to mitigate the effect of lignocellulose derived inhibitors during fermentation of synthetic lignocellulosic hydrolysate by Zymomonas mobilis. The individual inhibitors – vanillin,

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syringaldehyde, 5-hydroxymethylfurfural and 4-hydroxy-3-methoxycinnamaldehyde, exhibited toxicity on suspended cells, whereas simultaneous exposure to multiple inhibitors was

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detrimental for the cells. In contrast, the immobilized cells showed excellent growth and glucose uptake in the presence of these inhibitors, and the microorganisms achieved 95% of the theoretical ethanol yield under highly inhibitory conditions. ICHFMB performance could be

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improved significantly by increasing the flow rate of the hydrolysate over the membranes, and

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by increasing the number of membranes, indicating that diffusion through the membranes was an

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important factor during the fermentation. Z. mobilis showed gradual acclimatization to the

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presence of inhibitors as they were slowly released from the membrane pores into suspension,

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and enhanced glucose uptake rate and ethanol productivity. Cell immobilization within the membranes was also found to be very stable as the ICHFMB performance did not deteriorate

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during 20 consecutive runs under identical conditions. These results indicate that ICHFMB can

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be promising in lignocellulosic bioethanol production under inhibitory conditions.

Keywords: Bioethanol; Cell immobilization; Fermentation; Lignocellulose inhibitors;

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Zymomonas mobilis

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Introduction

Being one of the most abundant renewable feedstock material on Earth, lignocellulosic biomass has attracted considerable interest in the past few decade for production of a variety of energyrich compounds, including ethanol [1-3]. Lignocellulosic biomass comprises mainly of cellulose, hemicelluloses and lignin, of which lignin is not a cellulosic material and cannot yield

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fermentable sugars. The role of lignin is to cross-link the cellulosic material in a tight complex,

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providing stability to the biomass [4].

The transformation of lignocellulose into bioethanol requires several steps: thermo-chemical pretreatment, enzymatic hydrolysis, fermentation and purification [5]. Among these, the

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breakdown of lignin through pretreatment is critical to depolymerize the cellulosic material and

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make it more amenable to hydrolytic enzymes [6]. However, the use of harsh conditions during

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pretreatment to break down recalcitrant lignin also yields a variety of smaller undesirable

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molecules, such as furaldehydes, weak acids and phenolic compounds [7]. Many of these by-

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products exhibit inhibitory tendency to fermentative microorganisms, and their presence in the fermentation broth adversely affects cell growth, metabolism and ethanol productivity [8, 9].

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One approach to alleviate the toxicity of the lignocellulose-derived-inhibitors is the

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detoxification of the lignocellulosic hydrolysate, prior to fermentation. This can be achieved through suitable additives [10], or separation based on two-phase processes [5, 11, 12] or enzymatic treatment [13]. Alternatively, genetic engineering approach based on the use of

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recombinant Saccharomyces cerevisiae with enhanced resistance to inhibitors has also been demonstrated in lignocellulosic bioethanol production [14]. However, both of these approaches are targeted at a specific inhibitor or a set of inhibitors, and these may not be effective against structurally and chemically diverse group of inhibitors present in the hydrolysate. The detoxification strategy may decrease sugar yields as well leading to lower bioethanol production,

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whereas detoxification-based treatment and purification would increase bioethanol production costs [15].

Another approach to alleviate adverse effects of inhibitors on biological processes is cell immobilization within a polymeric matrix [16]. This technique has been widely used in

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alleviation of substrate and product inhibition in fermentation due to several advantages: (1) high cell density and productivity; (2) low shear stress; (3) continuous operation without cell washout

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concerns; (4) low exposure to inhibitory compounds, and; (5) regeneration and reuse of the cells for extended period [17, 18]. The efficacy of cell immobilization in alleviating substrate and product inhibition has been demonstrated in lignocellulosic bioethanol production using a variety

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of inorganic, organic and polymeric support [19, 20]. Cell immobilization on polymeric support,

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such as hollow fiber membranes, offers several advantages over conventional organic support

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materials such as alginate. While the polymeric membrane are characterized by high stability,

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tubular hollow fibers offer high surface area and porosity for cell encapsulation, and high rate of mass transfer for substrate diffusion [21]. Membrane-based immobilization does not require any

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covalent cross-linking or ionic bonding, and cells can simply diffuse in and out of the membrane

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walls, based on the prevailing condition in the bioreactor. Besides, the membranes can be fabricated prior to immobilization, thus allowing more flexibility in altering structural and

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transport properties of the support without compromising cell viability and productivity [22].

In this research, the objective was to design and operate an immobilized-cell hollow fiber

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membrane bioreactor (ICHFMB) for high-throughput bioethanol production in the presence of lignocellulose-derived inhibitors. Baseline experiments were conducted with suspended cells to investigate the inhibitory effects of inhibitors on cell growth and metabolism, and to study the sorption of inhibitors by the membranes. The ICHFMB was operated under various operating conditions and bioreactor stability was demonstrated under prolonged operation. Lignocellulosederived inhibitors may comprise of weak acids, furan derivatives or phenolic compounds, and 4

the toxicity of these inhibitors varies in the order of phenols > furans > acids [23]. Therefore, three common phenolic inhibitors – vanillin, syringaldehyde and 4-hydroxy-3methoxycinnamaldehyde (4-HMCA), and one furan derivative – 5-hydroxymethylfurfural (5HMF) were used as model inhibitors in this study.

Material and Methods

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Cell culture

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All chemicals used in this study were of analytical grade, purchased from Sigma- Aldrich (St

Louis, MO, USA) or Merck (Darmstadt, Hesse, Germany). Vanillin, syringaldehyde, 4-HMCA and 5-HMF were used as model inhibitors in this study. Apart from vanillin, inhibitors

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concentrations within 0.5-1.5 g/L were comparable to those reported in literature for low glucose

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concentration of 20 g/L [24].

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Zymomonas mobilis ATCC 31821 was used throughout this study. The cells were grown in Rich Medium (20 g/L glucose; 10 g/L yeast extract; 2 g/L KH2PO4) in 250 mL Erlenmeyer flask with

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100-150 mL effective volume. The flasks were incubated on a shaking water bath (GFL 1092,

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Germany) at 30°C and 150 rpm. Prior to inoculation, cells were induced by transferring stock culture from the agar slant to the liquid medium containing 20 g/L glucose. Activated cells in the

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late exponential growth phase were used as inoculum for all the experiments. All media, pipette tips, and Erlenmeyer flasks fitted with cotton plugs were autoclaved at 121°C for 20 min before

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use.

