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Keywords:

  • Clostridium acetobutylicum;
  • butanol;
  • adsorption;
  • in situ product recovery

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Although butanol is a promising biofuel, its fermentative production suffers from inhibition caused by end product toxicity. The in situ removal of butanol from cultures via expanded bed adsorption offers an effective strategy for mitigating the effects of product toxicity while eliminating the need to clarify cultures via microfiltration. The hydrophobic polymer resin Dowex Optipore L-493 was found to be both an effective butanol adsorbent and suitable for use in expanded bed adsorption. Recirculation rates through the adsorption column were strongly correlated with and ultimately controlled rates of butanol uptake from the media which, reaching as high as 41.1 g/L h, easily exceed those of its production in a typical fermentation. Vacuum application with vapor collection was found to be an effective means of adsorbent regeneration, with an average of 81% butanol recovery possible, with butanol concentrations in the cold trap reaching as high as 85.8 g/L. Integration of expanded bed adsorption with a fed-batch Clostridium acetobutylicum ATCC 824 fermentation and its continuous operation for 38.5 h enabled the net production (i.e., in solution and adsorbed) of butanol and total solvent products at up to 27.2 and 40.7 g/L of culture, respectively, representing 2.2- and 2.3-fold improvements over conventional batch culture. While adsorbent biofouling was found to be minimal, further investigation of biofouling in longer-term studies will provide useful and further insight regarding the robustness of the process strategy. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:68–78, 2014


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Faced with the ever-increasing global rate of consumption of total liquid fuels (89.17 million barrels per day in 2012[1]), sustainable solutions are needed to offset their demand. Biofuels, predominantly consisting of alcohols derived from the fermentation of renewable biomass, represent one such potential solution.[2, 3] While ethanol has traditionally served as the focal biofuel compound, n-butanol (hereafter referred to as butanol) perhaps represents a superior alternative.[4] Butanol is a natural fermentation product of several species of Clostridium, including the well characterized type strain C. acetobutylicum ATCC 824.[5, 6] Moreover, recent metabolic engineering efforts have also resulted in the creation of novel butanol-producing strains of Escherichia coli, Saccharomyces cerevisiae, and others.[7-10] However, all microbial butanol producers are hindered by end-product inhibition caused by the accumulation of toxic products. For example, butanol inhibits C. acetobutylicum ATCC 824 growth and productivity at final titers of approximately 13 g/L.[5, 11] Additionally, butanol's low toxicity threshold confines conventional bioprocesses to dilute fermentation conditions which represent ineffectual feeds for downstream separation and purification processes,[12] contributing to economically unviable and unsustainable bioprocesses.[13]

One approach to circumvent the inhibition caused by butanol is to separate it from cultures as it is produced. Several in situ product recovery strategies have been developed to this end (for comprehensive reviews see Schugerl,[14] Ramaswamy et al.,[15] and Ezeji et al.[16]). Said approaches have typically relied upon the use of liquid–liquid extraction,[17-19] gas stripping,[20, 21] membrane pervaporation,[22, 23] and adsorption.[24, 25] Whereas strengths and weaknesses are inherent to each strategy, adsorption offers notable advantages, including: high physical, chemical, and biological stability of the extraction phase, improved biocompatibility, phase immiscibility and elimination of emulsification (both of which improve downstream recovery), and increased potential for re-use.[26] Furthermore, it has been suggested that adsorption is superior with regards to overall energy efficiency and economy.[25] Numerous suitable butanol adsorbents have been developed and characterized in the literature, including activated[27] and mesoporous[28] carbons, silicalite[29, 30] and other zeolites,[31, 32] and polymer resins.[29, 33-35]

To date, adsorption has been most commonly integrated with bioprocesses for in situ product recovery by either (a) direct addition of the adsorbent to the fermentation media in a bioreactor or by (b) confining the adsorbent to within a column through which the medium is circulated. Whereas direct adsorbent addition represents perhaps the simplest configuration (as it requires no additional equipment) adsorbent removal for regeneration and recovery of products imposes a disruption to the process. Meanwhile, mechanical stresses caused by agitation can degrade the integrity of the adsorbent particles. The use of flow-through columns effectively eliminates such concerns by isolating the adsorbent external to the bioreactor, improving the modularity of the approach. As it offers optimal separation efficiency per unit mass of adsorbent,[36] packed-bed adsorption (PBA), which resembles traditional chromatography with tightly packed adsorbents, has been most commonly investigated to this end. However, with unclarified broths, PBA is prone to fouling by cells and ultimately clogging at the column inlet. Thus, cell removal by microfiltration upstream of the PBA column is typically performed,[37] which incurs additional capital and energy requirements. As an alternative to PBA, expanded bed adsorption (EBA) eliminates the need for media clarification by upstream microfiltration by increasing the interparticle voidage volume through fluidization of the adsorbent bed. This effect is easily realized by introducing the media at the bottom of a vertical column that accommodates a significant headspace above the adsorbent bed. This results in the facile passage of cells and cell debris, removing the need for prior cell filtration and reduced energy demand owing to reduced pressure drop in the column.

