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Preeminent productivity of 1,3-propanediol by Clostridium butyricum JKT37 and the role of using calcium carbonate as pH neutraliser in glycerol fermentation
Accepted Manuscript Preeminent Productivity of 1,3-Propanediol by Clostridium butyricum JKT37 and The Role of Using Calcium Carbonate as pH Neutraliser in Glycerol Fermentation Zhao Kang Tee, Jamaliah Md Jahim, Jian Ping Tan, Byung Hong Kim PII: DOI: Reference:
S0960-8524(17)30239-0 http://dx.doi.org/10.1016/j.biortech.2017.02.110 BITE 17682
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
7 December 2016 21 February 2017 22 February 2017
Please cite this article as: Tee, Z.K., Jahim, J.M., Tan, J.P., Kim, B.H., Preeminent Productivity of 1,3-Propanediol by Clostridium butyricum JKT37 and The Role of Using Calcium Carbonate as pH Neutraliser in Glycerol Fermentation, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.02.110
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Preeminent Productivity of 1,3-Propanediol by Clostridium butyricum JKT37 and The Role of Using Calcium Carbonate as pH Neutraliser in Glycerol Fermentation
Zhao Kang Tee1, Jamaliah Md Jahim1 *, Jian Ping Tan1, Byung Hong Kim2
1
Department of Chemical and Process Engineering, Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor, Malaysia. 2
Fuel Cell Institute, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,
Malaysia.
*Corresponding Author: [email protected]
Abstract Calcium carbonate was evaluated as a replacement for the base during the fermentation of glycerol by a highly productive strain of 1,3-propanediol (PDO), viz., Clostridium butyricum JKT37. Due to its high specific growth rate (µ max = 0.53 h-1), 40 g/L of glycerol was completely converted into 19.6 g/L of PDO in merely 7 hours of batch fermentation, leaving only acetate and butyrate as the by-products. The accumulation of these volatile fatty acids was circumvented with the addition of calcium carbonate as the pH neutraliser before the fermentation was inoculated. An optimal amount of 15 g/L of calcium carbonate was statistically determined from screening with various glycerol concentrations (20 – 120 g/L). By substituting potassium hydroxide with calcium carbonate as the pH neutraliser for fermentation in a bioreactor, a similar yield
(YPDO/glycerol = 0.6 mol/mol) with a constant pH was achieved at the end of the fermentation.
Keywords: 1,3-propanediol, isolation, fermentation, calcium carbonate, pH neutraliser
1.0
Introduction
Over the last two decades, many researchers have been performing the bioconversion of glycerol into 1,3-propanediol. The most common microorganisms used are Clostridium sp. and Klebsiella sp. Under batch fermentation, Clostridium sp. can produce a yield ranging from 0.62 to 0.66 mol of PDO /mol glycerol, while the yield from Klebsiella sp.ranges from 0.53 to 0.77 mol/mol (Liu et al., 2010; Saxena et al., 2009; Yang et al., 2017; Zhang et al., 2007). However, Clostridium sp. with its lower yield, has the advantage over Klebsiella sp. due to the non-pathogenicity and independent characteristic of vitamin B-12 on the enzyme glycerol dehydratase. Biebl et al. (1992) started their work by isolating Clostridium diolis DSM 15410 (previously known as C. butyricum DSM 5431) from soil samples. They were able to obtain a yield of 0.62 mol/mol of PDO. Papanikolaou et al. (2000) attained a yield of 0.66 mol/mol of PDO from isolated C. butyricum F2b. Furthermore, C. butyricum AKR102a was screened out as the best isolate from the soil sample from a palm oil refinery, with an efficiency of 0.404 mol/mol in 100 g/L of glycerol fermentation (Ringel et al., 2012). Recently, C. butyricum DSP1 was found to have achieved a yield of 0.66 mol/mol in a 5-litre bioreactor fermentation (Szymanowska-Powałowska et al., 2013).