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ICHFMB Design and Operation 2.2.1 Membrane synthesis and module preparation Polysulfone (PS) hollow fiber membranes of 450 µm inner diameter and 225 µm thickness were prepared through wet-spinning. Detailed procedure and conditions to fabricate the membranes are available elsewhere [21]. A hollow glass tube of 0.7 cm inner diameter with four outlets was

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used for module preparation. The membranes were packed in the tubes and secured at both ends using quick drying Araldite epoxy adhesive resins. The excess fiber protruding from the ends of

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the module were snipped, such that the module resembled a shell and tube heat exchanger. The effective membrane length in the module was 30 cm. Two sets of modules containing 8 and 16

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fibers were prepared.

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2.2.2 ICHFMB setup

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Fig. 1 shows the schematic diagram of the ICHFMB. A peristaltic pump (L/S modular pump,

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Masterflex, Vernon Hill, IL, USA) was used to pump the medium from a 500 mL Erlenmeyer

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flask with an effective volume of 250 mL to the membrane module. The liquid was recirculated from the flask to the tube side of the module, then to the shell side of the module, before

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returning to the flask. A flowmeter was used to monitor the liquid flow rate. Samples were

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periodically withdrawn to measure the concentrations of cells, glucose and ethanol in the ICHFMB. Prior to immobilization, 70% ethanol was pumped into the bioreactor at 10 mL/min

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for sterilization. The bioreactor was then washed with autoclaved ultrapure water.

2.2.3 Cell immobilization One hundred fifty mL of the suspended cells in late exponential phase were harvested through centrifugation at 8,000 rpm for 10 min (Eppendorf 5810R, Germany). The pellet was resuspended in 150 mL 0.3% NaCl solution and recirculated in the membrane module using a peristaltic pump operating at a flow rate of 10 mL/min for 6 h. The cell suspension was then 6

drained out and the module was rinsed with sterile ultrapure water to remove the loosely attached cells from the module and the tubing.

2.2.4 ICHFMB operation During ICHFMB operation, glucose concentration was maintained constant at 20 g/L in each

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experiment. To assess the effects of inhibitors on ICHFMB performance, separate experiments were conducted using 20 g/L glucose and a mixture of the inhibitors at two different levels: (1)

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0.5 g/L each and; (2) 1 g/L each of vanillin, syringaldehyde, 4-HMCA and 5-HMF. To

investigate the effects of membrane area, the number of membranes in the module were doubled from 8 to 16. The effects of flow rates on ICHFMB performance was investigated by varying the

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flow rates between 10-40 mL/min. All the experiments were conducted for 24 h. In order to

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investigate the stability of the ICHFMB, 20 consequent experimental runs were conducted under

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identical conditions. At least two batch runs were conducted under each experimental conditions. At the end of each experimental run, the ICHFMB was washed with 0.5 N NaOH. It was

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followed by thorough rinsing with sterile ultrapure water.

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Analysis

Suspended cell concentration was determined by measuring the optical density at 600 nm

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(OD600) using a UV-Visible Spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) using 1-cm path length cuvettes. The following correlation was used to compute dry cell weight (DCW)

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from OD600 values: DCW (g/L) = 0.307*OD600. Ethanol productivity and glucose uptake rate were calculated by dividing final ethanol concentration and initial glucose concentration, respectively, by duration in which glucose dropped to zero. Since it was not possible to determine the concentration of immobilized cells, all DCW measurements in the ICHFMB were based on suspended cell concentrations.

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Ethanol concentration was measured using gas chromatography (Clarus 600, Perkin Elmer, Waltham, MA, USA) equipped with a headspace sampler and flame ionization detector. Glucose concentration was analyzed using a biochemical analyzer (YSI 2700, Yellow Springs, OH, USA). The inhibitors were assayed through HPLC (Waters, Milford, MA, USA) using a C-18 column. Water and methanol mixture (70/30) was used as the mobile phase at a flow rate of 1

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mL/min. The peaks were detected using a UV detector at 280 nm. For Scanning Electron Microscopy (JEOL JSM-5600LV, Tokyo, Japan), the membrane samples were soaked in

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glutaraldehyde for 6 h. These were subsequently dehydrated using a graded series of ethanol

(25%, 50%, 75%, 95% and 100%) for 10 min each and finally air-dried for 2 h. The samples

Results & Discussion

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were coated with platinum before observation.

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Effect of inhibitors on suspended cells

In order to investigate the effects of lignocellulose-derived inhibitors on cell growth and ethanol

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production kinetics of Z. mobilis, experiments were conducted in synthetic hydrolysate containing 20 g/L glucose supplemented with 0.5-1.5 g/L of the inhibitors - vanillin,

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syringaldehyde, 4-HMCA and 5-HMF. A control experiment was conducted with 20 g/L without

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any inhibitors. The results have been summarized in Table 1.

Fig. 2a-b shows the effects of inhibitors on cell growth and glucose uptake by suspended cells at inhibitors concentrations of 1 g/L. In the absence of inhibitors, the microorganisms exhibited

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high growth rate of 0.38 h-1 and the biomass yield was 0.057 g/g. On the other hand, ethanol yield and productivity were computed as 0.49 g/g and 0.832 g/L-h, respectively. Both cell growth and ethanol production were adversely affected by the presence of inhibitors. Although Z. mobilis did not exhibit lag phase in the presence or absence of inhibitors, there was substantial decrease in cell growth, glucose uptake and ethanol production rates in the presence of 8

inhibitors. Cell growth rates in the presence of syringaldehyde was higher and glucose was completely fermented in about 13 h. Cell growth rate was moderately slower in the presence of 5-HMF and 4-HMCA and glucose was fermented within 16 h. However, final biomass concentration achieved in the presence of syringaldehyde, 5-HMF and 4-HMCA were nearly same. On the other hand, cell growth rate and glucose uptake were slowest in the presence of

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vanillin. Overall, the presence of 0.5-1.5 g/L inhibitors resulted in 25-350% decrease in cell

growth rate and the lowest cell growth rate of 0.11 h-1 was observed in the presence of 1.5 g/L

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vanillin (Table 1).

Similar trends were observed in ethanol production, where rate of ethanol production was

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associated with the changes in cell growth and glucose uptake (Fig. 2c). Both ethanol

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productivity and ethanol yield varied in the order of syringaldehyde > 5-HMF and 4-HMCA >

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vanillin (Table 1). Clearly, vanillin had the strongest inhibitory effect on cell growth. In the

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presence of 1 g/L vanillin, both cell growth rate and biomass yield decreased by about 60%, as compared to that in the control experiment. The slow growth rate affected glucose uptake and

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ethanol production. The resulting ethanol yield and productivity were 0.362 g/g and 0.477 g/L-h,

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respectively (Table 1). On the other hand, syringaldehyde had the least inhibitory effects on cells, and the cell growth rate decreased by 25-45% at initial syringaldehyde concentrations of

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0.5-1.5 g/L (Table 1). The effects of 4-HMCA and 5-HMF were of moderate toxicity, and their effects on cell growth and ethanol yield were comparable. Nevertheless, the effects of inhibitors on ethanol yield were not so severe, and changes in ethanol yield were <20% in most of the

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cases.