EBA has long served as a staple technology for the purification of therapeutic proteins,[38, 39] however, its adaptation for the in situ recovery of fermentation products has thus far been minimal. To date, applications of EBA have been limited to the purification of succinic acid and propionic acid from cultures of Actinobacillus sp.[40] and Propionibacterium sp.,[41] respectively, with anion exchange resins used as adsorbents in both cases. To date, however, this promising strategy has not yet been investigated as a means to enhance the bioproduction of butanol. Accordingly, this study is the first to explore the systematic development of an integrated bioprocess using EBA for the in situ recovery of butanol and other solvent products from C. acetobutylicum ATCC 824 fermentations.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Strains, media, and culture conditions

Clostridium acetobutylicum ATCC 824 was purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were routinely cultured at 37°C in reinforced clostridial medium (BD Difco, Franklin Lakes, NJ). Media was reduced by heating in a boiling water bath for 30 min and then sparged with ultra high purity (UHP) nitrogen gas until cooled. Reduced media (10 mL) was dispensed into 15 mL Hungate tubes (Bellco Biotechnology) and then immediately sealed. These cultures also served as seeds for bioreactor inoculation. Bioreactor fermentations were performed using clostridial reactor media (CRM), prepared according to Mermelstein et al.[42] with the following composition (all per L): KH2PO4, 1.5 g; K2HPO4, 1.5 g; MgSO4, 0.70 g; MnSO4·H2O, 20 mg; FeSO4·7H2O, 20 mg; p-aminobenzoic acid, 8 mg; NaCl, 2 g; asparagine, 4 g; yeast extract, 10 g; and (NH4)2SO4, 2 g. Glucose was supplemented into the media, as described below.

Adsorbent and chemicals

The macroporous and hydrophobic poly(styrene-co-divinylbenzene) resin Dowex Optipore L-493 (hereafter referred to as L-493), which was previously characterized as an effective butanol adsorbent,[43, 44] was selected as the adsorbent phase. L-493 was purchased from Sigma-Aldrich (St. Louis, MO). Butanol, acetone, ethanol were also purchased from Sigma-Aldrich. Media components and glucose were obtained from VWR.

Adsorption equilibrium studies

The equilibrium adsorption behavior of butanol, acetone, and ethanol on L-493 resin was investigated through a series of model adsorption studies. The three solvents were added to deionized water at range of known initial solute concentrations, either individually or together at a constant initial concentration ratio of 6:3:1 (representing the ratio by which they are produced by fermentation). Then, to 40 mL glass vials containing 25 mL of said prepared solutions, preweighed samples of fresh, dried resin (1–4 g) were added and allowed to equilibrate at 37°C while shaking at 120 rpm. After 24 h, a sample (1 mL) was removed and analyzed via high performance liquid chromatography (HPLC) to determine the equilibrated solvent concentration(s) (note: from preliminary experiments it was determined that equilibrium was typically reached within 2 h; data not shown). For each species in each solution, i, the specific loading capacity was determined by the following mass balance relationship:

  • display math(1)

where qi is the specific loading capacity of species i, Ci,0 and Ci are the initial and final concentration of species i in the aqueous solution, respectively, and m and V are the adsorbent mass and the volume of the aqueous solution, respectively. From these data, adsorption isotherms were then determined for each species and subsequently fit to the Freundlich isotherm model45:

  • display math(2)

The Freundlich model parameters k and n were estimated using nonlinear least squares regression via the nlinfit function in MATLAB. It should be noted that other isotherm models were also initially considered,[45] however, the Freundlich model provided the best overall fit in all cases, as is consistent with prior studies.[43, 44] All experiments were repeated in triplicate to estimate standard error.

Bioreactor operation and fermentation conditions

A schematic of the bioreactor with integrated EBA column is provided in Figure 1. Fermentation conditions were modeled after those used by Mermelstein et al.[42] Fermentations were carried out using an aqueous working volume 1 L in a 2 L Biostat A Plus bioreactor (Sartorius Stedim Biotech, Bohemia, NY), with BioPAT MFCS/DA 3.0 software used for control and data acquisition. Dissolved oxygen and pH were continuously measured using Oxyferm FDA225 and Easyferm Plus K8 200 probes (Hamilton, Reno, NV), respectively. Glucose-free CRM broth was prepared and autoclaved in the bioreactor. Concentrated glucose solution, autoclaved separately, was added to the bioreactor at an initial concentration of 140 g/L for the control fermentation or 120 g/L for the EBA fermentation. In the case of the EBA fermentation, additional glucose was twice added (30 g each) to the bioreactor in a fed-batch manner, for a total of 180 g/L. The bioreactor was inoculated with a 1% (v/v) seed culture, prepared as above. Fermentations were performed at a constant temperature of 37°C, with agitation at 300 rpm, with continuous sparging of UHP nitrogen gas at 100 mL/min to maintain anaerobic conditions, and Antifoam 204 (Sigma Aldrich) addition, as needed. To minimize stripping of both solvents and water, a water-cooled condenser was used at the off-gas port. While the initial pH was 5.5, a shift to 4.9 was introduced when the biomass concentration reached approximately 0.5 g/L, as this strategy has been reported to aid in facilitating the shift to solventogenesis.[46] Adjustment of pH was performed by automated addition of 5 M NH4OH or 1 M HCl, as appropriate. Samples were periodically drawn for analysis of biomass, glucose, and relevant fermentation product concentrations.

image

Figure 1. Experimental apparatus, consisting of: (a) EBA column containing Dowex Optipore L-493 resin, (b) bioreactor, (c) heating jacket for temperature control, (d) nitrogen purge line with flowmeter and sparger, (e) computer for control and data acquisition, (f) acid/base addition (note: single source/pump/inlet shown for space).