Although there have been few reports on the use of a mixed culture in glycerol fermentation, one common problem was the difficulty of maintaining microbial communities in a mixed culture. In 2013, Liu et al. studied the effect of fresh soil and old soil as the inoculum for the production of PDO. It was found that after 6 months of soil storage, the microbial community had changed, where the yield of PDO was reduced from 0.49 mol/mol to 0.44 mol/mol. Besides, Selembo et al. (2009) reported that a different substrate can significantly affect which bacteria become predominant, and the yield of products. As glycerol waste from biodiesel plants varies according to its composition, the use of crude glycerol in a mixed culture fermentation has to be studied in detail. The effects of impurities and pre-treatments on crude glycerol vary with the microorganism sources used (Samul et al., 2014). Recently, the study of the regulation of the pH in glycerol fermentation through the use of a mixed culture was carried out (Moscoviz et al., 2016). However, the experiment was conducted with only pure glycerol in order to reduce other variabilities that resulted from a mixed culture fermentation.
Due to the formation of acetate and butyrate as the bacteria grow, glycerol assimilation by Clostridium sp. resulted in low pH after the fermentation. During the glycerol fermentation without any pH control, the pH of the extracellular medium dropped below the dissociation constants of acetate (pKa = 4.76) and butyrate (pKa = 4.8) (Monot et al., 1984), which resulted in the undissociated form of the acids in equilibrium. Consequently, the undissociated acetic and butyric acids were diffused across the bacterial membrane into the cell, and were dissociated at the more alkaline region (Russell & Diez-Gonzalez, 1997). However, the anionic species of the acids
were unable to pass across the membrane, thus causing them to remain in the intracellular fluids. These anionic species acted as an uncoupler, where the proton motive force was not dissipated in a cyclic manner as more energy was utilized for the maintenance of the pH in the cell (Van Ginkel & Logan, 2005). As a result, the fermentation was inhibited and the PDO production was halted. A research conducted by Colin et al. (2001) showed that the undissociated acetate and butyrate acids caused a reduction in the PDO and biomass, respectively.
It is more preferable to use a bioreactor approach in conducting fermentation due to the automatic adjustment of crucial parameters, such as the pH of the media (Wu et al., 2008). Nevertheless, the addition of diluted acids or bases to control the pH can dilute the fermentation media, where the dilution factor has to be considered. The use of a pH-buffer in glycerol fermentation serves as an alternative in the pH control, particularly in shake flasks or serum vials. Calcium carbonate is widely used as a pHbuffer due to its low cost and non-hazardous properties (Ai et al., 2014). During the screening of Clostridium diolis 15410, 1 g/L of calcium carbonate was added as the pH buffer (Otte et al., 2009). However, Ringel and co-workers used 30 g/L of CaCO3 in their screening work to select the best PDO producer (Ringel et al., 2012). The addition of CaCO3 maintained the pH of the media within the range of 5 – 9 during the fermentation, even when added in excess. As calcium carbonate is insoluble, a higher concentration of the pH buffer in the media provided a larger buffer capacity to neutralise the volatile fatty acids (Ai et al., 2014). A proteomic analysis showed that the addition 4 g/L of CaCO3 had higher positive effects than a concentration of 2 g/L in
acetone-butanol-ethanol (ABE) fermentation, but there was no further improvement with more than 4 g/L of calcium carbonate (Han et al., 2013).
The aim of this study was to isolate a pure culture of a local1,3-propanediol producer that was able to utilise glycerol as the sole carbon source. It was then evaluated for its glycerol consumption, PDO yield and productivity by using a bioreactor for batch fermentation. Calcium carbonate was added to the fermentation medium, which was then screened for optimal buffer loading with different glycerol concentrations. A statistical analysis was used to further investigate the effect of calcium carbonate on the PDO production. Finally, batch fermentation was performed in a bioreactor by using the glycerol concentration with the highest productivity and optimal buffer loading, which was regarded to replace potassium hydroxide as the pH neutraliser.
