Microsoft Word - 2012_Dr_Bodor_Endre_HJIC.doc HUNGARIAN JOURNAL OF INDUSTRIAL CHEMISTRY VESZPRÉM Vol. 39(3) pp. 359-362 (2011) EXPERIMENTS FOR D-LACTIC ACID PRODUCTION WITH FERMENTATION Á. NÉMETH , Á. KISS, B. SEVELLA Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science 1111 Budapest Szt Gellért tér 4., HUNGARY E-mail: naron@f-labor.mkt.bme.hu Lactic acid is one of the oldest known chiral chemicals. Its L-enantiomer is widely produced and used both in the nature and in human society. The renaissance of the L-lactic acid production was forced by the biodegradable plastic production on lactic acid basis which produces PLA. This polymer has good biodegradability, but poor mechanic properties, thus its characteristic is under strong improvements. During this investigations it was established that the stereocomplex polymers from L and D enantiomers has higher melting point resulting in broader application. Unfortunately the production of D-lactic acid was not solved since it is not the natural form of lactic acid. For this reason we wanted to examine the possibilities for D-lactic acid fermentation with Lactobacillus coryniformis. We adapted a new technique for cultivation in microtiterplates. First we successfully optimized the media composition in term of carbon source (glucose) and nitrogen source (yeast extract) and obtained an optimal glucose=67.5 g/L and YE=27 g/L concentration by Gauss-surface fitting. Next we noticed (on observation that the final product titer never surpassed 65 g/L but a lot of glucose remained) that this fermentation is under strong product inhibition. We also examined the inhibitory effect of different form of product lactate (i.e. with Na+, Ca2+ and NH4 +). We obtained that all the examined products cause total inhibition on cell growth at 90–120 g/L concentration but they differ in their effect between 30–60 g/L: the strongest inhibition occurred at NH4-LA (both 30 and 60 g/L), the Na-LA has lower inhibition range, but its effect is also equal at 30 and 60 g/L and finally Ca-LA showed hardly inhibition at 30 g/L but strong inhibition at 60 g/L Keywords: D-lactic acid, Lactobacillus coryniformis, product inhibition, microtiter fermentation Introduction Lactic acid is one of the basic molecules in chiral chemistry. Four different ligands are connected onto its central carbon atom, of which ligands’ stereoscopic orientation results in two isomers, which are of reflected by mirror form of each other. In the nature the presence of the L-form is universal starting from simple organism (like prokaryotes) to higher order organisms (like humans). Furthermore it is also produced industrially and used in large scale mostly by the food industry. The real massproduction demand on lactic acid arised in the ‘90s, when the price of crude oil dramatic increased, resulting also in the price elevation of oil based polymers, beside the upcoming of their environmental-toxic effect. Although lactic acid can also be produced synthetically (resulting in racemic mixture of the D and L form) in large scale, but the fermentative production has been most widespread since early of the last century. The homopoylmers resulted by the polymerisation of the L-enantiomer is easily biodegradable, but its mechanic properties are disadvantageous from application point of view (brittle and easy to break). The situation was quite the same in case of polymerisation of D-enantiomer. The poor polymer characteristics were investigated with different additives, meanwhile it became clear, that with simultaneous polymerisation of the two isomers at appropriate polymerisation conditions a stereocomplex polymer can be formed, which has higher melting point, thus the application possibilities were widened [1]. However the industrial production of D-lactic acid has not been solved yet, thus the bottleneck of a widely applicable and at the same time easily biodegradable polymer manufacture is the production of the D-lactic acid. Although in nature the L-form is common, but between microbes it is possible to find also D or racemic (D, L) lactic acid producers. The chirality of the microbiologically produced lactic acid is depending on the stereospecifity of lactate dehydrogenase (LDH, EC 1.1.1.27) of the producer strain. In our research group fermentative production of lactic acid and related researches have quite long history [3-6], in the frame of which recently we investigate