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The EBA column consisted of a glass column (450 mm length, 37 mm ID, Ace Glass, Vineland, NJ) fitted with polypropylene end caps (#25 thread, 1/4" NPT inlet, Ace Glass, Vineland, NJ). The column-facing side of both end caps included a 350-µm polypropylene mesh to allow cell passage but not L-493 particles. Under nominal conditions, the column contained 75 g of L-493 resin (or an adsorbent fraction of 50 g/L), leaving approximately 360 mL of headspace (∼75% of the total column volume) available for bed expansion. At this adsorbent fraction up to a threefold expansion in bed height could be accommodated before packing at the column outlet (top) would occur; a situation avoided throughout the study. Bioreactor contents were circulated through the EBA column at a constant flow rate of 102.4 mL/min (or a superficial velocity of 9.5 cm/min), as initiated following the detection of solvents in the culture media. A sample of L-493 was removed from near the column inlet following fermentation for imaging by scanning electron microscopy (SEM).

Characterization of EBA under simulated culture conditions

A series of model experiments were performed in the absence of cells to investigate parameters influencing EBA operation and performance. The bioreactor was first filled with 1.5 L of a 14 g/L butanol in deionized water solution. The impact of circulation rate was investigated by continuously recirculating the media between the bioreactor and the EBA column (which contained L-493 at a constant adsorbent fraction of 53.3 g/L) at superficial velocities between 2.1 and 18.5 cm/min. To test the impact of adsorbent mass, the experiment was repeated. However, the media was instead recirculated at a constant superficial velocity of 9.5 cm/min through EBA columns containing L-493 in adsorbent fractions between 26.6 and 80 g/L. Butanol fermentation was finally simulated by the addition of pure butanol to a bioreactor initially containing 1.5 L deionized water at the constant rate of 0.37 g/L h (representing the wild-type production rate[22]) using a programmable syringe pump (Auto Syringe AS40A, Baxter, Deerfield, IL). Bioreactor contents were continuously recirculated between the bioreactor and an EBA column containing 33.0 g/L L-493 at a constant superficial velocity of 9.5 cm/min. In all experiments, samples were periodically drawn from the bioreactor and analyzed for butanol content by HPLC.

The impact of flow rate on bed expansion was investigated by recirculating deionized water at a range of constant superficial velocities (up to 21 cm/min) through EBA columns containing 26.6, 40, or 53.3 g/L L-493. Upon reaching steady state, the relative change in bed height was recorded. The experiment was modeled using the following expression, derived from the Richardson-Zaki equation[47, 48]:

  • display math(3)

where u is superficial velocity, ut is the terminal settling velocity, n is the Richardson-Zaki exponent, H is the height of the expanded bed, and H0 and ε0 are the height and porosity of the packed bed (i.e., with no circulation), respectively. This expression is applicable to flows with low Reynolds numbers, as are typical of EBA applications. It should be noted that under all conditions examined in this study, estimated Reynolds number remained <1, well within the laminar flow regime.

Butanol desorption and adsorbent regeneration

An EBA column containing 75 g L-493 was equilibrated with a solution initially containing between 13 and 15 g/L butanol in water by circulating the solution through the column overnight. Following equilibration, the column inlet was sealed and a vacuum was applied to the outlet port. An Alcatel 2021i vacuum pump (Pfeiffer Vacuum, Asslar, Germany) was used to reduce the absolute pressure in the column to <1 kPa. An cold trap immersed in an acetone dry ice bath was used to collect the vapors desorbed from the resin. After 4 h, the vacuum was stopped and the trap contents collected. This process was repeated up to six consecutive times resulting in up to six “fractions.” This adsorption–desorption process was repeated for three consecutive cycles.

Analytical methods

Glucose, acetone, acetate, butyrate, ethanol, and n-butanol levels were quantified via HPLC (1100 series Agilent) using a refractive index detector. Analytes were separated on an Aminex HPX-87H anion exchange column (Bio-Rad Laboratories) according to the method of Buday et al.[49] while external standards provided calibration. Biomass concentration was determined through optical density measurements at 600 nm (OD600) using a DU800 spectrophotometer (Beckman Coulter, Brea, CA). Serial dilution was performed to ensure samples were measured in the linear range of the instrument. Dry cell weight (DCW) was predicted using the conversion: 1 OD600 = 0.26 g/L.[46]

SEM analysis

Resin samples were separated from spent media by decanting and were dried in a desiccator for 2 h before then being pressed onto carbon tape adhered to pin stubs. Samples were sputter coated with gold on a Technics Hummer V sputter coater (Anatech, Union City, CA) before being imaged on a JEOL JSM6300 Scanning Electron Microscope (Peabody, MA).