Fig. 2d shows degradation of the lignocellulose-derived inhibitors by Z. mobilis. The results indicated poor degradation of vanillin and 4-HMCA, and only a marginal amount (< 10%) of these compounds were removed by the cells during the operation. Even syringaldehyde, which exhibited relatively lower toxicity, was poorly assimilated by Z. mobilis and the removal 9

efficiency was only 13% at the initial concentration of 1 g/L. However, at a lower concentration of 0.5 g/L, the removal efficiency for vanillin, 4-HMCA and syringaldehyde were 27%, 11% and 20%, respectively. These results indicated that at low concentrations of inhibitors, Z. mobilis was able to withstand the toxicity and they could metabolize the inhibitors partially [25]. In contrast, 5-HMF was degraded quite well by Z. mobilis irrespective of the initial concentration. More than

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>90% 5-HMF was removed at initial concentrations of 0.5-1 g/L, whereas >80% 5-HMF was removed at an initial concentration of 1.5 g/L. These results are consistent with literature and

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furfural assimilation by Z. mobilis has been reported earlier [25].

Although the mechanism of inhibition by lignocellulose-derived inhibitors are quite diverse, it is

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generally believed that these compounds may disturb the membrane potential of the

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microorganisms and acidify the cytoplasm [26]. The aldehydes are known to react with various

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organelles, enzymes and proteins within the cell wall, which could disrupt metabolic activity and

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reduce cell viability [8]. On the other hand, furan derivatives may inhibit enzymes that are essential for the central carbon metabolism. These are also known to cause damages to nucleic

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acid and cell membrane even at relatively low concentrations. [27]. Many phenolic and

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hydrophobic compounds dissolve readily into the cell membrane, causing an increase in the fluidity and permeability of the membrane, which may result in loss of cell integrity, and hinder

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transport through the cell membrane [7].

Although the inhibitors exhibited toxicity and affected growth and productivity of Z. mobilis,

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their effects on the cells were not detrimental at concentrations below 1.5 g/L. Consequently, the microorganisms could grow slowly but steadily, and the glucose supplied could be transformed into ethanol within a relatively short span of 24 h. However, when Z. mobilis was exposed to a mixture of inhibitors, comprising of 0.5-1 g/L each of vanillin, syringaldehyde, 4-HMCA and 5HMF, the cells succumbed to the high toxicity. No cell growth, glucose removal or ethanol production were detected in the medium, even after 24 h of operation, as shown in Fig. S1 (in 10

supplementary materials). The drastic differences observed on cell growth and ethanol production in the presence of individual and mixed inhibitors indicate that the effects of the presence of inhibitors on Z. mobilis was synergistic. Such synergistic inhibition and their adverse effects have been reported in lignocellulosic bioethanol fermentation [28].

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ICHFMB operation 3.2.1 Abiotic sorption and desorption of inhibitors

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Since hydrophobic polymeric membranes are known to exhibit high affinity for a large variety of organic compounds, abiotic experiments were conducted to investigate the sorption and desorption of the inhibitors by the PS hollow fiber membranes. Fig. 3 shows the sorption of the

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inhibitors at two different initial concentrations. At initial concentration of 0.5 g/L, the amount of

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vanillin, syringaldehyde, 4-HMCA and 5-HMF removed were 13.7%, 14.9%, 6.5% and 3.3%,

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respectively. On the other hand, at initial concentration of 1 g/L, sorption of inhibitors were

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slightly higher, and the amounts removed for vanillin, syringaldehyde, 4-HMCA and 5-HMF were 23.5%, 18%, 13% and 9.4%, respectively. It was also observed that sorption equilibrium

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was achieved within 10 min, which could be attributed to the high surface area to volume ratio of

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the hollow fiber membranes, although hydrophobic interactions between the membranes and the inhibitors could have contributed to the rapid mass transfer [29]. Contrary to sorption, desorption

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of the inhibitors from the membranes into water was slow (results not shown) and equilibrium was achieved after 6 h of contact. While more than 70% vanillin, 4-HMCA and 5-HMF could be desorbed from the membranes after 6 h of contact, only 10% syringaldehyde was removed from

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the membranes.

These results demonstrate one of the advantages of hollow fiber membranes in lignocellulosic bioethanol fermentation. Since the membranes could remove inhibitors through sorption, the membranes not only were acting as a support for cell immobilization, but these were also reducing the effective concentration of the inhibitors in the aqueous medium [30]. Such 11

partitioning phenomenon between two immiscible phases have been widely used in preventing substrate or product inhibition in bioprocesses [31]. Sorption-based lowering of inhibitors concentrations may enhance the diffusion of microorganisms from the membranes into the aqueous phase, as the hydrolysate would be more suited for the growth of suspended cells at

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lower concentrations of the inhibitors [22].

3.2.2 Cell immobilization and characterization

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In order to investigate the efficacy of cell immobilization process, the ICHFMB was operated

with 20 g/L glucose containing 0.5 g/L of each of the inhibitors for 24 h. The membrane module was then rinsed with water and the module casing was broken to collect membrane samples with

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immobilized cells. The samples were then analyzed under SEM, and the results have been shown

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in Fig. 4.

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The SEM images showed immobilized microorganisms dispersed non-uniformly within the

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membrane walls and surfaces. However, the cells were predominantly distributed within the

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macrovoids of the membranes, and very little biofilms were observed on the inner and outer surfaces of the membranes. This is different from some of the previous studies in hollow fiber

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membrane bioreactors, where cells have been found to attach quite well to the inner membrane surfaces [22]. There can be three reasons for the absence of cells on the membranes surface: (1)

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poor biofilm formation and EPS production by Z. mobilis; (2) short operating period which did not result in mature biofilm formation, and; (3) high inhibitory conditions in the ICHFMB. It is

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likely that the macrovoids offered better protection against the lignocellulosic inhibitors due to the thick membrane walls, and therefore, microorganisms immobilized primarily in the membrane pores [21].

