Ind. Eng. Chem. Res. 2010, 49, 5981–5985
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Optimization of Green Synthesis of Potassium Diformate and Its Potential as a Mold Inhibitor for Animal Feed Wenshuang Lin,†,‡,§ Huixuan Wang,†,‡,§ Qingbiao Li,*,†,‡,§ Jiale Huang,† Yao Zhou,†,‡,§ Jinbao Zheng,†,‡ Daohua Sun,† Ning He,† and Yuanpeng Wang† Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniVersity, Xiamen, 361005, P. R. China, National Engineering Laboratory for Green Chemical Productions of Alcohols, Ethers and Esters, Xiamen UniVersity, Xiamen, 361005, P. R. China, and Key Lab for Chemical Biology of Fujian ProVince, Xiamen UniVersity, Xiamen, 361005, P. R. China
Potassium diformate (KDF) has been regarded as an alternative to antibiotic growth promoters for animals. This paper delineates a 100% atom economical process for the preparation of KDF from formic acid and potassium formate. Parametric optimization of the synthesis was conducted with respect to reaction time, reaction temperature, and molar ratio of the reactants by employing orthogonal design of experiment method. The results manifested a molar ratio of HCOOH to HCOOK of 1.3, reaction temperature of 65 °C, and reaction time of 30 min as the optimal conditions with a KDF product yield of 94.0%. Efforts were also made to explore the antimold performance of KDF on animal feed using the plate count method. Compared with sodium diacetate (SDA), the widely used mold inhibitor, KDF exhibited even better antimold performance for animal feed. To our knowledge, this work first proved the applicability of KDF as a mold inhibitor for animal feed. 1. Introduction Potassium diformate (KH(HCOO)2, KDF), a conjugated acid salt, has been approved in the European Union as a nonantibiotic growth promoter for animals (Commission Regulation (EC) No. 492/2006). Extensive research has proved that KDF improves growth performance of pigs,1-4 chickens,5 and tilapia,6 based mainly on its significant antimicrobial effect in the gastrointestinal tract of animals.3,6-8 More interestingly, cooling mediums containing aqueous solution of KDF for fish preservation9 and a composition comprising KDF and carriers for controlling fungal growth on building materials10 have been demonstrated. Thus, KDF serving as an alternative to both mold inhibitors for feed and antibiotic growth promoters for animals could be anticipated. Although Kendall et al.11 demonstrated the preparation of acid potassium formates including KDF as early as 1921, commercial production of KDF was not reported until it was achieved by Norsk Hydro ASA.12 Other important synthesis techniques of KDF were conducted by reacting aqueous methyl formate13 or formic acid14,15 with basic compounds of potassium. To date, little information about the process optimization on synthesis of KDF by formic acid and potassium formate is available. Moreover, to the best of our knowledge, comparative study on applicability of KDF as a mold inhibitor for animal feed has not been carried out hitherto. Thus, this work aimed to conduct a parametric investigation of KDF synthesis, and special attention was also paid to explore the potential application of KDF as a mold inhibitor for animal feed. 2. Experimental Section 2.1. Materials. Formic acid (88%), potassium formate (99%), and other reagents were commercial analytic reagents. Sodium * To whom correspondence should be addressed. E-mail: kelqb@ xmu.edu.cn. Tel: (+86) 592-2189595. Fax: (+86) 592-2184822. † Department of Chemical and Biochemical Engineering. ‡ National Engineering Laboratory for Green Chemical Productions of Alcohols, Ethers and Esters. § Key Lab for Chemical Biology of Fujian Province.