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Characterization of solvent adsorption from single-component and multicomponent mixtures

To overcome the limitations caused by butanol accumulation, the development of an integrated bioprocess for in situ butanol removal via EBA was systematically explored. To reliably predict the adsorption behavior of butanol, acetone, and ethanol from fermentations, equilibrium adsorption isotherms on L-493 were characterized through a series of model studies. The adsorption behavior of both butanol and ethanol on L-493 has previously been the subject of extensive characterization,[43, 44] however, that of acetone and the solvent mixture has not yet been reported. Table 1 compares the resultant best-fit, Freundlich model parameter estimates for the individual solvents and the mixture. Despite variations between parameter estimates from this study and previous reports, the resultant predicted and experimental isotherms nevertheless strongly agreed over the concentration range of interest (see Supporting Information). We previously found that relative differences in octanol–water partitioning coefficients (logKO/W) could be used to predict relative differences in adsorption of butanol (logKO/W = 0.8) and ethanol (logKO/W = −0.26) on L-493,[43] and from this work appears that acetone (logKO/W = −0.16) likewise follows a similar trend. Overall, butanol, acetone, and ethanol were effectively adsorbed from all solutions; however, competitive effects may have reduced the adsorption of each in multicomponent mixtures but the changes observed were not statistically significant. This lack of selectivity was not unexpected as nonspecific hydrophobic interactions are responsible for the adsorption all three solvents on L-493. Thus, although butanol recovery was the principal objective of this study, the simultaneous recovery of acetone and ethanol by EBA should likewise be expected (albeit to a lesser extent), and must accordingly be accounted for. It is worth noting that glucose, the most abundant species in the bioreactor, has previously been shown to not adsorb appreciably to L-493.44 Here, we further found its presence to insignificantly impact butanol adsorption (see Supporting Information). Similarly, as its surface is not functionalized (i.e., it is not an ion exchange resin), we have also previously found that L-493 does not significantly interact with any ionic species in the medium, including both acetate and butyrate,[44] as well as inorganic nutrient salts (unpublished data). Thus, L-493 has been found to behave rather inertly with respect to all but the solvent products in the fermentation media, meaning that its presence should not be expected to inhibit growth or productivity.

Table 1. Freundlich Adsorption Isotherm Model Parameters for Acetone-Butanol-Ethanol (ABE) Fermentation Products and Dowex Optipore L-493
SolutionSpeciesk (mmol/kg)nSource
Single componentn-Butanol362 ± 252.00 ± 0.06This study
n-Butanol446 ± 1152.22 ± 0.26Nielsen et al.[43]
Acetone66 ± 111.46 ± 0.07This study
Ethanol18 ± 51.26 ± 0.09This study
Ethanol23 ± 121.25 ± 0.29Nielsen et al.[43]
Tertiary mixturen-Butanol328 ± 212.03 ± 0.06This study
Acetone55 ± 191.82 ± 0.26This study
Ethanol17 ± 51.60 ± 0.20This study

Development and characterization of an EBA process

A series of model characterizations were next performed to analyze the impact of select parameters of postulated importance to the EBA design. For instance, as seen in Figure 2A, butanol was removed from solution more rapidly as recirculation rates (in terms of superficial velocity) through the column were increased. Therefore, in practice, recirculation rates through the EBA column should be sufficiently high so as to suppress butanol accumulation during its peak production. Initial rates of butanol uptake were estimated as 5.6 ± 0.2, 12.6 ± 0.4, 19.9 ± 3.4, and 41.1 ± 4.4 g/L h for superficial velocities of 2.1, 5.7, 9.5, and 18.5, respectively, all of which are well above the typical volumetric productivity C. acetobutylicum ATCC 824. Moreover, as a strong linear correlation between superficial velocity and rates of butanol uptake was observed (R2 = 0.997; see Supporting Information), this implies that bulk transport and not intraparticle diffusion limited the kinetic performance of the EBA process over the range of conditions examined. Note, however, that at further higher recirculation rates this relationship is ultimately expected to fold as intraparticle diffusion then becomes rate limiting. Whereas higher recirculation enables faster butanol uptake, excessive pumping incurs higher utility costs and can impose detrimental shear stresses against the culture[50] and should thus be avoided. Therefore, the midpoint circulation rate of 9.5 cm/min was selected for all subsequent studies.

image

Figure 2. Investigating relevant operating parameters and performance of the EBA design through a series of cell-free experiments.

