Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

Immobilized preparations for the biotransformation of daunomycinone

Summary Biotransformation of daunomycinone into 13-dihydrodaunomycinone was performed using immobilized cells, immobilized cell homogenate and immobilized enzymes, extract of the microorganism Streptomyces aureofaciens B-96. The whole cells and the homogenate were incorporated into a gelatine matrix by cross-linking with glutaraldehyde, while the enzyme extract was immobilized on modified bead cellulose. The highest level of conversion of daunomycinone into 13-dihydrodaunomycinone was achieved with the immobilized enzyme extract.     

Immobilized catalase: deactivation and reactor stability

The quasi-steady behavior of a continuous flow reactor in which hydrogen peroxide is decomposed by immobilized catalase is investigated. Under certain conditions, reactors involving such substrate-inhibited, self-poisoning reactions are susceptible to suddne failure and the reactor moves catastrophically from high- to low-conversion quasi-steady states. This exchange-of-steady-states phenomenon is ex-amined in the light of experimental evidence for the enzyme catalase from bovine liver. (c) 1993 John Wiley & Sons, Inc.     

Pervaporation for simultaneous product recovery in the butanol/isopropanol batch fermentation

Summary In the butanol/isopropanol batch fermentation the substrate conversion can be raised by simultaneous product recovery using pervaporation with silicon tubing as a membrane.This investigation was supported (in part) by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

An immobilized cell reactor with simultaneous product separation. I. Reactor design and analysis

A four-phase reactor-separator (gas, liquid, solid, and immobilized catalyst) is proposed for fermentations characterized by a volatile product and nonvolatile substrate.In this reactor, the biological catalyst is immobilized onto a solid column packing and contacted by the liquid containing the substrate.A gas phase is also moved through the column to strip the volatile product into the gas phase. The Immobilized Cell Reactor-Separator (ICRS) consists of two basic gas-liquid flow sections: a cocurrent "enricher" followed by a countercurrent-"stripper".In this article, an equilibrium stage model of the reactor is developed to determine the feasibility and important operational variables of such a reactor-separator. The ICRS concept is applied to the ethanol from whey lactose fermentation using some preliminary immobilized cell reactor performance data. A mathematical model for a steady-state population based on an adsorbed monolayer of cells is also developed for the reactor. The ICRS model demonstrated that the ICRS should give a significant increase in reactor productivity as compared to an identically sized Immobilized Cell Reactor (ICR) with no separation. The gas-phase separation of the product also allows fermentation of high inlet substrate concentrations. The model is used to determine the effects of reactor parameters on ICRS performance including temperature, pressure, gas flow rates, inlet substrate concentration, and degree of microbial product inhibition.     

Optimization of a stagewise biochemical reactor system with product feedback

A consecutive, first-order, irreversible, biochemical reaction, \documentclass{article}\pagestyle{empty}\begin{document}$ A{\textstyle{{k(\theta)} \over {{\rm Enzyme }1}}} \to B{\textstyle{{k(\theta)} \over {{\rm Enzyme 2}}}} \to C $\end{document}, taking place in a series of N reactors with product recycle is considered. A discrete version of the maximum principle is used to derive general equations necessary for maximizing the production of (1) the final product, C, by choosing the temperature or the pH value in each reactor, and (2) the intermediate product, B, by choosing the reactor volume. A numerical computation for a series of three reactors with recycle is illustrated. The effects of varying the recycle rates on the optimal state and decision variables are also presented.     

