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Two-stage fermentation process for the production of calcium magnesium acetate and propionate road deicers
Enzyme and Microbial Technology 36 (2005) 953–959
Two-stage fermentation process for the production of calcium magnesium acetate and propionate road deicers Wenge Fu, Alexander P. Mathews ∗ Department of Civil Engineering, Kansas State University, Fiedler Hall, Manhattan, KS 66506, USA Received 19 May 2004; accepted 26 January 2005
Abstract Calcium magnesium acetate (CMA) and propionate (CMP) are environmentally benign deicing chemicals that can replace sodium chloride that is widely used on roads and highways at present for snow and ice control to provide safe driving conditions during winter. The price of CMA from petroleum-derived acetic acid is quite expensive. Anaerobic fermentations have not proven economical due to the low acid productivity and concentrations. A novel method for the production of CMA and CMP from lactose and whey permeate via a two-stage anoxic fermentation system, with calcium hydroxide for pH control is described in this paper. A homolactic bacterium Lactobacillus plantarum is used to convert lactose to calcium magnesium lactate (CML) in the first stage, and Propionicibacterium acidipropionici P200910 is used to convert CML to CMA and CMP in the second stage. In both stages, the conversion rates were ∼90% (w/w). Lactic acid productivity was 2.03 g/L/h in the first stage at a dilution ratio of 0.06 h−1 . Propionic and acetic acid yield was 1.79 g/L/h at a dilution rate of 0.05 h−1 . Calcium hydroxide addition did not significantly alter the overall yield of acids in either stage. However, the ratio of concentration of propionate to acetate in the final product changed from ∼3.0 when NaOH is used to ∼2.0 when lime is applied for pH control. After separation of the biomass, the liquid with a total concentration of 48–55 g/L of CMA and CMP can be processed to obtain a solid road deicer product. © 2005 Elsevier Inc. All rights reserved. Keywords: CMA; Propionic acid; Lactic acid; Lactose; Whey permeate
1. Introduction The application of deicing chemicals, principally sodium and calcium chlorides, is commonly practiced in the western countries to provide safe driving conditions in winter. Salt is beneficial in reducing storm related accidents during winter by 75% and reducing associated costs by 90% [1]. In the U.S. alone, about 20 million t/year of rock salt or sodium chloride is applied on roads and highways in winter [2]. In snow belt areas, roads and highways may receive more than 60 t of salt per kilometer during the winter season [3]. Such massive application of salt can cause long-term damages to the highway infrastructure, automobiles, and underground utilities from corrosion, and damage to the environment from contamination of soil, surface water and ground water. It is estimated that the average life of bridge decks is reduced by more than ∗
Corresponding author. Tel.: +1 1785 532 1582; fax: +1 1785 532 7717. E-mail address: [email protected] (A.P. Mathews).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.01.031
60% from salt induced corrosion [4]. While rock salt costs only about $40 t−1 , the associated highway infrastructure repair and replacement costs from accelerated corrosion is estimated to be $1200–$2400 t/year [5]. The application of salt increases automobile corrosion rate by about 15%, costing an estimated $16 billion in annual damage [6,20,21]. Salt accumulation in the soil alters soil properties, thereby impairing the normal growth of roadside vegetation. Calcium magnesium acetate (CMA) has been identified as one of the most viable substitute based on its deicing effectiveness, minimal environmental and corrosion related damages, and the potential for large-scale production [7]. CMA is currently being produced by reacting acetic acid with lime and is marketed at about $1100 t−1 . The cost of CMA is about 30 times that of salt, and hence its application has been limited so far to new bridges and environmentally sensitive areas. Acetic and propionic acids that are required for CMA/P production are also important feedstocks in the chemical
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industry. Commercial production of acetic acid is currently via acetaldehyde oxidation, methanol carboxylation, or natural gas oxidation. Production of these acids via fermentation using low value agricultural and industrial wastes such as molasses, whey permeate, beet pulp and bakery wastes has been studied by numerous researchers [8–12]. Whey permeate has BOD values as high as 60,000 mg/L, and the treatment and disposal of this waste is both capital and energy intensive. Hence, the utilization of whey permeate in the production of acetic and propionic acids via anaerobic fermentation has received considerable attention in the past (see, e.g., [11,13]). The commonly used bacteria for acetic and propionic acid fermentation from lactose in whey include Clostridium and Propionibacterium. Clostridium is an obligate anaerobe, and requires a strict anaerobic environment for growth. This is accomplished commonly by continuously passing pure CO2 or N2 through the fermentation medium during operation. The genus Propionibacterium are facultative anaerobes, and fermentations involving these bacteria are simpler, and do not require inert gas purge for the complete removal of oxygen. This is advantageous in reducing process costs. Propionibacterium can produce acetic and propionic acids in a single stage fermentation process from almost all sugars derived from low-grade biomass [12]. The conversion rate is low (<60%) because part of the carbon source is lost as carbon dioxide. The sugar in whey is mainly lactose, and it can be easily converted to lactic acid by Lactobacillus bacteria such as Lactobacillus plantarum. It has been reported that lactic acid is preferred over lactose as substrate by Propionibacterium [14,15,19]. Therefore, a two-stage fermentation process utilizing L. plantarum and Propionicibacterium acidipropionici can be envisaged for the conversion of lactose to acetic and propionic acids. In the first stage lactose would be converted to lactic acid by L. plantarum, and in the second stage acetic and propionic acids would be produced from lactic acid by P. acidipropionici. Batch fermentation studies have shown that the first stage homolactic fermentation process can be conducted at a high conversion efficiency of ∼0.9 and acid yield of ∼0.9 [16]. The second stage of operation can also be conducted at a high conversion rate and yield due to the minimal loss of carbon as CO2 . One of the problems associated with the anaerobic fermentation processes that have been reported for CMA production, is the high cost of NaOH used for pH control during fermentation and the subsequent need to add acid to reduce the pH to extract the organic acids. Lime was used to control pH in the two-stage fermentation process reported herein, since one of the objectives of this work is to minimize raw material costs in the production of the deicing salts. The use of lime can reduce the product deicer cost since lime is about a third the cost of sodium hydroxide. Moreover, since high product purity is not required for deicing applications, technical grade lime or dolomite is adequate. In this paper, a two-stage fermentation process using lime containing calcium and magnesium hydroxides is examined for pH
control and subsequent production of CMA and CMP. Comparative data are given for CMA/CMP production via batch and continuous fermentation with NaOH and lime for pH control.