diacetate (SDA) was prepared as described.16 Raw materials of animal feed including maize, wheat bran, and soybean meal were kept in sealed polyethylene bags. 2.2. Synthesis of Potassium Diformate. Synthesis experiments were performed by batch-type in a three-necked roundbottom flask of 50 mL equipped with a thermometer and a reflux condenser. The carefully weighed potassium formate was added to the flask, as well, 2.25 mL of deionized water and 10.0 mL of formic acid were then added in tandem. Afterward, the flask was immersed in a glycerin bath maintained at a constant temperature (45, 55, 65, 75, 85, and 95 °C). The reactant mixture was heated for a period of time (30, 60, 90, 120, 180, and 300 min) under magnetic stirring condition at a constant speed throughout the experiment. The reaction mixture obtained was cooled to room temperature under static conditions for crystallization. The crystallized product was subsequently dried at 60 °C for 48 h, and finally ground into fine grains. 2.3. Characterization of Potassium Diformate. The chemical structure of the as-prepared products was determined by Fourier transform infrared spectroscopy (FT-IR). Spectra were recorded employing a Nicolet Avatar 660 (Nicolet, USA) FTIR spectrometer in the 500-4000 cm-1 frequency range on translucent discs obtained by pressing the ground samples and potassium bromide. Formic acid content determination was based on acid-base titration of formic acid with sodium hydroxide using phenolphthalein as an indicator. Titrate solution was prepared by dissolving KDF samples with deionized water in a conical flask. Fresh NaOH titrant was added dropwise to the titrate solution employing a conventional buret, while titrate solution was constantly shaken throughout the titration operation, and a “blank titration” of the deionized water was also performed. The determinations were performed in duplicates. Moisture content of the KDF products was determined according to the following procedures: The carefully weighed sample was placed into a ceramic crucible, which was thereafter heated in an oven at 250 °C for 2 h to completely volatilize H2O in the sample and HCOOH which was derived from KDF
10.1021/ie100332m 2010 American Chemical Society Published on Web 05/25/2010
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Figure 1. KDF yield as a function of molar ratio of HCOOH to HCOOK. Conditions: reaction temperature 75 °C, reaction time 30 min.
decomposition. The substance thus obtained was cooled to room temperature in a silica gel desiccator, and subsequently weighed. The moisture content of the sample was finally obtained by the difference of weight loss (expressed in wt %) and the formic acid content. Duplicates were also conducted. 2.4. Mold Inhibition Assays. Animal feedstuffs including maize, wheat bran, and soybean meal were crushed and mixed with a blender. Moisture of the mixed feed was determined by heating samples at 105 °C to constant weight. The mixed feed was divided into lots of 3 kg. Then KDF or SDA was added to each lot except the control to reach subsequent concentrations (in grams per kilogram feed, g kg-1) of KDF 1.0, 3.0, 6.0, 12, 18, and 24, and of SDA 6.0. After mixing completely by a blender, each lot was divided into 1.0-kg portions and packed into polyethylene bags, which were stored in a storeroom with average temperature of 22.5 °C and relative humidity of 63.3% for 90 days. Samples (each of 25 g) of feed were taken from the bags with sterile spoons on days 15, 29, 43, 64, 78 and 90, containing both surface and inner material. Total molds were enumerated by a pour plate method (National Standard of China, GB/T13092-2006) with Salt Czapek Dox Agar Medium containing the following (in grams per liter): NaNO3, 2.0; KH2PO3, 1.0; MgSO4 · 7H2O, 0.5; KCl, 0.5; FeSO4, 0.01; NaCl, 60; sucrose, 30; agar, 20. Each of the samples (25 g) taken at selected time intervals was mixed with 225 mL of 0.85 wt % sterile NaCl solution and shaken vigorously for 30 min. Serial dilutions of the samples were separately plated with the Salt Czapek Dox Agar mediums. The plates were incubated for 5 days at 25 °C before total molds were counted. Total molds count was determined for duplicate plates from each treatment at each sampling time. The average number of molds from the duplicate plates was reported as number of colony-forming units (cfu) g-1 sample. 3. Results and Discussion 3.1. Effect of Reactant Molar Ratio. For the sake of examining the effect of reactant molar ratio on the KDF yield, synthetic reactions were performed under the same reaction conditions (reaction temperature 75 °C and reaction time 30 min), except that the molar ratio of HCOOH to HCOOK (n(HCOOH)/n(HCOOK)) in the feed reactants was varied. As observed in Figure 1, raw material composition has a significant influence on the KDF yield. It is worth pointing out that the products obtained were off quality when the molar ratio of HCOOH to HCOOK was less than 1.0, which was a consequence of stoichiometrically insufficient amount of formic acid in the reactants for the KDF synthesis. However, while the formic acid was added over the theoretically equivalent-needed amount, the
Figure 2. KDF yield as a function of reaction time. Conditions: n(HCOOH)/ n(HCOOK) 1.2, reaction temperature 75 °C.