Genetically engineered Escherichia coli FBR5: Part II. Ethanol production from xylose and simultaneous product recovery

In these studies, concentrated xylose solution was fermented to ethanol using Escherichia coli FBR5 which can ferment both lignocellulosic sugars (hexoses and pentoses). E. coli FBR5 can produce 40–50 g L^?1 ethanol from 100 g L^?1 xylose in batch reactors. Increasing sugar concentration beyond this level results in the loss of sugar with the reactor effluent thus affecting the process yield adversely. In a nonintegrated system without simultaneous product removal more than 120 g L^?1 xylose was left unused of the 220 g L^?1 that was fed into the reactor. In contrast to this, application of simultaneous product removal by gas stripping was able to relieve product inhibition and the culture was able to use 216.6 g L^?1 xylose thus producing 140 g L^?1 (based on reactor volume) ethanol resulting in a product yield of 0.48. The product stream achieved an ethanol concentration up to 148.41 g L^?1. This process has potential for greatly improving the performance of E. coli FBR5 where the strain can ferment all the lignocellulosic sugars to ethanol and gas stripping can be applied to recover product. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Immobilized lipase-mediated acidolysis of butteroil with conjugated linoleic acid: batch reactor and packed bed reactor studies

Six commercial lipases, in either free or immobilized forms, were screened for their ability to catalyze acyl exchange between the triacylglycerols of butteroil (milkfat) and conjugated linoleic acid (CLA) in an organic solvent-free medium. Immobilized lipase preparations from Candida antarctica and Mucor miehei demonstrated the ability to increase the CLA content of the milk fat acylglycerols from the native value of 0.6 g/100 g fat to values which were at least an order of magnitude higher. Comparable increases were also obtained with a free enzyme from Candida rugosa.

In addition to the screening studies, the effects of the weight ratio of milkfat to CLA on the product distribution and of the water content on the kinetics and maximum extent of this acidolysis reaction were systematically investigated in a batch reactor: The fatty acids liberated from the butteroil triacylglycerols were primarily short chain fatty acids, especially butyric and caproic acids.

Modified butteroils were also produced via acidolysis of butteroil with CLA in a packed bed reactor containing an immobilized lipase preparation from C. antarctica. Significant enrichment of the butteroil in CLA residues was accomplished at reactor space times (fluid residence times) of 2–4 h at 40–60°C. Under these conditions, approximately 80–90% of the free CLA fed to the reactor is (inter)esterified.     

Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures

Four immobilized forms of glucose oxidase (GOD) were used for biotransformation removal of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 °C) in comparison with those of free GOD. The decrease of V_max values after immobilization was observed, from 256.8 ± 7.0 μmol min⁻¹ mg_GOD⁻¹ for free enzyme to 63.8 ± 4.2 μmol min⁻¹ mg_GOD⁻¹ for GOD-ConA-TBC and 45 ± 2.7 μmol min⁻¹ mg_GOD⁻¹ for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.8–15.6% of its initial amount The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose).     

Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37 °C using a 14-L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L. The bioreactor was connected in series with a condensation system and vacuum pump. Vacuum was applied continuously or intermittently with 1.5 h vacuum sessions separated by 4, 6, and 8 h intervals. A control ABE fermentation experiment was characterized by incomplete glucose utilization due to butanol toxicity to C. beijerinckii P260, while fermentation coupled with in situ recovery by both continuous and intermittent vacuum modes resulted in complete utilization of glucose, greater productivity, improved cell growth, and concentrated recovered ABE stream. These results demonstrate that vacuum technology can be applied to integrated ABE fermentation and recovery even though the boiling point of butanol is greater than that of water.     

Purification of bioethanol effluent in an UASB reactor system with simultaneous biogas formation

In this study, the prospect of using an Upflow Anaerobic Sludge Blanket (UASB) reactor for detoxification of process water derived from bioethanol production has been investigated. The bioethanol effluent (BEE) originated from wet oxidized wheat straw fermented by Saccharomyces cerevisiae and Thermoanaerobacter mathranii A3M4 to produce ethanol from glucose and xylose, respectively. In batch experiments the methane potential of BEE was determined to 529 mL-CH(4)/g-VS. In batch degradation experiments it was shown that the presence of BEE had a positive influence on the removal of the inhibitors 2-furoic acid, 4-hydroxyacetophenone, and acetovanillone as compared to conversion of the inhibitors as sole substrate in synthetic media. Furthermore, experiments were carried out treating BEE in a laboratory-scale UASB reactor. The results showed a Chemical Oxygen Demand (COD) removal of 80% (w/w) at an organic loading rate of 29 g-COD/(L. d). GC analysis of the lignocellulosic related potentially inhibitory compounds 2-furoic acid, vanillic acid, homovanillic acid, acetovanillone, syringic acid, acetosyringone, syringol, 4-hydroxybenzoic acid, and 4-hydroxybenzaldehyde showed that all of these compounds were removed from the BEE in the reactor. Implementation of a UASB purification step was found to be a promising approach to detoxify process water from bioethanol production allowing for recirculation of the process water and reduced production costs.     