2. Materials and methods 2.1. Microorganism and medium L. plantarum (ATCC 21028) used in the first stage of fermentation, was obtained from American Type Culture Collection, Rockville, MD. The culture medium used and the experimental protocol have been described elsewhere [16]. P. acidipropionici (P200910), used in the second stage was obtained from the Department of Food Science and Human Nutrition, Iowa State University, Ames, IA. The culture medium contained: glucose (AR grade): 20 g/L; trypticase peptone (BBL): 20 g/L; yeast extract: 10 g/L; K2 HPO4 : 0.5 g/L; KH2 PO4 : 0.5 g/L; Resazurin (0.1%): 1 ml; The chemicals were dissolved in one liter of distilled water under vigorous stirring conditions and boiled for ∼10 min to remove the dissolved oxygen by flushing with N2 . After the color of the medium had changed from blue-purple (completely oxidized) to rose-pink (half reduced), 0.5 g/L cysteine–HCl was added into the medium to completely reduce the O2 in the medium (colorless). The final pH of the medium was adjusted to 6.5–7 with 1N HCl or 1N NaOH solutions. The experimental medium for P. acidipropionici was the filtered broth from the first stage fermentation. This medium contained lactic acid at a concentration of ∼30–50 g/L. After adjustment of pH and lactic acid concentration additional nutrients were added and the medium was sterilized at 121 ◦ C for 30 min. The nutrients added were: trypticase peptone (BBL), 5 g/L; yeast extract, 5 g/L. The sterilized medium was fed to the fermenter for second stage fermentation using P. acidiproprionici. The lime powder used in this study was obtained from Vulcan Materials Company, Countryside, IL. The composition of the supplied lime in (% w/w) is: Fe2 O3 , 0.53%; Al2 O3 , 2.4%; CaO, 55.4%; MgO, 40.04%; SiO2 , 0.32%; and S, 0.013%. The mole ratio of Ca to Mg is ∼1:1.
3. Experimental methods The two-stage fermentation operation was initiated in batch mode, and changed to continuous mode when the residual substrate concentration dropped to ∼1–6 g/L. The two stages can be arranged to work jointly, in series, or separately. The fermenters used in the experiments have the same working volume, but the microorganisms have markedly different growth rates. Hence, the reactors needed to be operated at different flow rates if the two stages are to be run in series. In this study the two fermentation stages
W. Fu, A.P. Mathews / Enzyme and Microbial Technology 36 (2005) 953–959
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Fig. 1. Batch fermentation of lactose by L. plantarum (NaOH for pH control).
were run independently for convenience in scheduling monitoring, sampling and analysis events. Batch fermentation experiments were conducted under aseptic conditions in a 2 L fermenter (The Virtis OmniCulture Bench-Top Fermenter, The Virtis Co., Inc., Gardiner, NY), at the agitation speed of 150 RPM and controlled temperature of 35 ◦ C. Anoxic conditions were maintained during operation without any N2 sparging. The pH was maintained at desired values by using a pH controller (PHCN-410, Omega, Engineering, Inc., One Omega Drive, Box 4047, Stamford, CT 06907) with the automatic addition of 3N or 6N lime solution containing Ca(OH)2 and Mg(OH)2 at 1:1 mole ratio. Comparative studies were conducted using 12N NaOH solution also. Continuous fermentation experiments were carried out using the same procedures as noted above. Fermentation was turned to continuous mode at the end of batch operation by pumping feed medium in and withdrawing product
out simultaneously at the desired dilution rate. The inflow and outflow rates were kept at the same level using a computerized pump with the same size pump heads (Cole Parmer Co., Chicago, IL). The analytical methods used for determining cell, lactose, and lactic, acetic, and propionic acid concentrations have been previously reported [16].
4. Results and discussion 4.1. Batch fermentation for the production of calcium magnesium lactate (CML) The results from batch fermentation of lactose to lactic acid by L. plantarum are shown in Fig. 1, wherein pH was controlled using 12N NaOH, and Fig. 2, wherein pH was controlled using 3N lime solution. Since the solubility of lime is quite low and the supplied material also contained some
Fig. 2. Batch fermentation of lactose by L. plantarum (Ca(OH)2 for pH control).