Figure 3. KDF yield at different reaction temperatures. Conditions: n(HCOOH)/ n(HCOOK) 1.2, reaction time 30 min.
KDF yield was improved significantly with the increase of the quantity of HCOOH. In addition, when the ratio n(HCOOH)/ n(HCOOK) was enhanced above 1.3, the KDF yield increased inconspicuously. 3.2. Effect of Reaction Time. To investigate the effect of reaction time upon the KDF yield, experiments were carried out by adjusting the reaction time with a constant n(HCOOH)/ n(HCOOK) of 1.2 at a fixed temperature of 75 °C. The results are shown in Figure 2 as the KDF yield versus reaction time. It could be observed that initially the KDF yield increased responding to the increase of the reaction time and reached a maximum value in 60 min, indicating a quick synthetic process of this system. However, afterward the KDF yield dramatically decreased with the extension of reaction time due to volatilization loss of formic acid. 3.3. Effect of Reaction Temperature. Given the constant n(HCOOH)/n(HCOOK) of 1.2 and reaction time of 30 min, alteration of the KDF yield via variation of reaction temperature within the range of 45-95 °C is illustrated in Figure 3. Among the six values presented, the highest yield of KDF was achieved at the temperature of 65 °C. In general, a higher reaction temperature favored accordingly a higher yield when the temperature was below 65 °C, owing to the thermal effect on the kinetics. However, when the temperature was above 65 °C, a notable decrease of the yield resulted, which could be ascribed to severe volatilization loss of formic acid at the temperature of 75-95 °C. 3.4. Optimization of Synthetic Process through Orthogonal Experimental Design. To further evaluate the effects of reactant molar ratio, reaction temperature, and reaction time on the KDF yield and estimate the optimal conditions for the synthesis process, an orthogonal array L9 (34) was adopted on
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Table 1. Factors and Levels of Orthogonal Design factors levels
(A) reactant molar ratio n(HCOOH)/n(HCOOK)
(B) reaction temperature /°C
(C) reaction time/min
1 2 3
1.1 1.2 1.3
55 65 75
30 60 90
Table 2. Layout and Result of Orthogonal Experiment no.
A
B
error
C
yield/%
1 2 3 4 5 6 7 8 9 k1a k2 k3 Ra
1 1 1 2 2 2 3 3 3 85.1 87.6 90.9 5.8
1 2 3 1 2 3 1 2 3 89.7 90.3 83.5 6.8
1 2 3 2 3 1 3 1 2 88.5 88.5 86.6 1.9
1 2 3 3 1 2 2 3 1 88.2 87.3 88.1 0.9
87.9 87.6 79.8 90.3 89.1 83.3 90.9 94.2 87.5
a
ki, average of every factor yield, %; R, range.
Table 3. Analysis of Variance of Orthogonal Experiment source of variation
sum of squares
degree of freedom
A B C error total
50.2 84.2 1.5 7.0 142.9
2 2 2 4 10
a
mean square 25.1 42.1 0.77
F statistic 11.7 19.6a 0.36
Significant parameter, F0.1, (2, 2) ) 9, F0.05, (2, 2) ) 19.
the basis of previous experiments. The process parameters and their selected levels are presented in Table 1. The experimental layout for KDF synthetic process parameters using L9 (34) orthogonal array is shown in Table 2, as well, in which the results are also presented. Nine separate experiments with specified levels were involved in the design, and the sequence in which the experiments were carried out was randomized to avoid any personal or subjective bias, such that the validity of test results was soundly ensured. The interactive effect of parameters was assumed to be negligible and thus not taken into consideration. This assumption was validated by confirmation experiments conducted at the optimum conditions. The significance of the three major parameters, reactant molar ratio (A), reaction temperature (B), and reaction time (C), can be evaluated by range analysis. The larger range (R, ki, max. - ki, min.) a factor presents the more significant influence it has on a target index. As shown in Table 2, the range of symbol B is the highest. That is, the reaction temperature is the most influencing parameter affecting the KDF yield. It should be noted that the R-value obtained for reaction time was smaller than that for the error column or any other parameters, indicating that the KDF yield was little affected by the reaction time in the investigated range of 30-90 min. Furthermore, results of the orthogonal experiments were statistically analyzed by the analysis of variance (ANOVA) technique to assess the influence and relative contributions of input variables on the process responses. The results acquired from the ANOVA analysis are summarized in Table 3, revealing that rather than the parameter reaction time, the reaction temperature and reactant molar ratio as the main parameters affected the KDF yield. The significance of the three parameters on the KDF yield can be described in the order of B > A > C. The best level should be selected for
Figure 4. Typical FT-IR spectrum of the KDF sample. Conditions for the KDF sample preparation: n(HCOOH)/n(HCOOK) 1.3, reaction temperature 65 °C, reaction time 30 min.