2.0
Materials and Methods
2.1
Source of Microorganisms
The bacteria were isolated from palm oil mill effluent (POME), which was collected from Sime Darby East Oil Mill, Selangor, Malaysia.
2.2
Isolation of Local Producer
The bacteria were isolated by using a synthetic agar medium containing the following components per litre of distilled water: K2HPO4, 3.4 g; KH2PO4, 1.3 g; (NH4)2SO4, 2.0 g; MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.02 g; yeast extract, 1.0 g; and agar powder, 20 g. Pure glycerol at a concentration of 20g/L was solely used as the carbon source for the
isolation. The enrichment was conducted by inoculating the isolate with Reinforced Clostridium Medium, RCM (BD Difco), with the following composition in one litre of distilled water: peptone, 10g; beef extract, 10 g; yeast extract, 3 g; dextrose, 5 g; sodium chloride, 5 g; soluble starch, 1 g; L-Cysteine HCl, 0.5 g, sodium acetate, 3 g; and agar. 0.5 g. All the media were autoclaved at 121ºC for 20 minutes prior to being dispensed onto petri plates. The pH of the autoclaved media was 6.8 ± 0.2.
A sample of 0.2 mL of non-sterilized POME was cultured in Hungate tubes containing 5 ml of a glycerol-based culture medium. The tubes were heat-shocked at 80ºC for 10 minutes to remove any methanogens in the samples. The tubes were then incubated for 48 hours at 37ºC with nitrogen gassing for 10 minutes. Next, 1 mL of the suspension was then re-inoculated into a Hungate tube containing 9 mL of melted synthetic medium agar. Roll tube method was used for the cultivation, where the inoculum was solidified on the surface of the tube. The colonies that were found on the surface of tube after three days of incubation were then transferred to an agar plate containing RCM for enrichment. All the agar plates were incubated in an anaerobic jar for three days. A purified Clostridium colony was then further identified using the 16S rRNA coding sequence.
2.3
Bacteria Identification
DNA from the extracted isolate was amplified using the reverse primer, 1492R (5-GGT TACCTTGTTACGACTT-3’), and the forward primer, 27F (3’AGAGTTTGATCMTGGCTCAG-5’). A polymerase chain reaction (PCR) was carried out with a reaction volume of 25 µl containing crude bacterial lysate DNA, 10 pmol of
each primer, deoxynucleotides triphosphates (dNTPs, 400 µM each), 0.75 U Taq DNA polymerase, and the supplied buffer. The PCR was performed as follows: one cycle for the initial denaturation at 95ºC for 5 minutes, 30 cycles for annealing and extension (95ºC for 45 seconds; 51ºC for 15 seconds; 72ºC for 2 minutes), and one cycle for the final extension of the amplified DNA at 72ºC for 10 minutes. The products were purified and sequenced with primers 518F and 800R using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).
The sequences encoding the 16S rRNA of the isolate were identified using NCBI Nucleotide BLAST programme. A phylogenetic tree was constructed using the Top 10 Hit Blast results against the NCBI 16S rRNA sequences (bacteria only) database, excluding the uncultured bacterium (tax id: 77133). The tree was inferred using the neighbour-joining method, with the percentage of replicated trees, in which the associated taxa clustered together in the bootstrap test (500 replicates), being shown next to the branches. The evolutionary distances were computed using the Jukes-Cantor method.
Microbial observations were made using a field emission scanning electron microscope (FESEM) (SUPRA 55VP, Carl Zeiss AG). The isolate was harvested by the microcentrifugation of the culture at 2,000 rpm for 5 minutes. It was then mixed with 2 % glutaraldehyde for 20 hours and refrigerated at 4ºC. Next, 0.1 M phosphate buffer solution was used to wash the pellets three times for 10 minutes each time. Dehydration was carried out by consequent immersion in 30, 50, 70, 80, and 90% ethanol, and finally three times in 100% (v/v) ethanol, for 10 minutes each time. The dehydrated samples
were then immediately dried in a critical point dryer (Leica EM CPD 300) for 1.5 hours. The dried samples were sputter-coated with platinum, and subsequently, analysed using FESEM.