Hydrolyzed polyacrylamide biotransformation in an up-flow anaerobic sludge blanket reactor system: key enzymes,functional microorganisms,and biodegradation mechanisms

Hydrolyzed polyacrylamide (HPAM) biotransformation in an up-flow anaerobic sludge blanket reactor including biodegradation performances, biodegradation mechanisms, key enzymes, and functional microorganisms was explored. Response surface methodology was applied to further improve HPAM degradation. The predicted degradation ratios of HPAM and COD_Cr were 46.2% and 83.4% under the optimal conditions. HPAM biodegradation ratio and total organic carbon removal ratio reached 40.5% and 38.9%. Total nitrogen concentration was dramatically decreased with the increasing fermentation time during the fermentation, while low ammonia nitrogen (NH₄⁺–N) and nitrite nitrogen (NO₂⁻–N) were generated. NH₄⁺–N and NO₂⁻–N increased slightly on the whole. Enzyme activity change was correlated with HPAM biodegradation. Dehydrogenase activity had a decline of 21.3–41.0%, and the minimum value occurred at 300 mg/L of HPAM. Urease activity was varied from 28.7 to 78.7% and the maximal inhibition ratio occurred at 200 mg/L of HPAM. Mechanisms for the biodegradation of HPAM were also explored by FT-IR, HPLC, and SEM. The results indicated that long-chain HPAM was broken into micromolecule compounds and the amide groups of HPAM were transformed into carboxyl groups. Based on the sequencing results on an Illumina MiSeq platform, Proteobacterias, Bacteroidetes, and Chloroflexi were turned out to be the critical microorganisms involved in HPAM degradation. This work lays a basis for HPAM-containing wastewater treatment and offers a support for water saving and emission reduction. It is of great significance to the sustainable development of oilfield.

     

High solid fed‐batch butanol fermentation with simultaneous product recovery: Part II—process integration

In these studies, liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed‐batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 g L^?1 SSB hydrolysis, a fed‐batch reactor with in situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 g L^?1 h^?1 and 0.36 were obtained, respectively. In the fed‐batch reactor fed with SSB hydrolyzates, these productivity and yield values were 0.44 g L^?1 h^?1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 g L^?1) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:967–972, 2018     

Association of microcystin-LR and its biotransformation product with a hepatic-cytosolic protein

Microcystin-LR (MCYST-LR), a cyclic peptide hepatotoxin, associates with high-molecular-weight, liver cytosolic components. Repetitive cycles of heat denaturation and pronase digestion released 80 ± 6% of the bound radiolabel from these components, parent toxin (22%), and two biotransformation products, with high-performance liquid chromatography (HPLC) retention times of 6.7 (52%) and 5.6 (13%) min. Both parent and the biotransformed (6.7 min) toxin appeared to be covalently bound to a monomeric protein of molecular weight 40,000 (protein plus radiolabeled toxin). Binding and biotransformation reactions were time- and temperature-dependent and did not require endogenous molecules <6,000 daltons. The binding appeared to be saturable with a maximum of 20 pmol MCYST-LR bound per mg protein. The binding protein(s) and biotransformation activity were present in rat liver, brain, kidney, heart, lung, small intestine, large intestine, testes, skeletal muscle, and to a lesser extent, in fat. Okadaic acid, a specific protein phosphatase inhibitor, showed a concentration-dependent inhibition of [³H]MCYST-LR binding to hepatic cytosol. The molecular weight and organ distribution of the binding protein(s), and inhibition of binding by okadaic acid were consistent with one of the binding sites being the catalytic subunit of protein phosphatase type 2A.