also the technology of a Biorefinery realizing in Hungary. In a biorefinery (that is a complex raw material processing bio-combinate) it would be possible to utilize different non-food grade crops (wheat (also with fusarium infections), corn or sweet sorghum) for both L-lactic acid and D-lactic acid production, which latter has higher price (value) causing benefits for the investment of the biorefinery. On the basis of these, our goal in the present work was to establish pre-experiments for D-lactic acid fermentation, and to compare these results with the outputs of L-isomer fermentation. During these experiments we adapted a 360 new high throughput method, which made it possible to carry out simultaneously high number of experiments on microtiterplates (MTP’s). Materials and methods We used Lactobacillus coryniformis DSM20007 strain previously described in the literature [2] as D-lactic acid producer. For strain maintenance MRS agar slants specially developed for lactobacilli were used. For fermentation we used MRS broth with different yeast extract (YE) and glucose content both in shaking flasks and in round lowwell (24 cells) microtiter plates (MTP). The applied temperature was 37 °C, whereas 150 rpm shaking was used, and the pH was adjusted to 5.8 twice on every day with 12% ammonium-hydroxide with the help of pH- electodes in the case of shaking flasks (from sample) and with pH-indicator strips in case of MTP cells. For shaking MTPs in a rotary shaker incubator we used ‘System Duetz’ from Enzyscreen, which consisted of a fixing adapter (clamp) and a ‘sandwich cover’ that is crucial for aerobic fermentation. The latter one has little holes above filter cloth for gas exchange, and on the other hand it can prevent the individual MTP cells from (cross)contaminations [7]. The glucose and lactic acid content of fermentation broths were analysed on BioRad Aminex HPX-87H column at 65 °C with 0.5 ml/min 5 mM H2SO4 eluent and RI detection in a Waters Breeze HPLC system. In case of shaking flasks biomass growth was followed with OD600 photometric measurements, while in the MTP’s cells we determined cell density with scanning the transparent MTP, and the obtained grayscale images were evaluated with the calibration curve on Fig. 1. y = 0,1568x R² = 0,9284 0 1 2 3 4 5 6 7 8 9 10 0 10 20 30 40 50 60 Cell Dry Weight (g/L) Gray scale Figure 1: Calibration curve for cell dry weight determination on the basis of grayscale To obtain the above calibration curve we drove fermentation in shaking flask, and samples was measured both with photometer (OD600), and after placing them into MTP cells with scanner (Epson stylus SX-600FW). For image evaluation we used Bell MicroImage Analyser software, with which we were able to determine the gray grade of the sample between 0 and 256 (0-black, no Lactobacillus cells, 256-white, full light reflection). Results and evaluation In the first shaking flask experiments the culture grew well beside 20 g/L glucose in 24 h and the decreasing pH also indicated the produced lactic acid. On the basis of this, in the first experiment on MTP we examined the more economical higher substrate concentration (30-60- 90-120 g/L), which may cause substrate inhibition or according to the resulted higher amount of lactic acid product inhibition can also occur. Each different substrate concentration was tested with different YE (i.e. nitrogensource) supplementations (5-15-25-35 g/L) which resulted 16 experimental set up. Beside these we designed a central point with 75 g/L glucose and 20 g/L YE running with three repeats and furthermore three “central point’s” were also created, but not inoculated to test (cross)contamination. To evaluate the obtained results we defined a combined parameter (objective function) (Q) to find the optimal media composition with smaller amount of remaining glucose and higher amount of lactic acid yield simultaneously. This was necessary, since the final lactic acid titer could be correlated with the different starting amount of substrate making impossible to compare them. The obtained Q = YLA/Gluremained values are presented on Fig. 2 as the function of glucose and YE concentrations. 