(A) Butanol removal from the bioreactor by circulation through an EBA column at superficial velocities of rates (in cm/min) of: 1.0 (solid squares), 2.7 (solid circles), 4.7 (open squares), and 8.9 (open circles). (B) Butanol removal from the bioreactor using EBA columns containing L-493 at an adsorbent fraction (in g/L) of: 26.7 (solid circles), 53.3 (open squares), and 80 (open diamonds). (C) Bed expansion as a function of flow rate for EBA columns containing L-493 at an adsorbent fraction (in g/L) of: 26.7 (solid circles), 53.3 (open squares), and 80 (open diamonds). (D) Accumulation of butanol in a bioreactor when added exogenously at a constant rate of 0.37 g/L h without (solid line) and with EBA operation (solid line with solid circles). Error bars represent standard error from triplicate measurements.

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The extent of butanol removal, meanwhile, is controlled by the net adsorption capacity of the adsorbent or in this case the mass of the adsorbent bed used in the EBA column. As seen in Figure 2B, increasing the adsorbent fraction from 26.6 to 53.3, and then to 80 g/L, the residual butanol concentration remaining in bioreactor at steady-state dropped from 10.8 ± 0.1 to 9.4 ± 0.1, and finally to 8.0 ± 0.2 g/L. Meanwhile, specific loadings of butanol on L-493 of approximately 4,340, 4,060, and 3,780 mmol/kg, respectively, were achieved at steady state; which strongly agree with the predicted and experimental equilibrium isotherms (see Table 1 and Supporting Information). Note that in all cases the butanol concentration in the bioreactor was effectively reduced from a titer above its inhibitory threshold (∼14 g/L) to a level below this mark. Whereas using a larger adsorbent bed increases the extent by which the aqueous accumulation of inhibitory products can be reduced, practical trade-offs also exist and should be considered. For example, in addition to increased cost, increasing the adsorbent fraction reduces the headspace available for expansion (assuming a fixed column volume) and thus suitably limits the maximum possible superficial velocity through the column (i.e., circulation rate). As seen in Figure 2C, bed height expansion as a function of superficial velocity of the circulating fluid was found to be well-modeled in this study using a form of the Richardson-Zaki equation (Eq. (3)), with best-fit estimates of the terminal settling velocity (ut) and the Richardson-Zaki exponent (n) found to be 56.7 cm/min and 4.38, respectively. Relative bed expansion was largely insensitive to the absolute mass of adsorbent used, indicating that the entire bed was effectively fluidized under all conditions studied. With respect to the EBA process developed in this study, at adsorbent fractions of 26.6, 40, and 53.3 g/L, for example, the headspace available in the EBA column represented 86.7%, 80%, and 73.4% of its total volume and would accommodate maximum height increases of 7.5-, 5-, and 3.8-fold, respectively. Thus, whereas the threefold maximum increase in expanded bed height (H) experienced in Figure 2C was readily accommodated in the present EBA design, careful and concurrent consideration of bed size and recirculation rate must be made to ensure that excessive expansion does not result in bed packing at the effluent (i.e., top) of the column.

The ability of the proposed design to simultaneously remove butanol from the bioreactor as it is produced was at last assessed through simulation. Butanol was continuously added to a well-mixed bioreactor (originally containing water) at a constant rate of 0.37 g/L h to approximate the wild-type production rate of C. acetobutylicum ATCC 824.22 The bioreactor contents were continuously recirculated through an EBA column containing 33 g/L L-493 at a constant superficial velocity of 9.5 cm/min. As seen in Figure 2D, butanol accumulation in the aqueous phase was effectively reduced via rapid adsorption onto L-493. In the control, the would-be inhibitory butanol threshold of 13 ± 0.1 g/L was reached in approximately 36 h, whereas with EBA the aqueous butanol levels reached a maximum of only 5.2 ± 0.1 g/L.

Of course, for EBA to constitute an effective strategy it must also be possible to efficiently desorb butanol from the resin and ultimately recover it. This process simultaneously results in regeneration of the resin and EBA column for their subsequent reuse, minimizing process costs. We have previously shown that regeneration of L-493 is possible by heating it to 100°C and collecting the resultant vapors.[44] Recovery of up to 85% of adsorbed butanol was possible by this approach with almost no deviation in adsorption behavior. However, there was concern that the repeated heating and cooling of a polymeric resin could prove detrimental in the long-term. Thus, here we have alternatively investigated the prospect of vacuum regeneration of L-493 at ambient temperatures, as described above. Table 2 presents the results from a series of three consecutive adsorption–desorption cycles, wherein it can be seen that butanol was effectively desorbed and collected. In most cases, two liquid phases were present in the condensed fraction as the butanol content exceeded its solubility limit in water (∼73 g/L at 25oC[51]), a feature that may further aid in downstream processing. In total, the butanol recovered by vacuum regeneration averaged about 81 ± 10% of that, which was originally adsorbed, a mark that compares well with thermal regeneration. However, noting that all final fractions collected still contained appreciable levels of butanol, it is expected that further optimization of this regeneration protocol to include extended periods of vacuum application will only add to this mark. Finally, an additional benefit of note here is that the resin was never required to be removed from the EBA column throughout the experiment, a feature that bodes well for overall process modularity.