2.4
Batch Fermentation in Bioreactor
The fermentation media used in this study contained (per litre of water) K2HPO4, 3.4 g; KH2PO4, 1.3 g; (NH4)2SO4, 2.0 g; MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.02 g; yeast extract, 1.0 g; L-Cysteine HCl, 0.5 g; 5 g/L resazurin solution, 1 ml; Fe solution, 1 ml; and trace elements solution, 2 ml (Biebl, 1991; Günzel et al., 1991). The Fe solution consisted of FeSO4·5H2O, 5 g/L; and 37% hydrochloric acid, 4 ml/L. The composition of the trace solution (per one litre of water) included ZnCl2, 70 mg; MnCl2.4H2O, 100 mg; H3BO3, 60 mg; CoCl2·2H2O, 200 mg; CuCl2·2H2O, 20 mg; NiCl2·6H2O, 20 mg; and Na2MoO4·2H2O, 40 mg. Pure glycerol with a concentration ranging from 20-120 g/L was added to the medium as the sole carbon source. In addition, crude glycerol obtained from a biodiesel plant was also evaluated on its performance in PDO production. 20 g/L of crude glycerol was used for comparison with pure glycerol.The fermentations were carried out in a 3.6-litre Labfors 5 bioreactor (Infors HT), with a working volume of 1 litre. The pH was set at 7.0, and it was controlled by adding 2M potassium hydroxide automatically. Samplings of the fermentation were taken at hourly intervals.
2.5
Effect of Calcium Carbonate as pH Neutraliser
The pH-buffered fermentations were run with identical fermentation media, but with additional 10 to 30 g/L of calcium carbonate (CaCO3). The 10% v/v inoculum used in
the experiment was transferred into 130-mL serum vials with a working volume of 50 mL by using sterile syringes. Anaerobic conditions were maintained by capping the vials in a Bactron I-2 anaerobic chamber (Shel Lab), with a gas mixture (80% N2, 10% CO2, and 10% H2) and using resazurin solution as an indicator. The fermentation was carried out at 35ºC with an agitation speed of 200 rpm for 24 hours. The experiments were conducted in triplicate, where independent samples were taken at the initial stage (T= 0 h) and at the end of the fermentation process (T=24 h).
A statistical analysis of the data was performed using the IBM SPSS Statistics 21.0. One-way ANOVA was used to investigate the effects of glycerol and the buffer loading on the pH, the PDO titre, acetate and butyrate, glycerol consumption, and yield of PDO. Univariate ANOVA was used to investigate the relationship between these two effects. A significance level of P < 0.05 was used (Saratale et al., 2016). Turkey’s HSD Post-hoc test was conducted right after the ANOVA test to confirm the significant difference between the levels in calcium carbonate loading and glycerol concentration, respectively. As a final verification, the optimal calcium carbonate and glycerol loadings were used in the batch fermentation using a bioreactor.
2.6
Analytical Methods
Glycerol, 1,3-propanediol, acetic acid and butyric acid were analysed using high performance liquid chromatography (Thermo Scientific UltiMate 3000), equipped with a Phenomenex RoA 300 mm × 7.8 mm column and a refractive index detector (RID). In addition, 5 mN of H2SO4 was used as the mobile phase, which was eluted isocratically at a flow rate of 0.6 ml/min. The column temperature was set at 60ºC while it was
detected by using the refractive index at 40ºC. The retention time for each component was as follows: glycerol, 11.94; acetic acid, 16.67; 1,3-propanediol, 20.53; and butyric acid, 25.27 minutes, respectively.