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3.2.3 Effects of inhibitors on ICHFMB Since the suspended cells experienced severe inhibition only exposed to a mixture of inhibitors, the ICHFMB was operated only with mixture of inhibitors exhibiting synergistic inhibition. Total concentrations of the inhibitors used during ICHFMB operation was 2 g/L (0.5 g/L each of the inhibitors) or 4 g/L (1 g/L each of the inhibitors), as these concentrations were detrimental

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for Z. mobilis. A control experiment was also conducted in the absence of any inhibitors. These experiments were performed with membrane modules containing 8 hollow fiber membranes. The

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initial glucose concentration was 20 g/L and the synthetic hydrolysate was pumped into the module at 10 mL/min.

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Fig. 5 shows temporal concentration profiles of cells, glucose and ethanol during ICHFMB

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operation at low (0.5 g/L each of the inhibitors) and high (1 g/L each of the inhibitors)

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concentration mix of the inhibitors along with the control experiment conducted in the absence

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of any inhibitors. Unlike suspended cells, the immobilized cells were able to grow and metabolize glucose under low and high inhibitors concentrations. Glucose assimilation by Z.

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mobilis started right from the onset of the experiment, indicating that the cells did not need to

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adapt to the inhibitory conditions, again signifying the excellent protection imparted by the membranes. At low concentrations of the inhibitors in the ICHFMB (Fig. 5b), glucose was

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completely consumed within 13 h, whereas ethanol yield and productivity were 0.489 g/g and 0.613 g/L-h, respectively. Although ethanol yield and productivity obtained during ICHFMB operation were lower than those obtained using suspended cells in the absence of inhibitors, the

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rate of glucose assimilation was comparable to that of the suspended cells in the absence of inhibitors. This could be the result of a high amount of immobilized cells in the ICHFMB at the beginning of operation. While the large inoculum size can influence rate of glucose assimilation, a dense bacterial culture is also more resistant to substrate or product inhibition [32]. It was also observed that the performance of the ICHFMB in the presence of the inhibitor mixture was better 13

than the performance of the suspended cells under individual inhibitors. The performance of the ICHFMB under low concentrations of the inhibitors was also comparable, and in some cases better than those achieved using suspended cells in the presence of individual inhibitors (Table 1). For example, in the presence of 1 g/L syringaldehyde and vanillin, the suspended cells metabolized 20 g/L glucose in 15 h and > 25h, respectively. Consequently, it can be concluded

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that cell immobilization within the PS hollow fiber membranes was effective in protecting the bacterial cells from acute toxicity emanating from the synergistic interactions between the

they could also achieve high ethanol yield and productivity.

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inhibitors. The immobilized cells were not only able to tolerate high concentration of inhibitors,

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A comparison of ICHFMB operation in the presence and absence of inhibitors has been

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presented in Table 2. In the absence of the inhibitors, immobilized cells exhibited high rate of

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glucose assimilation and 20 g/L glucose could be consumed completely within 9 h of operation

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(Fig. 5a). The rate of glucose uptake was faster than that observed by the suspended cells, owing to a high inoculum size in case of immobilization. Since a higher inoculum size in the ICHFMB

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could negate the effects of diffusion limitation, it is possible that the effects of mass transfer

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limitations were rather low on ICHFMB performance due to the large surface area and high porosity [32]. It was also observed that the final biomass yield in the ICHFMB varied from

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0.016-0.053 g/g, depending on the concentration of the inhibitors. In the absence of inhibitors, the biomass yield in the ICHFMB was 0.053 g/g, which was comparable to the biomass yield of 0.057 g/g during suspension culture under identical conditions. These results imply the dynamic

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nature of cell immobilization in the hollow fiber membranes. Since Z. mobilis was not bound to the support through chemical bonding, the cells could simply diffuse out of the membrane pores, when aqueous medium in the ICHFMB was favorable for growth, resulting in high cell growth (in suspension) and high glucose uptake rate [21]. On the other hand, in the presence of inhibitors, the biomass yield decreased dramatically to 0.019 and 0.016 g/g, at low and high 14

concentrations of the inhibitors. These results indicate that under inhibitory concentrations, the microorganisms preferred staying within the membrane pores, protected by the diffusion barrier provided by the membrane walls. Under these conditions, the decrease in suspended cell yield with increasing inhibitors concentration was not due to cell death, but due to the low rate of release of the cells from the membrane pores. This is supported by the high ethanol yield, which

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decreased by < 10% in the presence of the inhibitors, despite the dramatic changes in suspended cell concentrations. There was a drastic change in ethanol productivity though, which could be

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attributed to slower glucose uptake and ethanol production by the immobilized cells due to mass transfer limitations. These results are consistent with literature, where partial immobilization in hollow fiber membranes have been reported to enhance the efficiency of the bioreactor systems

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[22, 33-36]. At higher concentration of inhibitors, there was substantial decrease in the removal

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efficiency for vanillin, syringaldehyde and 5-HMF (Table 2). However, net removal of these

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inhibitors was higher at higher concentrations, owing primarily to their enhanced sorption on the

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membranes.

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Fig. 6 shows the changes in the concentration of the inhibitors when the ICHFMB was operated

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at high concentrations of the inhibitors (1 g/L each). It can be seen that vanillin and 4-HMCA concentrations decreased by 35% and 13%, respectively. Considering the fact that sorption on

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the membranes (Fig. 3) resulted in 23% vanillin removal and 13% 4-HMCA removal, it could be inferred that these changes during ICHFMB operation were caused predominantly by membrane sorption, rather than microbial metabolism. On the other hand, the removal of syringaldehyde

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and 5-HMF were 47% and 89%, respectively. Since membrane sorption could remove 18% syringaldehyde and 9.4% 5-HMF, microbial assimilation was responsible for removal of at least 29% syringaldehyde and 79% 5-HMF. While 5-HMF degradation and possible assimilation by the microorganisms were expected, syringaldehyde removal was surprising. It is possible that when cells were present in large number within the membranes, they were able to overcome its 15

toxicity and they could metabolize this compounds. It is also possible that due to the high affinity for the membranes, syringaldehyde was more accessible to the immobilized cells, and the cells gradually adopted to its presence and could also facilitate its removal from the medium. It was also noted that assimilation of 4-HMCA and 5-HMF by Z. mobilis in ICHFMB was lower as compared to suspended cells (Table 1), which could have been caused by the presence of a

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mixture of inhibitors in the ICHFMB.

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Although ICHFMB exhibited excellent performance in toxicity alleviation and achieve high ethanol yield and productivity, it should be noted that the experimental conditions were

relatively mild in this study. In lignocellulosic hydrolysate, total fermentable sugar concentration

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may reach above 100 g/L, whereas concentration of phenolic inhibitors may exceed 20 g/L [24].

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Under these conditions, microorganisms may experience severe inhibitory effects from sugars,

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ethanol and lignocellulosic inhibitors, which may require changes in membrane structure and

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microorganisms to the inhibitors [22].