Table 2. Comparing Butanol Recovery via Vacuum Regeneration of L-493 Following Three Repeated Adsorption–Desorption Cycles
CycleC0 (g/L)Ce (g/L)Butanol Adsorbed (g)FractionCtrap (g/L)Vtrap (mL)Butanol Trap (g)Butanol Recovered (%)
  1. Up to six fractions of condensed vapor products were collected each cycle to monitor the progress of the desorption process. Ce is the butanol concentration in the aqueous phase at equilibrium, Ctrap is the mean concentration of all liquid phases collected in the condensate trap, Vtrap is the volume of condensate collected in the trap, and all other parameters are as defined in the text.

115.0 ± 0.110.6 ± 0.14.7 ± 0.2197.7 ± 0.48.5 ± 0.20.83 ± 0.0278.9 ± 1.3
293.7 ± 0.48.0 ± 0.20.75 ± 0.02
391.9 ± 0.410.0 ± 0.20.92 ± 0.02
477.8 ± 0.48.5 ± 0.20.66 ± 0.02
565.9 ± 0.48.0 ± 0.20.53 ± 0.01
6
Total85.8 ± 0.543.0 ± 1.03.69 ± 0.09
214.2 ± 0.19.6 ± 0.14.6 ± 0.2179.5 ± 0.48.5 ± 0.20.68 ± 0.0272.5 ± 1.4
287.7 ± 0.48.0 ± 0.20.70 ± 0.02
382.8 ± 0.47.0 ± 0.20.58 ± 0.02
464.0 ± 0.47.0 ± 0.20.45 ± 0.01
583.2 ± 0.47.0 ± 0.20.58 ± 0.02
663.2 ± 0.45.5 ± 0.20.35 ± 0.01
Total77.5 ± 0.643.0 ± 1.23.33 ± 0.10
313.2 ± 0.19.6 ± 0.13.6 ± 0.2189.0 ± 0.48.0 ± 0.20.71 ± 0.0292.4 ± 1.4
278.8 ± 0.48.0 ± 0.20.63 ± 0.02
384.4 ± 0.47.0 ± 0.20.59 ± 0.02
468.8 ± 0.47.0 ± 0.20.48 ± 0.01
579.6 ± 0.47.2 ± 0.20.57 ± 0.02
660.5 ± 0.45.5 ± 0.20.33 ± 0.01
Total77.8 ± 0.642.7 ± 1.23.32 ± 0.10
         

Traditional batch fermentation of C. acetobutylicum ATCC 824

To establish a baseline for comparison, a traditional batch fermentation of C. acetobutylicum ATCC 824 was first performed, with products allowed to accumulate to their maximum achievable levels. The collective results from the 72-h fermentation are shown in Figure 3 and summarized in Table 3. Following a brief (6–8 h) lag phase, the culture entered acidogenesis wherein production of acetic and butyric acids was predominant, reaching maximum titers of 3.4 ± 0.1 and 4.9 ± 0.1 g/L by 28.6 h, respectively. Solvent production began at approximately 24 h and peaked after 52 h as biomass accumulation began to slow (note: DCW reached a maximum concentration of 4.99 ± 0.05 g/L at an overall glucose yield of 0.045 ± 0.001 g/g). The butanol titer reached a maximum of 11.5 ± 0.2 g/L, approaching the reported toxicity limit of approximately 13 g/L.[5] The average (estimated between 24 and 52 h) volumetric productivity for butanol was 0.41 ± 0.02 g/L h, which compares well with prior reports of 0.32,[52] 0.35,[16] 0.37,[22] and 0.48[53] g/L h. Acetone and ethanol titers, meanwhile, reached up to 3.9 ± 0.4 and 2.7 ± 0.1 g/L, respectively, giving a total solvent titer 17.7 ± 0.7 g/L, which agrees well with prior reports.[54, 55] Note that whereas ethanol and acetone can contribute to overall solvent stress, as is typical of C. acetobutylicum ATCC 824 fermentations,[5] their accumulation did not approach their reported inhibitory thresholds (50–60 and 70 g/L, respectively),[5] leaving butanol as the primary stressor in this case. Thus, of the 140 g/L glucose initially added, only 77% was ultimately consumed, likely as a result of butanol inhibition which prevented its further and complete consumption. The yield of butanol in this case was found to be 0.11 ± 0.01 g/g with that of total solvents being 0.17 ± 0.01 g/g. These values notably compare well with those of Mermelstein et al. (calculated as 0.12 and 0.18 g/g, respectively), a study performed using the same media and otherwise similar fermentation conditions (i.e., temperature, pH, and agitation). However, relative to the established literature as a whole, these yields are generally low. For example, for batch fermentations of C. acetobutylicum ATCC 824 at pH 4.5, Li et al.[56] reported butanol and total solvent yields of 0.23 and 0.32 g/g, respectively, outputs more typical of Clostridium sp. and closer to the theoretical maximum yield of butanol on glucose of 0.41 g/g. Nevertheless, as the present objective was to demonstrate that EBA could be used to enhance butanol production by preventing its accumulation to inhibitory levels—a threshold that the maximum achievable butanol titers were here able to approach—the present conditions were deemed suitable for this purpose. Further optimization of the fermentation and overall process conditions will surely be key to achieving higher and more representative yields and maximizing the performance.

image

Figure 3. C. acetobutylicum ATCC 824 control fermentation (i.e., without EBA).