the significant factor or general factor; while for the nonsignificant factor, the value of level should be decided by the economical, environmental, and convenience principle. Therefore, a recommended combination was B2-A3-C1, representing reaction temperature 65 °C, n(HCOOH)/n(HCOOK) 1.3, and reaction time 30 min. Furthermore, three confirmation experiments were carried out under these optimal conditions with KDF yields of 94.1%, 94.4%, and 93.6%, respectively (an average of 94.0 ( 0.4%). 3.5. Physicochemical Properties of KDF. 3.5.1. FT-IR Analysis Results. The possibility of directly stating the structural groups constitutes the essence of IR spectroscopy and substantiates it as one of the most important methods of instrumental analysis.17 The FT-IR absorption spectrum of KDF sample obtained at the optimal conditions, as shown in Figure 4, can offer information regarding the structural groups of KDF molecule. Some absorption bands centered at 1223, 1349, 1384, 1593, and 1631 cm-1 were observed in the region 1000-2000 cm-1. Among them, the absorbance bands located at 1631 and 1593 cm-1 in the curve could be associated with the antisymmetric CdO-stretching vibrations, and two bands thus presented reveal that two CdO were involved in the KDF molecule. Whereas the two bands at 1349 and 1384 cm-1 corresponded to the symmetric CdO-stretching vibrations. If an organic carboxylic acid converted to its salt, it would result in an ideal bonding equilibrium between the CdO-double and the CdOsingle bond due to the complete dissociation of the anion and cation.17 Because both oscillators have the same mass and force constant, there is a defined coupling of the vibrations,17 lying at 1593 cm-1 for the antisymmetric vibrational mode and 1349 cm-1 for the symmetric mode as delineated in Figure 4. In addition, the band at 1223 cm-1 could be assigned mainly to the C-O-stretching vibration. To a larger extent, the band with a very strong, broad, and obtuse peak at 3421 cm-1 could be attributed to the -OH group, and the character of the peak indicates that there is hydrogen-bond association in the molecule.17 Weak but characteristic shoulders at 2810 and 2725 cm-1 originated from an interaction of the aliphatic C-Hstretching with the overtone of the H-CdO-bending.17 The structure information depicted in Figure 4 coincided well with that of KDF molecule which possesses two hydrogen bonds with the HCOOH molecule and the HCOO- anion as proton donor and proton acceptor, respectively.18 3.5.2. Formic Acid and Moisture Content. To evaluate the amount of HCOOH existing in the KDF sample via hydrogen bonding, the titration procedure mentioned above has been undertaken, and the content of formic acid in the KDF product obtained at the optimal synthetic conditions was as high as 33.7
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Table 4. Total Molds Count in Feed Samples with Additives total molds count (log cfu g-1) additives
15 d
29 d
43 d
64 d
78 d
90 d
control KDF, 1.0 g kg-1 KDF, 3.0 g kg-1 KDF, 6.0 g kg-1 KDF, 12 g kg-1 KDF, 18 g kg-1 KDF, 24 g kg-1 SDA, 6.0 g kg-1
4.5 3.7 3.8 2.9 2.3 2.3 2.0 3.3
4.6 4.1 3.6 3.2 3.4 3.2 2.8 4.1
4.5 4.0 4.1 3.2 3.3 2.8 2.3 3.8
4.8 4.2 4.2 3.3 3.1 2.7