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membrane properties, or a higher number of membranes to reduce the exposure of the

3.2.4 Effects of operating conditions on ICHFMB

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The performance of the ICHFMB could be affected by several different factors. One of the key factors was the number of hollow fiber membranes in the membrane module, as it would affect

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both the mass transfer area and the surface available for cell immobilization. Changing the number of fibers in the module would also affect the packing density and flow through the shell

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side of the module. Another key factor affecting the mass transfer in the membrane processes is the liquid flow rate by lowering the mass transfer resistance. In order to understand the mass transfer process in the ICHFMB, experiments were conducted at different packing density and flow rates. The experiments were conducted at initial glucose concentration of 20 g/L in the presence of low concentrations of the inhibitors. The results have been summarized in Table 3.

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Doubling the number of membranes, and thus the packing density of the membrane module, improved ICHFMB performance substantially. A higher number of membranes resulted in higher glucose uptake rate of 2.21 g/L-h, which shortened the fermentation duration by 30% to 9 h. Similarly, higher inhibitors removal were observed at higher packing density, which was attributed to the enhanced sorption of the inhibitors on the membranes. This improved

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performance of ICHFMB was a direct consequence of the increase in the amount of immobilized cells, although the process would have benefitted from a high rate of glucose diffusion to the

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immobilized cells, owing to the higher mass transfer area. It was also observed that suspended

cells concentration in the ICHFMN was 0.46 g/L, which was 25% higher than that observed at the lower packing density. Since suspended cell concentration was higher despite a higher

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surface area available for cells to attach, this could only be attributed to the higher number of

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cells in the membranes. When there was nutrient depletion, more cells were released from the

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membranes into suspension to obviate the effects of diffusion limitation. Ethanol yield was

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independent of the number of membranes, but ethanol productivity improved at higher packing

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density due to shorter fermentation time. These results indicates the effects of diffusion limitation on ICHFMB operation, especially during the initial phase, when suspended cells

PT

concentration was low and most of the glucose was assimilated by the immobilized cells [22]. These results also indicate one of the key advantages of using membranes as immobilization

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support, as the challenges arising from diffusion limitation can be easily obviated using higher number of membranes. The modular design also provides the advantages of connecting

A

additional modules in series and parallel to existing setup to improve performance or to increase capacity of the bioreactor [32].

Although the results imply that a higher number of membranes in the modules, and thus higher packing density, would be beneficial for the process, increasing the packing density of the module may result in various operational problems [29]. Since increasing the number of 17

membranes reduces the space in the shell side of the module for liquid flow, narrow space between the membranes may lead to channeling and poor contact between the liquid and the solid phases. In such a scenario, even cleaning of the membrane module may be challenging, as metabolic wastes and dying cells build up in certain sections of the module characterized by poor flow regimes. When the ICHFMB was operated with 16 membranes, some biomass build-up was

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seen within the module. Therefore, the packing density was not increased any further to avoid

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any operational challenges.

The effects of increasing flow rate on the ICHFMB was analogous to the effects of the high membrane area. Although ethanol yield remained unchanged, glucose uptake rate and ethanol

U

productivity improved significantly. At the flow rates of 10 mL/min, 20 mL/min and 40 mL/min,

N

glucose could be completely metabolized within 16 h, 13 h and 12 h, yielding glucose uptake

A

rates of 1.25 g/L-h, 1.56 g/L-h and 1.74 g/L-h, respectively. O the other hand, suspended cells

M

concentration increased gradually from 0.33 g/L to 0.43 g/L, when the flow rates were increased from 10 mL/min to 40 mL/min. these results again indicate the presence of diffusion limitation

ED

during ICHFMB operation. In contrast, there were no changes in inhibitors removal at different

PT

flow rates, indicating inhibitor assimilation by Z. mobilis or sorption on the membranes were not influenced by changes in flow rates.

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Contrary to the effects of increased membrane area, which could have been either a result of higher number of membranes or higher mass transfer area, the improved ICHFMB performance

A

at higher flow rate could only have been a result of higher mass transfer rate through the aqueous boundary layer at the liquid/solid interface [29]. Therefore, it is likely that glucose diffusion through the boundary layer was the rate limiting step during the mass transfer. This is augmented by the fact that the cells were present in the macrovoids within the membrane walls. Thus, the diffusion path was considerably shorter, as glucose molecules did not need to diffuse through the membranes, but only through the outer and inner walls of the membranes to reach the 18

microorganisms. The results also indicate that the boundary layer was not a prominent factor in glucose diffusion above flow rate of 20 mL/min, as the improvements in ICHFMB performance at 40 mL/min flow rate were marginal.

Apart from investigating the efficacy of ICHFMB in alleviating the adverse effects of

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lignocellulose-derived inhibitors on Z. mobilis, experiments were also conducted to assess the effectiveness of the ICHFMB in mitigating substrate inhibition from glucose and product

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inhibition from ethanol at glucose concentrations above 140 g/L. It can be seen from Fig S2 and Table S1 (in supplementary materials) that ICHFMB exhibited excellent performance at glucose concentrations of 140-200 g/L, and achieved ethanol yield of 0.35-0.40 g/g. These results

U

indicated the effectiveness of the membranes in protecting the microorganisms against substrate

A

N

and product inhibitions at very high sugar concentrations.

3.2.5 Stability of ICHFMB

M

Although cell immobilization based approaches have been used for ethanol fermentation for a

ED

long time, the real challenge lies in the stability of the immobilization support over a prolonged period of time [19]. Moreover, the support should have ample pores to accommodate the

PT

biomass proliferating over the operating period. In order to investigate the stability and sustainability of the membrane-based immobilization approach in lignocellulosic bioethanol

CC E

production in the presence of inhibitors, 20 consecutive batch experimental runs were performed at 20 g/L glucose and high concentration of the inhibitors. The bioreactor was closely monitored

A

for its performance, as well as for any operating challenges.

Fig. 7 shows the temporal concentration profiles of glucose and ethanol during the 20 experimental runs conducted over a period of 10 days. It can be seen that glucose was completely consumed within 12 h and ethanol yield was 0.47 ± 0.023 g/g during all these runs, and there was no drastic changes in the performance of the ICHFMB. As expected, glucose and 19

ethanol profiles showed complementary trends, and ICHFMB performance improved gradually over time. For example, during the first two runs, glucose was consumed in 12 h. However, glucose could be completely metabolized within 9 h during subsequent runs. Towards end of operation, glucose removal could be achieved within 6 h, which indicated about 60% increase in average glucose uptake rate and over 70% increase in ethanol productivity. The gradual

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enhancement in ICHFMB performance could be attributed to higher amount of immobilized

cells in the hollow fiber membranes. These results could also imply that the immobilized cells

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may have adapted to the presence of inhibitors, and exhibited higher efficacy in their presence [21].