Upper: DCW (open squares) and glucose (solid squares) concentrations. Middle: Concentrations of butanol (open diamonds), acetone (open circles), and ethanol (open inverse triangles). Lower: Concentrations of acetic acid (open stars) and butyric acid (left triangle) as well as pH (solid line). Error bars represent standard error from triplicate measurements.

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Table 3. Comparing the Performance of C. acetobutylicum Fermentations Operated in Batch Mode and Fed-Batch with In Situ Product Recovery by Expanded Bed Adsorption
Operating ModeAqueous/Net Accumulation* (g/L)Productivity (g/L h)Glucose Yield (g/g)
ButanolAcetoneEthanolTotal SolventsButanolTotal SolventsButanolTotal Solvents
  1. “Fed Batch + EBA experiments” only.

Batch11.5 ± 0.23.9 ± 0.42.7 ± 0.117.7 ± 0.70.41 ± 0.020.63 ± 0.030.11 ± 0.010.17 ± 0.01
Fed Batch + EBA12.1 ± 0.1/27.1 ± 0.28.2 ± 0.1/10.7 ± 0.12.5 ± 0.1/3.1 ± 0.122.8 ± 0.3/40.7 ± 0.40.48 ± 0.020.72 ± 0.030.19 ± 0.020.28 ± 0.02

EBA for in situ product recovery from a C. acetobutylicum ATCC 824 fermentation

The EBA column was at last integrated with a C. acetobutylicum ATCC 824 fermentation and evaluated for its ability to enhance butanol production. The collective results are depicted in Figure 4 and summarized in Table 3. Following a lag phase, cell growth and acidogenesis began after approximately 15 h. Once again, pH adjustment was used as a strategy to encourage the shift to solventogenesis, although this time it was accordingly performed after approximately 19 h. Acetic and butyric acids accumulated to similar maximum levels relative to the control fermentation (2.4 ± 0.2 and 4.5 ± 0.1 g/L, respectively). Following the appearance of butanol in the medium (first detected at 33.5 h) circulation through the EBA column was initiated. Note that circulation was not initiated earlier so as to minimize possible stress to the culture. This promptly resulted in a decrease in aqueous butanol concentration, from 10.1 ± 0.1 to 5.1 ± 0.1 g/L by 37 h. With an increased capacity for butanol accumulation in the system, the supplementation of additional glucose in a fed-batch manner was explored in an effort to increase its net production. At both 43.4 and 56.1 h, 30 g of additional glucose was added to the culture and quickly assimilated. This resulted in additional biomass production and, most importantly, the increased accumulation of butanol (together with acetone and ethanol). At the end of 72 h, butanol titers, which reached up to 12.1 ± 0.1 g/L, again ultimately approached their inhibitory threshold. Acetone and ethanol titers, meanwhile, peaked at 8.2 ± 0.1 and 2.5 ± 0.1 g/L, respectively. However, in addition to its accumulation in the aqueous media, butanol also accumulated significantly upon L-493, which, by use of the equilibrium isotherm (Table 1), was predicted to reach a final specific loading of 300 g/kg (or 3,850 mmol/kg). Thus, by accounting for all butanol accumulated in the system (i.e., aqueous and adsorbed), an effective titer of 27.1 ± 0.2 g/L was estimated by simple mass balance. Likewise, the adsorption of acetone and ethanol on L-493 were predicted as 48.5 and 10.0 g/kg (or 835 and 217 mmol/kg), respectively, boosting their effective titers to 10.7 ± 0.1 and 3.1 ± 0.1 g/L, and that of total solvent production to 40.7 ± 0.4 g/L. In this case, 90% of the total glucose added was consumed by the culture and the average (as determined between 22.8 and 72 h) butanol productivity was elevated to 0.48 ± 0.02 g/L h (17% greater). Meanwhile, butanol and total solvent yields on glucose were improved to 0.19 ± 0.02 and 0.28 ± 0.02 g/g, respectively; both of which were approximately 70% greater than achieved in conventional batch operation. DCW, however, reached a maximum of only 4.36 ± 0.07 g/L, or 13% less than the control. The growth reduction suggests that the EBA process may have negatively impacted culture fitness, perhaps as a result of its continuous pumping and recirculation through the column. Further optimization of the re-circulation rate may help to minimize such detrimental effects.

image

Figure 4. C. acetobutylicum ATCC 824 fermentation with EBA for in situ product recovery.

Upper: DCW (open squares) and glucose (solid squares) concentrations. Glucose additions indicated with gray arrows (note: samples taken after addition). Middle: Concentrations of butanol (open diamonds), acetone (open circles), and ethanol (open inverse triangles). Lower: Concentrations of acetic acid (open stars) and butyric acid (left triangle), as well as pH (solid line). Dashed vertical line indicates time at which EBA was initiated; continuous circulation through the EBA column resumed for the duration of the experiment. Error bars represent standard error from triplicate measurements.