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solid SiO2 , Fe2 O3 , etc., the delivery of lime solution in slurry form to the fermentation unit was quite difficult. Preparing the lime in 3–5% starch solution solved this problem. Starch or wheat flour was stirred in the lime solution and boiled to obtain a gel like semi-solid slurry, and this slurry was fed to the fermenter using a peristaltic pump. The normality of lime was increased to 6N for the continuous fermentation runs to minimize volume change due to base addition. In the case of 3N lime solution, the volume change is 13%, and for 6N lime solution the broth volume increase is reduced to 6.7%. The volume increase with 12N NaOH solution is 3.3%. The reactivity of CaO and MgO in the lime is greatly affected by pH. CaO can continue to dissolve rapidly up to pH ∼12, whereas MgO is essentially inert at pH > 6 [7]. If the fermentation pH is controlled at a pH ∼6, only calcium lactate will be produced. In the studies reported here, the pH was maintained at ∼5, and under these conditions both CaO and MgO can effectively neutralize the lactic acid produced and control the pH. The batch fermentation data shown in Figs. 1 and 2 indicate that lactose was almost completely consumed for both cases in about 40 h. The overall conversion rate is 97% (w/w) when NaOH was used for pH control. In the second case with lime for pH control, the lactose concentration decreased from 35.5 g/L to 0.63 g/L, in ∼43 h. The final lactic acid concentration was 28.2 g/L. Taking into account the volume change caused by lime solution addition (broth volume change from 1500 ml to 1700 ml), the overall conversion rate is ∼92%. This is slightly lower than that for pH control with NaOH. The acid yield in both cases was about 90%. After 43 h, the operation was changed from batch to continuous fermentation mode by pumping in medium with 34.5 g/L lactose, and simultaneously withdrawing the product broth at the dilution rate of 0.03 h−1 . As evident from Fig. 2, continuous operation with lime for pH control performed well. The substrate concentration was consistently reduced to low levels providing a high lactose conversion
rate. These results clearly show that lime solution can be used in lactose fermentation instead of NaOH without any significant adverse effects. 4.2. Continuous fermentation for CML production Continuous fermentation runs were made over an extended period using 12 N NaOH for pH control to determine the effects of dilution rate (D) on performance and acid productivity. NaOH was used for pH control for the sake of convenience since there are no significant differences in performance between NaOH and lime solution. Lactose, lactic acid, and cell concentrations are plotted for 7 days of operation at various dilution rates. Theoretically, the specific growth rate of bacterial cells should equal the dilution rate. For L. plantarum the maximum specific growth rate at pH 5 is 0.38 h−1 with lactose as the substrate [16]. Under the experimental conditions wherein the steady-state substrate concentration (S) was maintained at ∼3–10 g/L, the ratio of S/(S + Ks ) in Monod’s equation is ∼0.06–0.17 h−1 . Therefore, the actual specific growth rate is around 0.02–0.06 h−1 . The continuous fermentation data reported previously [17] is reproduced in Fig. 3. The data indicate stable operation at dilution rates 0.02, 0.04 and 0.06. When D was increased to 0.08 h−1 or 0.1 h−1 , “washout” conditions occurred. In these cases the substrate concentration in the fermenter began to increase and the product concentration decreased due to the decrease in cell density in the reactor. The results from continuous fermentation experiments are summarized Table 1. Lactic acid yield decreased slightly from 0.94 at D = 0.02 to 0.83 at D = 0.06, and the acid productivity increased from 0.62 to 2.03. In practice, using as high a D value as possible would be better in a two-stage operation to obtain high acid productivity. Any moderate amounts of lactose left from the first fermentation stage can be utilized by P. acidiproprionici in the second stage of operation. Alternatively, an intermediate storage vessel can be used for the reduction of residual lactose.
Fig. 3. Continuous fermentation of lactose by L. plantarum (reproduced with permission from World Scientific Publishers, 2001).
W. Fu, A.P. Mathews / Enzyme and Microbial Technology 36 (2005) 953–959
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Table 1 Batch and continuous fermentation of lactose by L. plantarum Dilution rate (L/h)
Lactic acid yield (w/w)
Productivity (g/L/h)
Conversion (%)
Feed (g/L)
Batch 0.02 0.04 0.06
0.88 0.94 0.86 0.83
0.48 0.62 1.35 2.03
100 97 98 94
33.8 30.8 38.4 38.4
4.3. Second stage batch fermentation for CMA/CMP production The CML broth collected from first stage was filtered to remove Lactobacillus bacteria and used as substrate in the second stage for CMA and CMP production using P. acidipropionici bacteria. Batch fermentation experiments were conducted to determine the effect of lime use for pH control. As shown in Fig. 4 cell growth and acid production occurs rapidly after an initial lag phase. Champagne et al. [18] have observed lower fermentative activity in whey fermentation by P. shermanii with NaOH as neutralizing agent compared to lime. At the same pH, fermentation rates were reported to be 20% higher when lime was used as the neutralizing agent. Since CML is converted to the salts CMA and CMP in the second stage of fermentation, the additional amount of lime required for neutralization and pH control is quite small. The lime requirement for the second stage was about 13% of the amount used in the first stage. The volume change caused by the addition of basic solution is less than 1% with 6N lime solution. The pH in both cases was initially maintained at 7. In the case of lime solution, at the end of batch fermentation, a small amount (<2 g/L) of CaCO3 solid slurry was formed. This is due to the reaction of lime with the CO2 produced in the fermentation process. The effect of pH on the accumulation of precipitates during CML fermentation with lime for pH control was studied by varying the pH. When pH was controlled at 7 and the carbonate precipitates were allowed to accumulate, the total precipitate concentration increased to ∼19.4 g/L. As pH
Fig. 5. Effect of pH on CaCO3 precipitation in the broth.
was decreased from 7 to 5.9, the concentration of participates decreased and at pH ∼5.9 the precipitates were completely dissolved. These results are shown in Fig. 5. In subsequent runs when pH was controlled between 5.9 and 6.2, there was no formation of CaCO3 precipitate in the broth. Thus, to avoid the formation of carbonate precipitates during CML fermentation, the pH must be controlled at less than ∼6.2. For P. acidipropionici, bacterial growth and metabolism, will not be significantly affected by maintaining a pH ∼6.0. Continuous fermentation experiments were conducted at different dilution rates to determine long-term operational stability of the system when lime is used for pH control. The results from operation over a period of 90 days are shown in
Fig. 4. Batch fermentation of lactic acid by P. acidipropionici P200910 (Ca(OH)2 for pH control).