U

At the end of each experimental run, the membrane module was only rinsed with water and no

N

chemicals were used to clean the membranes. However, this did not result in any loss in

A

performance, which indicated that membrane biofouling had no significant effects on the

M

ICHFMB operation during this period. That the ICHFMB was able to exhibit consistent performance over 20 operating runs also indicated that cell immobilization within the PS hollow

ED

fiber membranes was stable and it could be used to facilitate biochemical processes during

PT

prolonged runs. The stability of PS has been demonstrated in several previous studies and it is already used in many commercial applications [37, 38].

Conclusions

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4

The ICHFMB with immobilized Z. mobilis was designed and operated for bioethanol

A

fermentation in the presence of a total of 2-4 g/L lignocellulose-derived inhibitors. The membrane barrier was effective in protecting cells from acute toxicity, and resulted in high glucose uptake rate and ethanol yield. The ICHFMB allowed great flexibility in cell immobilization by allowing cells to freely diffuse in and out of the pores, while glucose mass transfer to the immobilized cells could be enhanced by changing flow rate and membrane surface

20

area. In addition, cell immobilization in the membranes was stable and ICHFMB performance could be sustained during long-term operation.

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Xie, S.K. Khanal, Anaerobic biorefinery: Current status, challenges, and opportunities, Bioresour. Technol., 215 (2016) 304-313.

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[2] R. Liguori, V. Ventorino, O. Pepe, V. Faraco, Bioreactors for lignocellulose conversion into fermentable sugars for production of high added value products, Appl. Microbiol. Biotechnol., 100 (2016) 597-611.

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[3] L. Viikari, J. Vehmaanperä, A. Koivula, Lignocellulosic ethanol: From science to industry,

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[4] Y. Sun, J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: A review,

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[5] B. Carter, P. Squillace, P.C. Gilcrease, T.J. Menkhaus, Detoxification of a lignocellulosic biomass slurry by soluble polyelectrolyte adsorption for improved fermentation efficiency,

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Biotechnol. Bioeng., 108 (2011) 2053-2060. [6] N. Mosier, C. Wyman, B. Dale, R. Elander, Y.Y. Lee, M. Holtzapple, M. Ladisch, Features

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of promising technologies for pretreatment of lignocellulosic biomass, Bioresour. Technol., 96 (2005) 673-686.

[7] H.B. Klinke, L. Olsson, A.B. Thomsen, B.K. Ahring, Potential inhibitors from wet oxidation

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of wheat straw and their effect on ethanol production of Saccharomyces cerevisiae: wet oxidation and fermentation by yeast, Biotechnol. Bioeng., 81 (2003). [8] M.P. Taylor, I. Mulako, M. Tuffin, D. Cowan, Understanding physiological responses to pretreatment inhibitors in ethanologenic fermentations, Biotechnol. J., 7 (2012) 14.

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[9] M.H. Thomsen, A. Thygesen, A.B. Thomsen, Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw, Appl. Microbiol. Biotechnol., 83 (2009) 447-455. [10] A. Cavka, L.J. Jonsson, Detoxification of lignocellulosic hydrolysates using sodium borohydride, Bioresour. Technol., 136 (2013) 368-376.

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[11] X. Liu, P. Fatehi, Y.H. Ni, Removal of inhibitors from pre-hydrolysis liquor of kraft-based dissolving pulp production process using adsorption and flocculation processes, Bioresour.

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[12] A. Saeed, P. Fatehi, Y. Ni, An integrated process for removing the inhibitors of the prehydrolysis liquor of kraft-based dissolving pulp process via cationic polymer treatment,

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Biotechnol. Prog., 28 (2012) 998-1004.

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[13] A.D. Moreno, D. Ibarra, I. Ballesteros, J.L. Fernández, M. Ballesteros, Ethanol from

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laccase-detoxified lignocellulose by the thermotolerant yeast Kluyveromyces-Effects of steam

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pretreatment conditions, process configurations and substrate loadings, Biochem. Eng. J., 79

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[14] L.N. Jayakody, K. Horie, N. Hayashi, H. Kitagaki, Engineering redox cofactor utilization

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for detoxification of glycolaldehyde, a key inhibitor of bioethanol production, in yeast Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol., 97 (2013) 6589-6600.

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[15] J.A. Zhang, Z.N. Zhu, X.F. Wang, N. Wang, W. Wang, J. Bao, Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae

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ZN1, and the consequent ethanol fermentation, Biotechnol. Biofuels, 3 (2010) 15. [16] X.Y. Cheng, Y.W. Tong, K.C. Loh, An immersed hollow fiber membrane bioreactor for enhanced biotransformation of indene to cis-indandiol using Pseudomonas putida, Biochem. Eng. J., 87 (2014) 1-7. [17] D. Borovikova, R. Scherbaka, A. Patmalnieks, A. Rapoport, Effects of yeast immobilization on bioethanol production, Biotechnol. Appl. Biochem., 61 (2014) 33-39. 22

[18] P. Praveen, K.-C. Loh, Photosynthetic aeration in biological wastewater treatment using immobilized microalgae-bacteria symbiosis, Appl. Microbiol. Biotechnol., 99 (2015) 1034510354. [19] Y. Kourkoutas, A. Bekatorou, I.M. Banat, R. Marchant, A.A. Koutinas, Immobilization technologies and support materials suitable in alcohol beverages production: a review, Food

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Microbiol., 21 (2004) 377-397.

[20] S. Nikolić, L. Mojović, D. Pejin, M. Rakin, M. Vukašinović, Production of bioethanol from

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corn meal hydrolyzates by free and immobilized cells of Saccharomyces cerevisiae var. ellipsoideus, Biomass Bioenerg., 34 (2010) 1449-1456.

[21] K.C. Loh, T.S. Chung, W.F. Ang, Immobilized-Cell Membrane Bioreactor for High-

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Strength Phenol Wastewater, J. Environ. Eng., 126 (2000) 75-79.

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[22] Y. Li, K.C. Loh, Activated carbon impregnated polysulfone hollow fiber membrane for cell

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J. Membr. Sci., 276 (2006) 81-90.