3.0
Results and Discussion
3.1
Phylogenetic Analysis
The partial 16S rRNA genetic sequence of the local isolates was compared with the sequence in GenBank. Figure 1 shows the optimal tree with the sum of the branch lengths being 0.0569. According to the phylogenetic tree, Clostridium butyricum VPI3266, Clostridium butyricum JCM1391 and Clostridium butyricum ATCC 19398 also produce 1,3-propanediol from glycerol fermentation. The isolate was named as Clostridium butyricum JKT37 (GenBank No.: KU513553).
A microbial observation was done on the JKT37 isolate under field emission scanning electron microscopy (FESEM). The images were taken after the culture had been incubated for 3 days, where the growth of the isolate was at the stationary phase. The rod-shaped isolates could be identified as pure cultures, where no contaminants were found on the images. The average size of the isolates was 0.62 × 0.62 µm, with lengths ranging from 3.1 to 5.9 µm. The elongation and division of cells could be seen clearly in the images (see Supplementary Materials), thereby indicating that healthy growth was taking place in the culture.
3.2
Fermentation in Bioreactor
The bioconversions of pure glycerol with concentrations ranging from 20 – 120 g/L are summarised in Table 1A, 1C, 1D, 1E and 1F. Table 1B represents the fermentation of crude glycerol at a concentration of 20 g/L as a comparison. Generally, glycerol fermentation by Clostridium butyricum JKT37 produces PDO as the main product, while co-producing acetate and butyrate throughout the fermentation. The fermentation profile for each parameter is shown in Figure 2.
In all the experiments, it was observed that the glycerol was almost completely utilized, with the average yield being 0.6 mol/mol. The fast-growing JKT37 isolate showed an outstanding productivity with regard to PDO, with the maximum productivity being 2.8 g/L.h during batch fermentation. However, the productivity of PDO decreased with an increase in the glycerol concentration, coupled with a decrease in the AA/BA ratio. The accumulation of butyrate during the fermentation inhibited the growth of JKT37, resulting in a lower biomass production and lower glycerol uptake. It was observed in Figure 2F that there was a slow down after 18 hours of fermentation, where the glycerol was not fully consumed at the end of the fermentation. A low AA/BA ratio of 0.30 at the stated experiment indicated a lower ATP yield and low biomass yield (X=1.31 g/L) (Zhang et al., 2009).
Glycerol fermentation in Clostridium butyricum JKT37 consists of oxidoreductive pathways, where glycerol is oxidized to dihydroxyacetone, and progressively to pyruvate (Xin et al., 2016). At the same time, glycerol is also converted to 3hydroxypropioaldehyde, and then reduced to 1,3-propanediol (Deckwer, 1995). The possible products and by-products formed by Clostridium sp. from glycerol metabolism can be mainly represented by biochemical reactions as follows:
(1) PDO production + 2 → +
(2) Acetate production → + 6 + 2 + −
(3) Butyrate production 2 → + 8 + 3 + 2
(4) Biomass generation 4 + 3 + 35 → 3 + 8 + 6
It was assumed that the molecular formula of the biomass was C4 H7O2N, with a yield of 8.6 g/mol of ATP (Zeng, 1996). The maximum theoretical yield for PDO production is 0.72 mol/mol due to the reducing cofactors in the reductive pathways that have to be regenerated by converting glycerol into pyruvate via the oxidative pathway, thus producing acetate as a by-product (Zeng, 1995; Zeng, 1996). Nevertheless, most of the experimental works on glycerol fermentation produced acetate and butyrate as byproducts. For the equal utilization of cofactors in the fermentation, the maximum theoretical yield for PDO production decreased to 0.69 mol/mol. In the current study, the experimental yield by JKT37 reached 0.60 mol/mol, which was equivalent to 87% of the maximum theoretical yield. Table 2 gives a summary of the PDO production from batch fermentation in a bioreactor scale. Surprisingly, the microorganism isolated
from this study was considered to have among the highest productivity compared to previous studies. One reason for this promising result was the rapid growth of JKT37 (µ max= 0.53 h-1), where no lag phase was observed during the fermentation. This result was comparable to the estimated µ max of 0.527 h-1 when the Contois model was used in a modelling study (Papanikolaou & Aggelis, 2003).