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Fig. 6. Continuous fermentation of lactic acid by P. acidipropionici P200910 (Ca(OH)2 for pH control). Table 2 Continuous fermentation of calcium magnesium lactate broth from first stage using P. acidipropionici Dilution rate (L/h)
Feed (g/L)
Total acid yield (w/w)
Conversion (%)
Productivity (g/L/h)
P/A ratio (w/w)
0.02 0.024 0.04 0.05
39.6 42.2 42.4 42.4
0.95 0.91 0.92 0.94
92 90 86 86
0.748 0.85 1.51 1.79
2.0 2.2 2.0 2.1
Fig. 6. The broth used in the experiment was obtained from first the stage fermentation operation and the CML concentration was adjusted to a constant level of ∼40 g/L lactic acid before use. Fig. 6 shows operation at four different dilution rates. The system was operated at each dilution level until steady values of substrate and product concentrations were achieved. Typically, operations were conducted at a given dilution rate for more than 24 h (6–24 residence times) to attain uniform substrate and product concentration values. Thereafter, the dilution rate was incrementally changed. The fermentation system reached steady state after about 48 h at the dilution rates of 0.02 h−1 and 0.04 h−1 . For D = 0.05 h−1 , the system reached “washout” conditions and the cell and product concentrations began to decrease. When D was decreased to 0.024, the system reached steady operation again within about 48 h. The acid productivity and yields at various dilution rates are shown in Table 2. In all cases, the total acid yield is greater than 90%. The acid productivity ranged from 0.75 g/L/h to 1.8 g/L/h for dilution rates 0.02 h−1 to 0.05 h−1 . Using as high a dilution rate as possible below washout conditions would be better for obtaining high acid productivity values. Longterm operational stability was excellent at the dilution rates of 0.01 h−1 and 0.02 h−1 as seen in Fig. 6. The system was operated for more than 50 days at D = 0.01 h−1 and for 10 days at D = 0.02 h−1 at a CML conversion rate greater than 90%. The ratio of propionate to acetate (P/A in w/w) was ∼2.0–2.3 when pH was controlled using lime (see Table 2).
This is different from the value of ∼3.0 obtained when NaOH is used for pH control. This is probably due to the high concentrations of Ca2+ and Mg2+ in the broth that could potentially alter the metabolic pathway. The final product broth contained mainly CMA and CMP at a total concentration of 48–55 g/L. The bacterial cells can be separated by filtration and recycled or used as animal feed. The product CMA and CMP in the clear liquid then can be separated by multiple effect evaporation to obtain the deicer in solid form. Alternatively, the solution can be concentrated to ∼25–28% (w/w) and used as deicing agent in liquid form.
5. Conclusions A novel two-stage bioconversion process has been developed for the production of deicing salts calcium magnesium acetate (CMA) and calcium magnesium propionate (CMP) from inexpensive raw materials such as whey lactose and lime. In the first stage of fermentation lactose is converted to lactic acid by L. plantarum at 92% conversion and acid yield of 0.9. Ca(OH)2 and Mg(OH)2 were used instead of NaOH to neutralize the acids produced during the fermentation, with no adverse effects on cell growth and acid yield. In the second stage of fermentation, calcium magnesium lactate was converted to acetate and propionate salts at ∼91% conversion and acid yield of ∼0.9. While the overall yield of total acids did not change when lime was used for pH control instead of NaOH, the propionate to acetate ratio changed from
W. Fu, A.P. Mathews / Enzyme and Microbial Technology 36 (2005) 953–959
∼3.0 to ∼2.0 (w/w) in the final CMA and CMP products. The pH needs to be maintained below ∼6.0 in the fermenter to prevent the formation of carbonate precipitates. Continuous fermentation experiments were conducted for over 90 days with lime for pH control, and showed excellent long-term operational stability. The maximum total acid productivity in the second stage was 1.8 g/L/h. The liquid with total CMA and CMP concentration around 48–55 g/L can be evaporated by multiple effect evaporation to obtain the deicer in solid form.
Acknowledgments This material is based upon work supported by the National Science Foundation under Grant No. 9613273. We wish to express our sincere gratitude to Dr. Bonita A. Glatz in Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, for providing us with P. acidipropionici P200910 cultures.
References [1] Kobelt PS. Winter highway deicing benefits detailed. Public Works 1991;7:72–4. [2] Salt Institute [http://www.saltinstitute.org/]; 1997. [3] Amrhein C, Mosher PA, Strong JE, Pacheco PG. Heavy metals in the environment: trace metal solubility in soil and waters receiving deicing salts. J Environ Qual 1994;23:219–27. [4] Transportation Research Board. America’s highways: accelerating the search for innovation. Special Report 202. Washington, DC: National Research Council; 1984. [5] Transportation Research Board. Highway deicing: comparing salt and calcium magnesium acetate. Special Report 235. Washington, DC: National Research Council; 1991. [6] Rendahl B, Hedlund S. Influence of deicing salts on motor vehicle corrosion. Mater Perform 1991;5:42–4. [7] Federal Highway Administration (FHWA). Process development for production of calcium magnesium acetate (CMA). FHWA/RD82/145; 1983.