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immobilization and cometabolic biotransformation of 4-chlorophenol in the presence of phenol,

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[23] S.J. Lee, J.H. Lee, X. Yang, S.B. Kim, J.H. Lee, H.Y. Yoo, C. Park, S.W. Kim, Phenolic compounds: Strong inhibitors derived from lignocellulosic hydrolysate for 2,3-butanediol

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production by Enterobacter aerogenes, Biotechnol. J., 10 (2015) 1920-1928. [24] Y. Yang, M. Hu, Y. Tang, B. Geng, M. Qiu, Q. He, S. Chen, X. Wang, S. Yang, Progress

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and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis, Bioresour. Bioprocess., 5 (2018) 6.

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[25] M.A. Franden, H.M. Pilath, A. Mohagheghi, P.T. Pienkos, M. Zhang, Inhibition of growth of Zymomonas mobilis by model compounds found in lignocellulosic hydrolysates, Biotechnol. Biofuels, 6 (2013) 99. [26] J.B. Russell, F. DiezGonzalez, The effects of fermentation acids on bacterial growth, in: R.K. Poole (Ed.) Advances in Microbial Physiology, Vol 39, Academic Press Ltd-Elsevier Science Ltd, London, 1998, pp. 205-234. 23

[27] T. Modig, G. Liden, M.J. Taherzadeh, Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase, Biochem. J., 363 (2002) 769-776. [28] A.J.A. van Maris, D.A. Abbott, E. Bellissimi, J. van den Brink, M. Kuyper, M.A.H. Luttik, H.W. Wisselink, W.A. Scheffers, J.P. van Dijken, J.T. Pronk, Alcoholic fermentation of carbon

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sources in biomass hydrolysates by Saccharomyces cerevisiae: current status, Anton. Leeuw. Int. J. G., 90 (2006) 391-418.

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[29] P. Praveen, K.-C. Loh, Solventless extraction/stripping of phenol using trioctylphosphine

oxide impregnated hollow fiber membranes – Experimental & modeling analysis, Chem. Eng. J., 255 (2014) 641-649.

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[30] H. Siegrist, A. Joss, Review on the fate of organic micropollutants in wastewater treatment

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and water reuse with membranes, Water Sci. Technol., 66 (2012) 1369-1376.

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[31] P. Praveen, K.-C. Loh, Trioctylphosphine oxide-impregnated hollow fiber membranes for

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removal of phenol from wastewater, J. Membr. Sci., 437 (2013) 1-6.

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[32] P. Praveen, K.C. Loh, Two-Phase Biodegradation of Phenol in a Hollow Fiber Membrane Bioreactor, J. Environ. Eng., 139 (2013) 654-660.

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[33] J.R. Lloyd, A.W. Bunch, The physiological state of an ethylenogenic Escherichia coli immobilized in hollow-fiber bioreactors, Enzyme Microb. Technol., 18 (1996) 113-120.

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[34] D.S. Inloes, D.P. Taylor, S.N. Cohen, A.S. Michaels, C.R. Robertson, Ethanol Production by Saccharomyces cerevisiae Immobilized in Hollow-Fiber Membrane Bioreactors, Appl.

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Environ. Microbiol., 46 (1983) 264-278. [35] L. Wang, K.C. Loh, Y.W. Tong, Immobilization of growing Sphingomonas sp HXN-200 to gelatin microspheres: Efficient biotransformation of N-Cbz-pyrrolidine and N-Boc-pyrrolidine into hydroxypyrrolidine derivatives, J. Biotechnol., 182 (2014) 74-82. [36] L.H. Liu, S.M. Liu, X.Y. Tan, Zirconia microbial hollow fibre bioreactor for Escherichia coli culture, Ceram. Int., 36 (2010) 2087-2093. 24

[37] M. Padaki, R.S. Murali, M.S. Abdullah, N. Misdan, A. Moslehyani, M.A. Kassim, N. Hilal, A.F. Ismail, Membrane technology enhancement in oil-water separation. A review, Desalination, 357 (2015) 197-207. [38] C. Charcosset, Membrane processes in biotechnology: An overview, Biotechnol. Adv., 24

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M

A

N

U

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(2006) 482-492.

25

Tables

Table 1 Effects of inhibitors on suspended cell growth and ethanol production. All points represent average of three replicates. Standard deviations varied between 3-8% for most of the parameters. Ethanol

Ethanol

Inhibitor

conc.

Rate

Yield

conc.

yield

productivity

removal

(g/L)

(h-1)

(g/g)

(g/L)

(g/g)

(g/L-h)

(%)

-

0.376

0.057

9.82

0.49

0.408

-

0.5

0.194

0.032

8.30

0.415

0.346

27.6

1.0

0.160

0.024

7.24

0.362

0.302

8.09

1.5

0.113

0.016

5.42

0.271

0.226

5.02

0.5

0.307

0.052

9.04

0.452

0.377

20.0

1.0

0.280

0.046

8.98

0.449

0.374

13.1

1.5

0.266

0.046

8.76

0.438

0.365

6.05

0.5

0.255

0.052

9.94

0.497

0.414

10.8

1.0

0.224

0.041

8.40

0.420

0.350

9.20

0.122

0.014

5.86

0.293

0.244

4.01

0.233

0.047

8.20

0.410

0.342

92.2

0.235

0.046

7.28

0.364

0.303

93.1

0.213

0.039

5.32

0.266

0.222

81.6

A

Syring-

1.5

1.0

CC E

5-HMF

PT

0.5

1.5

*

ED

4-HMCA

M

aldehyde

IP T

Ethanol

SC R

Vanillin

Biomass

*

U

Control

Growth

N

Experiment

Inhibitor

A

Ethanol productivity was calculated based on fermentation time of 24 h

26

Table 2 Effects of inhibitor mixture on ICHFMB performance. Data represent average and standard deviation of three replicates.

Parameters None

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Inhibitor conc. (g/L) Lower conc. mix

Higher conc. mix

(0.5 g/L each)

(1 g/L each)

9

13

Ethanol conc. (g/L)

10.0 ± 0.03

9.13 ± 0.06

9.81 ± 0.04

Ethanol yield (g/g)

0.506 ± 0.01

0.460 ± 0.01

0.489 ± 0.004

Productivity (g/L-h)

1.12 ± 0.0

0.702 ± 0.0

0.613 ± 0.002

Suspended cell conc. (g/L)

1.05 ± 0.02

0.369 ± 0.02

0.053 ± 0.0

Glucose uptake rate (g/L-h)

2.32 ± 0.06

4-HMCA

U

52.3 ± 1.6

35.1 ± 1.2

-

87.8 ± 0.9

47.4 ± 3.2

-

12.9 ± 0.6

13.1 ± 2.4

-

97.0 ± 2.3

89.2 ± 3.3

PT

5-HMF

yield was calculated based only on suspended cell concentrations

A

CC E

#Biomass

0.016 ± 0.0 1.25 ± 0.01

ED

Syringaldehyde

0.330 ± 0.005

1.53 ± 0.0

-

Vanillin

0.019 ± 0.0

M

Inhibitors removal (%)

