Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping

We examined the effect of gas-stripping on the in situ removal of acetone, butanol, and ethanol (ABE) from batch reactor fermentation broth. The mutant strain (Clostridium beijerinckii BA101) was not affected adversely by gas stripping. The presence of cells in the fermentation broth affected the selectivities of ABE. A considerable improvement in the productivity and yield was recorded in this work in comparison with the non-integrated process. In an integrated process of ABE fermentation-recovery using C. beijerinckii BA101, ABE productivities and yield were improved up to 200 and 118%, respectively, as compared to control batch fermentation data. In a batch reactor C. beijerinckii BA101 utilized 45.4 g glucose l^–1 and produced 17.7 g total ABE l^–1, while in the integrated process it utilized 161.7 g glucose l^–1 and produced total ABE of 75.9 g l^–1. In the integrated process, acids were completely converted to solvents when compared to the non-integrated process (batch fermentation) which contained residual acids at the end of fermentation. In situ removal of ABE by gas stripping has been reported to be one of the most important techniques of solvent removal. During these studies we were able to maintain the ABE concentration in the fermentation broth below toxic levels.     

Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19

Conventional acetone–butanol–ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L^?1 of ABE (17.6 g L^?1 butanol, 10.5 g L^?1 ethanol, and 4.4 g L^?1 acetone) from 85.2 g L^?1 glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell‐recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L^?1 h^?1, respectively, could be achieved at the dilution rate of 0.85 h^?1. Further cell recycling experiments were carried out with controlled cell‐bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h^?1 with the bleeding rate of 0.04 h^?1. Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L^?1 h^?1, and the yields of 0.17 and 0.34 g g^?1, respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known‐processes. Biotechnol. Bioeng. 2013; 110: 1646–1653. © 2013 Wiley Periodicals, Inc.     

Butanol production from corn fiber xylan using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L x h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L x h). CFAX was hydrolyzed by xylan-hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.     

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     

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.     

Enhancement of ABE fermentation through regulation of ammonium acetate and D–xylose uptake from acid-pretreated corncobs

Clostridium acetobutylicum TISTR 1462 and Clostridium beijerinckii TISTR 1461 were chosen to optimize acetone–butanol–ethanol (ABE) fermentation by using glucose as a carbon source. The enhancement in its productivity by adding various concentrations of ammonium acetate was studied. Then, the variation of glucose/xylose ratios in the pre-grown medium was investigated. The results showed that both increased ammonium acetate in the production medium and D–xylose in the pre-grown medium could produce more ABE. With these conditions, using corncob hydrolysate as a substrate, 20.58 g/L ABE was produced from C. beijerinckii TISTR 1461 with 0.44 g/L/h and 0.45 of ABE productivity and yield, respectively.     

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol‐butanol‐ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1083–1088, 2013     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Acetone–Butanol–Ethanol Production by Clostridium acetobutylicum ATCC 824 Using Sago Pith Residues Hydrolysate

Sago pith residues (58 % starch, 23 % cellulose, 9.2 % hemicellulose, and 4 % lignin) are one of the abundant lignocellulosic residues generated after starch extraction process in sago mill. In this study, fermentable sugars from enzymatic hydrolysis of sago pith residues were converted to acetone–butanol–ethanol (ABE) by Clostridium acetobutylicum ATCC 824. With an initial concentration of 30 g/L of concentrated sago pith residues hydrolysate containing 23 g/L of glucose and 4.58 g/L of cellobiose, 4.22?±?0.17 g/L of ABE were produced after 72 h of fermentation with yield and productivity of 0.20 g/g glucose and 0.06 g/L/h, respectively. Results are in agreement when synthetic glucose was used as a carbon source. Increasing sago pith residue hydrolysate to 50 g/L (containing 40 g/L glucose) and supplementing with 0.5 g/L yeast extract, approximately 8.84?±?0.20 g/L of ABE (5.41?±?0.10 g/L of butanol) were produced with productivity and yield of 0.12 g/L/h and 0.30 g/g glucose respectively, providing a 52 % improvement.     

Production of acetone butanol (AB) from liquefied corn starch,a commercial substrate,using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> coupled with product recovery by gas stripping

A potential industrial substrate (liquefied corn starch; LCS) has been employed for successful acetone butanol ethanol (ABE) production. Fermentation of LCS (60 g l⁻¹) in a batch process resulted in the production of 18.4 g l⁻¹ ABE, comparable to glucose: yeast extract based medium (control experiment, 18.6 g l⁻¹ ABE). A batch fermentation of LCS integrated with product recovery resulted in 92% utilization of sugars present in the feed. When ABE was recovered by gas stripping (to relieve inhibition) from the fed-batch reactor fed with saccharified liquefied cornstarch (SLCS), 81.3 g l⁻¹ ABE was produced compared to 18.6 g l⁻¹ (control). In this integrated system, 225.8 g l⁻¹ SLCS sugar (487 % of control) was consumed. In the absence of product removal, it is not possible for C. beijerinckii BA101 to utilize more than 46 g l⁻¹ glucose. A combination of fermentation of this novel substrate (LCS) to butanol together with product recovery by gas stripping may economically benefit this fermentation. Mention of trade names of commercial products in this article/publication is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Improving performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation

The effect of factors such as gas recycle rate, bubble size, presence of acetone, and ethanol in the solution/broth were investigated in order to remove butanol from model solution or fermentation broth (also called acetone butanol ethanol or ABE or solvents). Butanol (8 g L^–1, model solution, Fig. 2) stripping rate was found to be proportional to the gas recycle rate. In the bubble size range attempted (<0.5 and 0.5–5.0 mm), the bubble size did not have any effect on butanol removal rate (Fig. 3, model solution). In Clostridium beijerinckii fermentation, ABE productivity was reduced from 0.47 g L^–1 h^–1 to 0.25 g L^–1 h^–1 when smaller (<0.5 mm) bubble size was used to remove ABE (Fig. 4, results reported as butanol/ABE concentration). The productivity was reduced as a result of addition of an excessive amount of antifoam used to inhibit the production of foam caused by the smaller bubbles. This suggested that the fermentation was negatively affected by antifoam.Mention of trade names of commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Microbial production of a biofuel (acetone–butanol–ethanol) in a continuous bioreactor: impact of bleed and simultaneous product removal

Acetone butanol ethanol (ABE) was produced in an integrated continuous one-stage fermentation and gas stripping product recovery system using Clostridium beijerinckii BA101 and fermentation gases (CO₂ and H₂). In this system, the bioreactor was fed with a concentrated sugar solution (250–500 g L^?1 glucose). The bioreactor was bled semi-continuously to avoid accumulation of inhibitory chemicals and products. The continuous system was operated for 504 h (21 days) after which the fermentation was intentionally terminated. The bioreactor produced 461.3 g ABE from 1,125.0 g total sugar in 1 L culture volume as compared to a control batch process in which 18.4 g ABE was produced from 47.3 g sugar. These results demonstrate that ABE fermentation can be operated in an integrated continuous one-stage fermentation and product recovery system for a long period of time, if butanol and other microbial metabolites in the bioreactor are kept below threshold of toxicity.     

Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce

SO₂–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h⁻¹. The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h⁻¹. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.     

High-efficiency acetone-butanol-ethanol production and recovery in non-strict anaerobic gas-stripping fed-batch fermentation

Conventional acetone-butanol-ethanol (ABE) fermentation coupled with gas stripping is conducted under strict anaerobic conditions. In this work, a fed-batch ABE fermentation integrated with gas stripping (FAFIGS) system using a non-strict anaerobic butanol-producing symbiotic system, TSH06, was investigated for the efficient production of butanol. To save energy and keep a high gas-stripping efficiency, the integrated fermentation was conducted by adjusting the butanol recovery rate. The gas-stripping efficiency increased when the butanol concentration increased from 6 to 12 g/L. However, in consideration of the butanol toxicity to TSH06, 8 g/L butanol was the optimal concentration for this FAFIGS process. A model for describing the relationship between the butanol recovery rate and the gas flow rate was developed, and the model was subsequently applied to adjust the butanol recovery rate during the FAFIGS process. In the integrated system under non-strict anaerobic condition, relatively stable butanol concentrations of 7 to 9 g/L were achieved by controlling the gas flow rate which varied between 1.6 and 3.5 vvm based on the changing butanol productivity. 185.65 g/L of butanol (267.15 g/L of ABE) was produced in 288 h with a butanol recovery ratio of 97.36%. The overall yield and productivity of butanol were 0.23 g/g and 0.64 g/L/h, respectively. This study demonstrated the feasibility of using FAFIGS under non-strict anaerobic conditions with TSH06. This work is helpful in characterizing the butanol anabolism performance of TSH06 and provides a simple and efficient scheme for butanol production.

     

Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover

As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hydroscopicity. However, solvent production appeared limited by butanol toxicity. The strain of Clostridium acetobutylicum was subjected to mutation by mutagen of N-methyl-N'-nitro-N-nitrosoguanidine for 0.5?h. Screening of mutants was done according to the individual resistance to butanol. A selected butanol-resistant mutant, strain 206, produced 50?% higher solvent concentrations than the wild-type strain when 60?g glucose/l was employed as substrate. The strain was also able to produce solvents of 23.47?g/l in 80?g/l glucose P2 medium after 70?h fermentation, including 5.41?g acetone/l, 15.05?g butanol/l and 3.02?g ethanol/l, resulting in an ABE yield and productivity of 0.32?g/g and 0.34?g/(l?h). Subsequently, Acetone-butanol-ethanol (ABE) production from enzymatic hydrolysate of NaOH-pretreated corn stover was investigated in this study. An ABE yield of 0.41 and a productivity of 0.21?g/(l?h) was obtained, compared to the yield of 0.33 and the productivity of 0.20?g/(l?h) in the control medium containing 52.47 mixed sugars. However, it is important to note that although strain 206 was able to utilize all the glucose rapidly in the hydrolysate, only 32.9?% xylose in the hydrolysate was used after fermentation stopped compared to 91.4?% xylose in the control medium. Strain 206 was shown to be a robust strain for ABE production from lignocellulosic materials and has a great potential for industrial application.     