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[8] Anderson TM, Bodie EA, Goodman N, Schwartz RD. Inhibitory effect of autoclaving whey-based medium on propionic acid production by Propionibacterium shermanii. Appl Environ Microbiol 1986;51:427–8. [9] Bodie EA, Anderson TM, Goodman N, Schwartz RD. Propionic acid fermentation of ultra-high-temperature sterilized whey using mono- and mixed-cultures. Appl Microbiol Biotechnol 1987;25: 434–7. [10] Yang ST, Zhu H, Lewis VP, Tang IC. Calcium magnesium acetate (CMA) production from whey permeate: process and economic analysis. Resour Conserv Recycl 1992;7:181–200. [11] Colomban A, Roger L, Boyaval P. Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling. Biotechnol Bioeng 1993;42:1091–8. [12] Choi CH, Mathews AP. Continuous flow cell-recycle fermentation of biomass hydrolysates. In: Galindo E, Ramirez OT, editors. Advances in bioprocess engineering. Kluwer Academic Publishers; 1994. p. 75–80. [13] Yang ST, Huang Y. A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose. Biotechnol Bioeng 1995;45:379–86. [14] Begin A, Beaulieu Y, Goulet J, Castaigne F. Whey fermentation by Propionibacterium shermanii immobilized in different gels. Milchwissenschaft 1991;47:411–5. [15] Lewis VP, Yang ST. Continuous propionic acid fermentation by immobilized Propionibacterium acidipropionici in a novel packed-bed bioreactor. Biotechnol Bioeng 1992;40:465–74. [16] Fu W, Mathews AP. Lactic acid production from lactose by Lactobacillus plantarum: kinetic model and effects of pH, substrate and oxygen. Biochem Eng J 1999;3:163–70. [17] Mathews AP, Fu W. Fermentation kinetics in the production of lactic acid from high strength wastewaters. In: Hu X, Lok P, editors. Technology for a sustainable environment. World Scientific Publ; 2001. [18] Champagne CP, Cote CB, Goulet J. Whey fermentation by immobilized cells of Propionibacterium. J Appl Bacteriol 1989;66:175– 84. [19] Lewis VP, Yang ST. Propionic acid fermentation by Propionibacterium acidipropionici: effects of growth substrate. Appl Microbiol Biotechnol 1992;8:104–10. [20] Anonymous. Automotive corrosion by deicing salts. Mater Perform 1989;12:28. [21] McCrum RL. Calcium magnesium acetate and sodium chloride as highway deicing salts: a comparative study. Mater Perform 1989;12:23–8.
Two-stage fermentation process for the production of calcium magnesium acetate and propionate road deicers Wenge Fu, Alexander P. Mathews ∗ Department of Civil Engineering, Kansas State University, Fiedler Hall, Manhattan, KS 66506, USA Received 19 May 2004; accepted 26 January 2005
Abstract Calcium magnesium acetate (CMA) and propionate (CMP) are environmentally benign deicing chemicals that can replace sodium chloride that is widely used on roads and highways at present for snow and ice control to provide safe driving conditions during winter. The price of CMA from petroleum-derived acetic acid is quite expensive. Anaerobic fermentations have not proven economical due to the low acid productivity and concentrations. A novel method for the production of CMA and CMP from lactose and whey permeate via a two-stage anoxic fermentation system, with calcium hydroxide for pH control is described in this paper. A homolactic bacterium Lactobacillus plantarum is used to convert lactose to calcium magnesium lactate (CML) in the first stage, and Propionicibacterium acidipropionici P200910 is used to convert CML to CMA and CMP in the second stage. In both stages, the conversion rates were ∼90% (w/w). Lactic acid productivity was 2.03 g/L/h in the first stage at a dilution ratio of 0.06 h−1 . Propionic and acetic acid yield was 1.79 g/L/h at a dilution rate of 0.05 h−1 . Calcium hydroxide addition did not significantly alter the overall yield of acids in either stage. However, the ratio of concentration of propionate to acetate in the final product changed from ∼3.0 when NaOH is used to ∼2.0 when lime is applied for pH control. After separation of the biomass, the liquid with a total concentration of 48–55 g/L of CMA and CMP can be processed to obtain a solid road deicer product. © 2005 Elsevier Inc. All rights reserved. Keywords: CMA; Propionic acid; Lactic acid; Lactose; Whey permeate
1. Introduction The application of deicing chemicals, principally sodium and calcium chlorides, is commonly practiced in the western countries to provide safe driving conditions in winter. Salt is beneficial in reducing storm related accidents during winter by 75% and reducing associated costs by 90% [1]. In the U.S. alone, about 20 million t/year of rock salt or sodium chloride is applied on roads and highways in winter [2]. In snow belt areas, roads and highways may receive more than 60 t of salt per kilometer during the winter season [3]. Such massive application of salt can cause long-term damages to the highway infrastructure, automobiles, and underground utilities from corrosion, and damage to the environment from contamination of soil, surface water and ground water. It is estimated that the average life of bridge decks is reduced by more than ∗
Corresponding author. Tel.: +1 1785 532 1582; fax: +1 1785 532 7717. E-mail address: [email protected] (A.P. Mathews).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.01.031
60% from salt induced corrosion [4]. While rock salt costs only about $40 t−1 , the associated highway infrastructure repair and replacement costs from accelerated corrosion is estimated to be $1200–$2400 t/year [5]. The application of salt increases automobile corrosion rate by about 15%, costing an estimated $16 billion in annual damage [6,20,21]. Salt accumulation in the soil alters soil properties, thereby impairing the normal growth of roadside vegetation. Calcium magnesium acetate (CMA) has been identified as one of the most viable substitute based on its deicing effectiveness, minimal environmental and corrosion related damages, and the potential for large-scale production [7]. CMA is currently being produced by reacting acetic acid with lime and is marketed at about $1100 t−1 . The cost of CMA is about 30 times that of salt, and hence its application has been limited so far to new bridges and environmentally sensitive areas. Acetic and propionic acids that are required for CMA/P production are also important feedstocks in the chemical
954
W. Fu, A.P. Mathews / Enzyme and Microbial Technology 36 (2005) 953–959