N

Biomass yield (g/g)

A

#

16

SC R

Operation time (h)

27

Table 3 Effects of operational conditions on ICHFMB performance. Data represent average and standard deviation of three replicates. Flow rate (mL/min)

Parameters

No. of membranes

10

20

40

8

16

higher

higher

higher

lower

lower

16

13

12

13

9

9.81 ± 0.04

9.82 ± 0.33

10.2 ± 0.42

SC R

9.38 ± 0.03

U

0.613 ± 0.002 0.756 ± 0.02 0.848 ± 0.01

0.702 ± 0.0

1.04 ± 0.0

N

0.330 ± 0.005 0.390 ± 0.02 0.430 ± 0.03 0.369 ± 0.02 0.460 ± 0.05 0.019 ± 0.0

0.021 ± 0.0

0.019 ± 0.0

0.023 ± 0.0

1.25 ± 0.01

1.56 ± 0.0

1.74 ± 0.01

1.53 ± 0.0

2.21 ± 0.04

35.1 ± 1.2 47.4 ± 3.2 13.1 ± 2.4 89.2 ± 3.3

33.4 ± 0.8 49.6 ± 1.6 12.3 ± 2.7 89.5 ± 1.8

34.5 ± 0.6 47.8 ± 2.3 13.5 ± 1.1 90.9 ± 6.2

52.3 ± 1.6 87.8 ± 0.9 12.9 ± 0.6 97 ± 2.3

68.2 ± 4.3 96.3 ± 1.5 23.3 ± 1.1 98.3 ± 0.9

M

A

0.016 ± 0.0

PT

yield was calculated based only on suspended cell concentrations

A

CC E

#Biomass

9.13 ± 0.06

0.489 ± 0.004 0.483 ± 0.04 0.488 ± 0.02 0.460 ± 0.01 0.471 ± 0.02

ED

Operation time (h) Ethanol conc. (g/L) Ethanol yield (g/g) Productivity (g/L-h) Suspended cell conc. (g/L) # Biomass yield (g/g) Glucose uptake rate (g/L-h) Inhibitors removal (%) Vanillin Syringaldehyde 4-HMCA 5-HMF

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Inhibitor conc.

28

Figure Captions Figure 1 Schematic diagram of ICHFMB setup Figure 2 Effect of 1 g/L inhibitors on suspended cells of Z. mobilis: (a) Cell growth profile; (b) Glucose removal; (c) Ethanol production; (d) Degradation of inhibitors. All points represent average of three replicates, and the error bars represent corresponding standard deviation.

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Figure 3 Sorption of inhibitors on PS hollow fiber membranes at initial concentrations of 0.5 g/L and 1 g/L. All points represent average of three replicates, and the error bars represent

SC R

corresponding standard deviation.

Figure 4 SEM analysis of the hollow fiber membranes: (a) Cross section of hollow fiber

U

membrane; (b) Macrovoids; (c) Immobilized cells in macrovoids.

N

Figure 5 ICHFMB operation with 20 g/L glucose and inhibitors concentrations of: (a) 0 g/L each

A

(control); (b) 0.5 g/L each; (c) 1 g/L each. All points represent average of three replicates, and

M

the error bars represent corresponding standard deviation g/L. Figure 6 Temporal changes in inhibitors concentrations in the ICHFMB operated with initial

ED

inhibitors concentration of 1 g/L each. All points represent average of three replicates, and the error bars represent corresponding standard deviation.

PT

Figure 7 Long-term operation of ICHFMB at initial glucose concentration of 20 g/L and

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inhibitors concentration of 1 g/L each. All points represent average of three replicates, and the

A

error bars represent corresponding standard deviation.

29

A Figure 1

ED

PT

CC E

IP T

SC R

U

N

A

M

Figures

30

1.4

(a)

Control Vanillin 5-HMF 4-HMCA Syringaldehyde

1 0.8

IP T

DCW (g/L)

1.2

0.6

SC R

0.4 0.2

0

5

10

15

25

A

N

Time (h)

20

U

0

ED

15

10

Control Vanillin 5-HMF 4-HMCA Syringaldehyde

PT

Glucose conc. (g/L)

M

20

(b)

CC E

5

0

A

0

5

10

15

20

25

Time (h)

Figure 2

31

12

(c)

6 Control Vanillin 5-HMF 4-HMCA Syringaldehyde

4 2 0 5

10

15

25

(d)

A

N

Time (h)

20

U

0

IP T

8

SC R

Ethanol conc. (g/L)

10

M

1

ED

Inhibitor (g/L)

0.8 0.6

Vanillin

0.4 0.2

PT

5-HMF

4-HMCA

CC E

Syrinaldehyde

0

A

0

5

10

15

20

25

Time (h)

Figure 2

32

IP T SC R

0.8

U

0.6

0.2

N

0.4

0 0

5

5-HMF Syrinaldehyde

A

Vanillin 4-HMCA

M

Inhibitor conc. (g/L)

1

10

15

20

25

ED

Time (h)

A

CC E

PT

Figure 3

33

SC R

IP T

(a)

ED

M

A

N

U

(b)

A

CC E

PT

(c)

Figure 4

34

1.4 Cell

15

0.8

10

0.6 0.4

5

0.2

0

0 5

10

15

20

Time (h) Glucose

Ethanol

Cell

0.4

U

20

0.3

N

15

0.2

A

10

0.1

M

5 0 0

5

10

ED

Glucose/Ethanol conc. (g/L)

25

25

IP T

1

0

(b)

1.2

20

Cell conc. (g/L)

Ethanol

Cell conc. (g/L)

Glucose

SC R

(a)

Glucose/Ethanol conc. (g/L)

25

0 15

20

25

Time (h)

Ethanol

0.4

Cell

0.3

A

0.2

10

0.1

5 0

Cell conc. (g/L)

15

PT

20

Glucose

CC E

(c)

Glucose/Ethanol conc. (g/L)

25

0 0

5

10

15

20

25

Time (h)

Figure 5

35

IP T SC R

0.8 0.6 0.4

N

0.2

U

Vanillin 5-HMF 4-HMCA Syringaldehyde

0 0

5

10

A

Inhibitor conc. (g/L)

1

15

20

25

ED

M

Time (h)

A

CC E

PT

Figure 6

36

IP T

Glucose

Ethanol

SC R

20 16

U

12

N

8 4

A

Glucose/Ethanol conc. (g/L)

24

0 20

40

60

80

100 120 140 160 180 200 220 240

M

0

ED

Time (h)

A

CC E

PT

Figure 7

37