Applied in situ product recovery in ABE fermentation

The production of biobutanol is hindered by the product's toxicity to the bacteria, which limits the productivity of the process. In situ product recovery of butanol can improve the productivity by removing the source of inhibition. This paper reviews in situ product recovery techniques applied to the acetone butanol ethanol fermentation in a stirred tank reactor. Methods of in situ recovery include gas stripping, vacuum fermentation, pervaporation, liquid–liquid extraction, perstraction, and adsorption, all of which have been investigated for the acetone, butanol, and ethanol fermentation. All techniques have shown an improvement in substrate utilization, yield, productivity or both. Different fermentation modes favored different techniques. For batch processing gas stripping and pervaporation were most favorable, but in fed‐batch fermentations gas stripping and adsorption were most promising. During continuous processing perstraction appeared to offer the best improvement. The use of hybrid techniques can increase the final product concentration beyond that of single‐stage techniques. Therefore, the selection of an in situ product recovery technique would require comparable information on the energy demand and economics of the process. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:563–579, 2017     

Performance of batch, fed-batch, and continuous A-B-E fermentation with pH-control

Batch, fed-batch, and continuous A-B-E fermentations were conducted and compared with pH controlled at 4.5, the optimal range for solvent production. While the batch mode provides the highest solvent yield, the continuous mode was preferred in terms of butanol yield and productivity. The highest butanol yield and productivity found in the continuous fermentation at dilution rate of 0.1 h⁻¹ were 0.21 g-butanol/g-glucose and 0.81 g/L/h, respectively. In the continuous and fed-batch fermentation, the time needed for passing acidogenesis to solventogenesis was an intrinsic hindrance to higher butanol productivity. Therefore, a low dilution rate is suggested for the continuous A-B-E fermentation, while the fed-batch mode is not suggested for solvent production. While 3:6:1 ratio of acetone, butanol, and ethanol is commonly observed from A-B-E batch fermentation by Clostridium acetobutylicum when the pH is uncontrolled, up to 94% of the produced solvent was butanol in the chemostat with pH controlled at 4.5.     

Acetone-butanol-ethanol fermentation and isoflavone extraction using kudzu roots

The economics of Acetone-butanol-ethanol (ABE) fermentation is greatly affected by raw materials, and the use of readily available starchy materials from marginal farming lands could be a viable option for reducing costs. Kudzu, a rapidly growing perennial leguminous vine, has been planted on marginal farming land and widely distributed in Asia and America. This study investigated ABE fermentation by C. acetobutylicum ATCC 824 using kudzu roots and isoflavone extraction from kudzu fermentation residue (KFR). The kudzu roots could be used as a sole substrate for ABE fermentation without nutritional supplements. Batch culture containing 140 g kudzu/L produced 17.99 ± 1.08 g/L solvent (ABE), including 11.20 ± 0.79 g/L butanol, 5.54 ± 0.20 g/L acetone, and 1.15 ± 0.09 g/L ethanol, with a productivity of 0.19 g/(L/h) and a yield of 0.33 g solvent/g sugar after 96 h of fermentation. Isoflavone yield extracted from KFR was 1.90/100 g KFR, approximately 48% higher compared with that extracted from raw kudzu. A kinetic analysis of the extraction process showed that both the isoflavone yield and the extraction rate obtained from KFR were higher than the corresponding values obtained from raw kudzu. These results indicate that kudzu may provide a new potential raw material for ABE production and the process of ABE fermentation integrated with isoflavone extraction may provide a new way to reduce fermentable substrate costs.     

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone–butanol–ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.     

Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran

Wheat bran, a by-product of the wheat milling industry, consists mainly of hemicellulose, starch and protein. In this study, the hydrolysate of wheat bran pretreated with dilute sulfuric acid was used as a substrate to produce ABE (acetone, butanol and ethanol) using Clostridium beijerinckii ATCC 55025. The wheat bran hydrolysate contained 53.1 g/l total reducing sugars, including 21.3 g/l of glucose, 17.4 g/l of xylose and 10.6 g/l of arabinose. C. beijerinckii ATCC 55025 can utilize hexose and pentose simultaneously in the hydrolysate to produce ABE. After 72 h of fermentation, the total ABE in the system was 11.8 g/l, of which acetone, butanol and ethanol were 2.2, 8.8 and 0.8 g/l, respectively. The fermentation resulted in an ABE yield of 0.32 and productivity of 0.16 g l⁻¹ h⁻¹. This study suggests that wheat bran can be a potential renewable resource for ABE fermentation.