industry. Commercial production of acetic acid is currently via acetaldehyde oxidation, methanol carboxylation, or natural gas oxidation. Production of these acids via fermentation using low value agricultural and industrial wastes such as molasses, whey permeate, beet pulp and bakery wastes has been studied by numerous researchers [8–12]. Whey permeate has BOD values as high as 60,000 mg/L, and the treatment and disposal of this waste is both capital and energy intensive. Hence, the utilization of whey permeate in the production of acetic and propionic acids via anaerobic fermentation has received considerable attention in the past (see, e.g., [11,13]). The commonly used bacteria for acetic and propionic acid fermentation from lactose in whey include Clostridium and Propionibacterium. Clostridium is an obligate anaerobe, and requires a strict anaerobic environment for growth. This is accomplished commonly by continuously passing pure CO2 or N2 through the fermentation medium during operation. The genus Propionibacterium are facultative anaerobes, and fermentations involving these bacteria are simpler, and do not require inert gas purge for the complete removal of oxygen. This is advantageous in reducing process costs. Propionibacterium can produce acetic and propionic acids in a single stage fermentation process from almost all sugars derived from low-grade biomass [12]. The conversion rate is low (<60%) because part of the carbon source is lost as carbon dioxide. The sugar in whey is mainly lactose, and it can be easily converted to lactic acid by Lactobacillus bacteria such as Lactobacillus plantarum. It has been reported that lactic acid is preferred over lactose as substrate by Propionibacterium [14,15,19]. Therefore, a two-stage fermentation process utilizing L. plantarum and Propionicibacterium acidipropionici can be envisaged for the conversion of lactose to acetic and propionic acids. In the first stage lactose would be converted to lactic acid by L. plantarum, and in the second stage acetic and propionic acids would be produced from lactic acid by P. acidipropionici. Batch fermentation studies have shown that the first stage homolactic fermentation process can be conducted at a high conversion efficiency of ∼0.9 and acid yield of ∼0.9 [16]. The second stage of operation can also be conducted at a high conversion rate and yield due to the minimal loss of carbon as CO2 . One of the problems associated with the anaerobic fermentation processes that have been reported for CMA production, is the high cost of NaOH used for pH control during fermentation and the subsequent need to add acid to reduce the pH to extract the organic acids. Lime was used to control pH in the two-stage fermentation process reported herein, since one of the objectives of this work is to minimize raw material costs in the production of the deicing salts. The use of lime can reduce the product deicer cost since lime is about a third the cost of sodium hydroxide. Moreover, since high product purity is not required for deicing applications, technical grade lime or dolomite is adequate. In this paper, a two-stage fermentation process using lime containing calcium and magnesium hydroxides is examined for pH
control and subsequent production of CMA and CMP. Comparative data are given for CMA/CMP production via batch and continuous fermentation with NaOH and lime for pH control.
2. Materials and methods 2.1. Microorganism and medium L. plantarum (ATCC 21028) used in the first stage of fermentation, was obtained from American Type Culture Collection, Rockville, MD. The culture medium used and the experimental protocol have been described elsewhere [16]. P. acidipropionici (P200910), used in the second stage was obtained from the Department of Food Science and Human Nutrition, Iowa State University, Ames, IA. The culture medium contained: glucose (AR grade): 20 g/L; trypticase peptone (BBL): 20 g/L; yeast extract: 10 g/L; K2 HPO4 : 0.5 g/L; KH2 PO4 : 0.5 g/L; Resazurin (0.1%): 1 ml; The chemicals were dissolved in one liter of distilled water under vigorous stirring conditions and boiled for ∼10 min to remove the dissolved oxygen by flushing with N2 . After the color of the medium had changed from blue-purple (completely oxidized) to rose-pink (half reduced), 0.5 g/L cysteine–HCl was added into the medium to completely reduce the O2 in the medium (colorless). The final pH of the medium was adjusted to 6.5–7 with 1N HCl or 1N NaOH solutions. The experimental medium for P. acidipropionici was the filtered broth from the first stage fermentation. This medium contained lactic acid at a concentration of ∼30–50 g/L. After adjustment of pH and lactic acid concentration additional nutrients were added and the medium was sterilized at 121 ◦ C for 30 min. The nutrients added were: trypticase peptone (BBL), 5 g/L; yeast extract, 5 g/L. The sterilized medium was fed to the fermenter for second stage fermentation using P. acidiproprionici. The lime powder used in this study was obtained from Vulcan Materials Company, Countryside, IL. The composition of the supplied lime in (% w/w) is: Fe2 O3 , 0.53%; Al2 O3 , 2.4%; CaO, 55.4%; MgO, 40.04%; SiO2 , 0.32%; and S, 0.013%. The mole ratio of Ca to Mg is ∼1:1.
3. Experimental methods The two-stage fermentation operation was initiated in batch mode, and changed to continuous mode when the residual substrate concentration dropped to ∼1–6 g/L. The two stages can be arranged to work jointly, in series, or separately. The fermenters used in the experiments have the same working volume, but the microorganisms have markedly different growth rates. Hence, the reactors needed to be operated at different flow rates if the two stages are to be run in series. In this study the two fermentation stages
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Fig. 1. Batch fermentation of lactose by L. plantarum (NaOH for pH control).
were run independently for convenience in scheduling monitoring, sampling and analysis events. Batch fermentation experiments were conducted under aseptic conditions in a 2 L fermenter (The Virtis OmniCulture Bench-Top Fermenter, The Virtis Co., Inc., Gardiner, NY), at the agitation speed of 150 RPM and controlled temperature of 35 ◦ C. Anoxic conditions were maintained during operation without any N2 sparging. The pH was maintained at desired values by using a pH controller (PHCN-410, Omega, Engineering, Inc., One Omega Drive, Box 4047, Stamford, CT 06907) with the automatic addition of 3N or 6N lime solution containing Ca(OH)2 and Mg(OH)2 at 1:1 mole ratio. Comparative studies were conducted using 12N NaOH solution also. Continuous fermentation experiments were carried out using the same procedures as noted above. Fermentation was turned to continuous mode at the end of batch operation by pumping feed medium in and withdrawing product
out simultaneously at the desired dilution rate. The inflow and outflow rates were kept at the same level using a computerized pump with the same size pump heads (Cole Parmer Co., Chicago, IL). The analytical methods used for determining cell, lactose, and lactic, acetic, and propionic acid concentrations have been previously reported [16].