0 2 4 6 8 10 12 14 16 18 20 40 60 80 100 120 5 10 15 20 25 30 35 Q Gl uc os e (g /L ) YE (g/L) Figure 2: Examination of the media composition on the effectiveness of lactic acid fermentation Onto the calculated Q values obtained from the experiment we fitted a Gauss-surface (R2=0.98) with Sigma Plot 2001 software, and from the fitted constants of the function the location of the maximal Q can be established as follows: Glucose = 67.5 g/L and YE = 27 g/L. It have to be remarked, that we never found higher lactic acid concentration than 65 g/L, although the obtained more than 90% product yield at lower substrate concentration (for example 30 g/L) predicted very high lactic acid titer at 90 or 120 g/L starting glucose concentration. The problem is clearly demonstrated on Fig. 3, indicating product inhibition, since the final glucose remains around zero up to ca 60 g/L of final LA 361 titer, but above it the final remaining glucose concentration breaks out. 0 10 20 30 40 50 60 0 20 40 60 80 Remaining glucose (g/L) Final LA titer (g/L) Figure 3: Coming up the question of product inhibition To examine the product inhibition more detailed a new MTP experiment was designed as follows: 30-60- 90-120 g/L initial concentration of different (Na+, Ca2+, NH4 +) lactates were applied before inoculation with two replicates of every experiental set up. Becauuse in the existing LA technologies these alkali (NaOH, NH4OH, CaCO3) are applied for pH controlling during fermentation, we wanted to examine the effects of these. In the cases of 90 and 120 g/L initial lactates we never found any cell growth, indicating full product inhibition. The 30 and 60 g/L cases can be compared on Fig. 4. Time (h) 0 20 40 60 80 100 Cell Dry Weight (g/L) 0 1 2 3 4 5 6 NH-LA (R2=0,98) Na-LA (R2=0,99) Ca-LA (R2=0,99) Figure 4: Comparing the different kind of 30 g/L initial lactates If the two profiles on Fig. 4 and 5. are compared it can be obtained, that ammonium-lactate (NH-LA) has the strongest inhibitory effect and there is no difference between the levels of 30 and 60 g/L, thus already 30 g/L can dramatic decrease in the cell growth of L. coryniformis. The inhibitory effect of sodium-lactate (Na-LA) was lower, but also the same at 30 and 60 g/L concentrations. Finally, Ca-LA can hardly inhibit in 30 g/L amount, but it has strong inhibitory effect at 60 g/L concentration. Time (h) 0 20 40 60 80 100 Cell Dry Weight (g/l) 0 1 2 3 4 NH4-LA (R 2=0,95) Ca-LA (R2=1) Na-LA (R2=0,99) Figure 5: Comparing the different kind of 60 g/L initial lactates Conclusions Our preliminary results clearly show, that the lactic acid fermentation of L. coryniformis has high yield (>90 %) when applying lower substrate concentration, but the industrially acceptable level of substrate causes product inhibition. On the basis of our experiments we can start to build kinetic approaches to obtain inhibition constants in order to describe the lactic acid fermentation performance with this strain. However, most probably to introduce it to an industrial technology it needs thorough strain improvements. ACKNOWLEDGEMENTS This work is connected to the scientific program of the “Development of quality-oriented and harmonized R+D+I strategy and functional model at BME” project. This project is supported by the New Hungary Development Plan (Project ID: TÁMOP-4.2.1/B-09/1/KMR-2010-0002). REFERENCES 1. C. LEE: Production of D-lactic acid by bacterial fermentation of rice, Fibers and Polymers 8 (1) (2007) 571–578 2. R. YÁÑEZ, A. B. MOLDES, J. L. ALONSO, J. C. PARAJÓ: Production of D(-)-lactic acid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subsp. torquens, Biotechnology Letters 25 (2003) 1161–1164 3. K. HETÉNYI, Á. NÉMETH, B. SEVELLA: Use of sweet sorghum juice for lactic acid fermentation: preliminary steps in a process optimization, Journal of chemical technology 85 (2010) 872–877 362 4. K. HETÉNYI, Á. NÉMETH, B. SEVELLA: Optimization of wheat flour based medium for lactic acid fermentation, Chemical and Biochemical Engineering Quarterly 24(2) (2010) 195–201 5. T. TÁBI, Á. NÉMETH, A. SELMECZI, F. SZALAY: Keményítővel és cellulózzal töltött politejsavból előállított fröccsöntött lebomló polimer termékek laboratóriumi lebontásának és komposztálásának vizsgálata, Biohulladék 5(2-3) (2010) 30–34 6. K. HETÉNYI, Á. NÉMETH, B. SEVELLA, P. KOVÁCS, ZS. BODNÁR: Eljárás haszonnövények fermentációs felhasználására tejsav és származékainak előállítása céljából, patent (2010), P 09 00193 7. W. DUETZ: Growth in microtiterplates, http://www.enzyscreen.com/DB.html 20.03.2011. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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