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A comparison of actual solvent titers achieved in the control fermentation with effective titers achieved using EBA clearly demonstrates the impact of this strategy. As seen in Figure 5, total solvent production was increased by 2.3-fold compared to the control fermentation, whereas net butanol production was improved 2.2-fold. In contrast, in previous works we found that net butanol production could be improved by only approximately 1.8-fold when L-493 was added directly to a batch culture.[44] The net impact of EBA on butanol production was similar to that of previous reports for succinic acid, where 2.9- and 2-fold improvements were made in net production relative to batch and fed-batch operation, respectively.[40] Alternative strategies for the in situ recovery of butanol have also been explored including through solvent extraction. For example, Roffler et al.[57] reported that butanol productivity could be increased 1.7-fold (up to 1.0 g/L-h) in fed batch cultures by circulating the culture through a Karr reciprocating plate extraction column that contained oleyl alcohol as solvent. Meanwhile, to date, gas stripping has been found to provide perhaps the greatest enhancement to butanol and solvent production. For example, improvements in total solvent production and productivity of about 2.3- and 1.7-fold, respectively, have been reported in batch configurations with further and more significant improvements (up to 14- and 2.6-fold, respectively) possible through the adoption of continuous operating protocols.[16] It is likewise anticipated the further evolution of the proposed EBA design—for example, through incorporation of simulated moving-bed protocols to support semicontinuous or continuous operation[58]—will lead to additional and sizable gains in butanol production. Of course, whereas the focus of this study was predominantly directed toward in situ butanol recovery, the efficient recovery of ethanol and acetone will ultimately be of importance to the net viability and sustainability of the process. As the accumulation of these species to inhibitory levels is not the foremost concern (see above), this can be achieved through downstream processing also with the aid of adsorption (following butanol removal) or other suitable strategies (e.g., gas stripping, solvent extraction, distillation).

image

Figure 5. Comparing effective titers of acetone (circles), butanol (diamonds), ethanol (inverted triangle), and total solvent products (upright triangle) between control (solid shapes) and EBA (open shapes) fermentations. Error bars represent standard error from triplicate measurements.

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Assessing the prospects of biofouling

Biofouling is an important concern whenever biological and separation processes are interfaced. Here, for instance, fouling could reduce the surface area available for adsorption and thus the apparent capacity of the adsorbent. SEM imaging was performed on L-493 samples taken from the EBA column to provide a preliminary appraisal of biofouling. As seen from Figure 6, surface fouling by whole cells and cell debris was minimal on L-493. The low apparent density (∼2 ×·106 cells/cm2) and dispersed nature of the fouling suggests that the mechanism is predominantly random and not the result of active biofilm formation. Since it is known that Clostridium sp. can form biofilms on porous surfaces (e.g., brick and bone char[59, 60]) and that hydrophobic surfaces can be particularly susceptible to increased biofilm formation,[61] the limited degree of fouling observed here may have been due to the short duration of the experiment (72 h total with only 38.5 h of media contact with resin) and should be reinvestigated in subsequent, longer-term studies. The preliminary findings are, however, consistent with those of a prior report wherein the anion exchange adsorbent used to recover succinic acid from Actinobacillus succinogenes cultures via EBA was subjected to whole cell biofouling at a comparable level of approximately 1.85·106 cells/cm2 over the course of 126 h.[40] In that case, fouling was easily reversed through simple water washing, a strategy that should likewise be tested for its efficacy with L-493. Meanwhile, from Figure 6 it also appears that biofouling by noncellular debris—perhaps extracellular polymeric substances (EPS)[62]—may have occurred. EPS, which C. acetobutylicum ATCC 824 has been reported to produce as a prelude cell surface attachment and biofilm maturation,[63] may be indicating that significant whole cell fouling in longer fermentations is a distinct possibility. Further characterization, however, would be required to confirm the nature of the observed noncellular debris, as well as its influence on fouling, biofilm formation and, most importantly, its effect on butanol adsorption and EBA performance.

image

Figure 6. SEM images of fresh L-493 at ×500 magnification (A), and L-493 recovered from the EBA column following fermentation magnified at ×500 (B), ×1,000 (C), and ×2,500 (D).

Asterisk and “+” symbols included as points of reference. Inset rectangles show approximate regions imaged at next magnification level, as appropriate.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

These works provide the first demonstration of concept of the use of EBA for the in situ removal of butanol from C. acetobutylicum ATCC 824 fermentations. With the demonstrated ability to support a greater than twofold improvement in butanol and total solvent production, this strategy appears promising. However, further optimization of both the bioreactor conditions and expanded bed adsorption column design will be required before this approach can reach its full potential. Furthermore, future demonstration of its robustness in longer-term studies as well as the development of semicontinuous and/or continuous operating protocols will help to assess its practicality and suitability for industrial biofuel production.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

This work was supported by the U.S. Department of Energy, Office of ARPA-E (Award No. DE-AR0000011) and the National Science Foundation (Award No. CBET-1067684).

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  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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