4. Results and discussion 4.1. Batch fermentation for the production of calcium magnesium lactate (CML) The results from batch fermentation of lactose to lactic acid by L. plantarum are shown in Fig. 1, wherein pH was controlled using 12N NaOH, and Fig. 2, wherein pH was controlled using 3N lime solution. Since the solubility of lime is quite low and the supplied material also contained some
Fig. 2. Batch fermentation of lactose by L. plantarum (Ca(OH)2 for pH control).
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solid SiO2 , Fe2 O3 , etc., the delivery of lime solution in slurry form to the fermentation unit was quite difficult. Preparing the lime in 3–5% starch solution solved this problem. Starch or wheat flour was stirred in the lime solution and boiled to obtain a gel like semi-solid slurry, and this slurry was fed to the fermenter using a peristaltic pump. The normality of lime was increased to 6N for the continuous fermentation runs to minimize volume change due to base addition. In the case of 3N lime solution, the volume change is 13%, and for 6N lime solution the broth volume increase is reduced to 6.7%. The volume increase with 12N NaOH solution is 3.3%. The reactivity of CaO and MgO in the lime is greatly affected by pH. CaO can continue to dissolve rapidly up to pH ∼12, whereas MgO is essentially inert at pH > 6 [7]. If the fermentation pH is controlled at a pH ∼6, only calcium lactate will be produced. In the studies reported here, the pH was maintained at ∼5, and under these conditions both CaO and MgO can effectively neutralize the lactic acid produced and control the pH. The batch fermentation data shown in Figs. 1 and 2 indicate that lactose was almost completely consumed for both cases in about 40 h. The overall conversion rate is 97% (w/w) when NaOH was used for pH control. In the second case with lime for pH control, the lactose concentration decreased from 35.5 g/L to 0.63 g/L, in ∼43 h. The final lactic acid concentration was 28.2 g/L. Taking into account the volume change caused by lime solution addition (broth volume change from 1500 ml to 1700 ml), the overall conversion rate is ∼92%. This is slightly lower than that for pH control with NaOH. The acid yield in both cases was about 90%. After 43 h, the operation was changed from batch to continuous fermentation mode by pumping in medium with 34.5 g/L lactose, and simultaneously withdrawing the product broth at the dilution rate of 0.03 h−1 . As evident from Fig. 2, continuous operation with lime for pH control performed well. The substrate concentration was consistently reduced to low levels providing a high lactose conversion
rate. These results clearly show that lime solution can be used in lactose fermentation instead of NaOH without any significant adverse effects. 4.2. Continuous fermentation for CML production Continuous fermentation runs were made over an extended period using 12 N NaOH for pH control to determine the effects of dilution rate (D) on performance and acid productivity. NaOH was used for pH control for the sake of convenience since there are no significant differences in performance between NaOH and lime solution. Lactose, lactic acid, and cell concentrations are plotted for 7 days of operation at various dilution rates. Theoretically, the specific growth rate of bacterial cells should equal the dilution rate. For L. plantarum the maximum specific growth rate at pH 5 is 0.38 h−1 with lactose as the substrate [16]. Under the experimental conditions wherein the steady-state substrate concentration (S) was maintained at ∼3–10 g/L, the ratio of S/(S + Ks ) in Monod’s equation is ∼0.06–0.17 h−1 . Therefore, the actual specific growth rate is around 0.02–0.06 h−1 . The continuous fermentation data reported previously [17] is reproduced in Fig. 3. The data indicate stable operation at dilution rates 0.02, 0.04 and 0.06. When D was increased to 0.08 h−1 or 0.1 h−1 , “washout” conditions occurred. In these cases the substrate concentration in the fermenter began to increase and the product concentration decreased due to the decrease in cell density in the reactor. The results from continuous fermentation experiments are summarized Table 1. Lactic acid yield decreased slightly from 0.94 at D = 0.02 to 0.83 at D = 0.06, and the acid productivity increased from 0.62 to 2.03. In practice, using as high a D value as possible would be better in a two-stage operation to obtain high acid productivity. Any moderate amounts of lactose left from the first fermentation stage can be utilized by P. acidiproprionici in the second stage of operation. Alternatively, an intermediate storage vessel can be used for the reduction of residual lactose.
Fig. 3. Continuous fermentation of lactose by L. plantarum (reproduced with permission from World Scientific Publishers, 2001).
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Table 1 Batch and continuous fermentation of lactose by L. plantarum Dilution rate (L/h)
Lactic acid yield (w/w)
Productivity (g/L/h)
Conversion (%)
Feed (g/L)
Batch 0.02 0.04 0.06
0.88 0.94 0.86 0.83
0.48 0.62 1.35 2.03
100 97 98 94
33.8 30.8 38.4 38.4
4.3. Second stage batch fermentation for CMA/CMP production The CML broth collected from first stage was filtered to remove Lactobacillus bacteria and used as substrate in the second stage for CMA and CMP production using P. acidipropionici bacteria. Batch fermentation experiments were conducted to determine the effect of lime use for pH control. As shown in Fig. 4 cell growth and acid production occurs rapidly after an initial lag phase. Champagne et al. [18] have observed lower fermentative activity in whey fermentation by P. shermanii with NaOH as neutralizing agent compared to lime. At the same pH, fermentation rates were reported to be 20% higher when lime was used as the neutralizing agent. Since CML is converted to the salts CMA and CMP in the second stage of fermentation, the additional amount of lime required for neutralization and pH control is quite small. The lime requirement for the second stage was about 13% of the amount used in the first stage. The volume change caused by the addition of basic solution is less than 1% with 6N lime solution. The pH in both cases was initially maintained at 7. In the case of lime solution, at the end of batch fermentation, a small amount (<2 g/L) of CaCO3 solid slurry was formed. This is due to the reaction of lime with the CO2 produced in the fermentation process. The effect of pH on the accumulation of precipitates during CML fermentation with lime for pH control was studied by varying the pH. When pH was controlled at 7 and the carbonate precipitates were allowed to accumulate, the total precipitate concentration increased to ∼19.4 g/L. As pH
Fig. 5. Effect of pH on CaCO3 precipitation in the broth.
was decreased from 7 to 5.9, the concentration of participates decreased and at pH ∼5.9 the precipitates were completely dissolved. These results are shown in Fig. 5. In subsequent runs when pH was controlled between 5.9 and 6.2, there was no formation of CaCO3 precipitate in the broth. Thus, to avoid the formation of carbonate precipitates during CML fermentation, the pH must be controlled at less than ∼6.2. For P. acidipropionici, bacterial growth and metabolism, will not be significantly affected by maintaining a pH ∼6.0. Continuous fermentation experiments were conducted at different dilution rates to determine long-term operational stability of the system when lime is used for pH control. The results from operation over a period of 90 days are shown in
Fig. 4. Batch fermentation of lactic acid by P. acidipropionici P200910 (Ca(OH)2 for pH control).
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Fig. 6. Continuous fermentation of lactic acid by P. acidipropionici P200910 (Ca(OH)2 for pH control). Table 2 Continuous fermentation of calcium magnesium lactate broth from first stage using P. acidipropionici Dilution rate (L/h)
Feed (g/L)
Total acid yield (w/w)
Conversion (%)
Productivity (g/L/h)
P/A ratio (w/w)
0.02 0.024 0.04 0.05
39.6 42.2 42.4 42.4
0.95 0.91 0.92 0.94
92 90 86 86
0.748 0.85 1.51 1.79
2.0 2.2 2.0 2.1
Fig. 6. The broth used in the experiment was obtained from first the stage fermentation operation and the CML concentration was adjusted to a constant level of ∼40 g/L lactic acid before use. Fig. 6 shows operation at four different dilution rates. The system was operated at each dilution level until steady values of substrate and product concentrations were achieved. Typically, operations were conducted at a given dilution rate for more than 24 h (6–24 residence times) to attain uniform substrate and product concentration values. Thereafter, the dilution rate was incrementally changed. The fermentation system reached steady state after about 48 h at the dilution rates of 0.02 h−1 and 0.04 h−1 . For D = 0.05 h−1 , the system reached “washout” conditions and the cell and product concentrations began to decrease. When D was decreased to 0.024, the system reached steady operation again within about 48 h. The acid productivity and yields at various dilution rates are shown in Table 2. In all cases, the total acid yield is greater than 90%. The acid productivity ranged from 0.75 g/L/h to 1.8 g/L/h for dilution rates 0.02 h−1 to 0.05 h−1 . Using as high a dilution rate as possible below washout conditions would be better for obtaining high acid productivity values. Longterm operational stability was excellent at the dilution rates of 0.01 h−1 and 0.02 h−1 as seen in Fig. 6. The system was operated for more than 50 days at D = 0.01 h−1 and for 10 days at D = 0.02 h−1 at a CML conversion rate greater than 90%. The ratio of propionate to acetate (P/A in w/w) was ∼2.0–2.3 when pH was controlled using lime (see Table 2).
This is different from the value of ∼3.0 obtained when NaOH is used for pH control. This is probably due to the high concentrations of Ca2+ and Mg2+ in the broth that could potentially alter the metabolic pathway. The final product broth contained mainly CMA and CMP at a total concentration of 48–55 g/L. The bacterial cells can be separated by filtration and recycled or used as animal feed. The product CMA and CMP in the clear liquid then can be separated by multiple effect evaporation to obtain the deicer in solid form. Alternatively, the solution can be concentrated to ∼25–28% (w/w) and used as deicing agent in liquid form.
5. Conclusions A novel two-stage bioconversion process has been developed for the production of deicing salts calcium magnesium acetate (CMA) and calcium magnesium propionate (CMP) from inexpensive raw materials such as whey lactose and lime. In the first stage of fermentation lactose is converted to lactic acid by L. plantarum at 92% conversion and acid yield of 0.9. Ca(OH)2 and Mg(OH)2 were used instead of NaOH to neutralize the acids produced during the fermentation, with no adverse effects on cell growth and acid yield. In the second stage of fermentation, calcium magnesium lactate was converted to acetate and propionate salts at ∼91% conversion and acid yield of ∼0.9. While the overall yield of total acids did not change when lime was used for pH control instead of NaOH, the propionate to acetate ratio changed from
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∼3.0 to ∼2.0 (w/w) in the final CMA and CMP products. The pH needs to be maintained below ∼6.0 in the fermenter to prevent the formation of carbonate precipitates. Continuous fermentation experiments were conducted for over 90 days with lime for pH control, and showed excellent long-term operational stability. The maximum total acid productivity in the second stage was 1.8 g/L/h. The liquid with total CMA and CMP concentration around 48–55 g/L can be evaporated by multiple effect evaporation to obtain the deicer in solid form.
Acknowledgments This material is based upon work supported by the National Science Foundation under Grant No. 9613273. We wish to express our sincere gratitude to Dr. Bonita A. Glatz in Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, for providing us with P. acidipropionici P200910 cultures.
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