3.3
Effect of Calcium Carbonate as pH Neutraliser
Calcium carbonate was added to serve as an acid buffer pool to neutralise the volatile fatty acids produced by the bacteria. An increase in buffer concentration enabled the accommodation of more acetate and butyrate, thereby, allowing a higher conversion of glycerol. The experiments were carried out with calcium carbonate loadings ranging from 10 to 30 g/L. The experimental data are shown in Table 3. Note that for glycerol fermentation at a concentration of 20 g/L, the carbonate loading was tested only between 10 – 20 g/L due to the complete conversion of glycerol under the abovementioned conditions, where further additions of calcium carbonate did not show any additional positive effect on the fermentation (data not shown). Therefore, the investigation of the calcium carbonate loadings focused on the higher initial glycerol concentrations, i.e. ranging from 40 g/L to 120 g/L. Generally, the final pH for all the samples was slightly acidic due to the presence of a buffer in the fermentation medium. An obvious improvement was observed when the carbonate loading was from 10 to 15 g/L, as shown in Figure 3. Further increments in the carbonate loading did not have much effect on the fermentation. The best PDO result recorded was a concentration of 19 g/L that was obtained from 40 g/L of glycerol with a carbonate loading of 15 g/L.
Nearly all the glycerol at concentrations of 20, 40 and 60 g/L was consumed within 24 hours by the fermentation process. In contrast, the consumption of glycerol declined to half at concentrations higher than 80 g/L. Note that there was not much change in the final pH after 24 hours of fermentation, and therefore, the effect of the dissociated butyrate and acetate could be neglected. It was presumed that at high glycerol concentrations, the osmotic and hydrogen gas pressure may have inhibited bacterial growth (Szymanowska-Powałowska, 2015).
In order to investigate the effect of glycerol and calcium carbonate on the effectiveness of the fermentations, the data in Table 3 were used for the one-way ANOVA calculations. The response parameters, including the pH, PDO titre, acetate and butyrate, glycerol consumption and PDO yield, were set as dependent variables, whereas the glycerol and calcium carbonate concentrations were set as factors in each analysis, as shown in Table 4. The glycerol concentration acted as a direct factor on the pH, PDO, acetate and butyrate, and glycerol consumption with a p-value of less than the significance level at 0.05. In contrast, the calcium carbonate loading did have a significant effect on the yield of PDO production (p = 0.027). However, the one-way ANOVA tests did not show the specified levels of glycerol or calcium carbonate concentrations that were significant.
As such, Turkey’s HSD post-hoc tests were conducted to further investigate the matter through multiple comparison tables (see Supplementary Materials). The results showed that fermentation of 20 g/L of glycerol had a significant effect on the pH compared to glycerol concentrations at 40 g/L and above. Additionally, there was no
significant difference at 40 g/L and higher concentrations of glycerol. In this case, 40 g/L was the optimal glycerol concentration in the batch fermentation. On the other hand, there was a significant difference in the PDO titre when 20g/L of glycerol was used in the fermentation compared to higher concentrations of glycerol. However, by using the same comparison method, 60 g/L of glycerol was found to be the optimal concentration for the highest PDO titre. While acetate and butyrate are dependent on the consumption of glycerol, the results of the glycerol consumption in the post-hoc tests could be used to confirm the optimal concentration of glycerol. The results showed that 20 g/L of glycerol consumption had no significant effect as compared to 40 – 120 g/L of glycerol. However, there were significant differences between 40 g/L and 60, 80, 100, and 120 g/L of glycerol. It was noted that at a concentration of 80 g/L of glycerol, there were no significant differences in the consumption with 100 and 120 g/L. As 20 and 40 g/L of glycerol fermentation can achieve almost complete consumption, further increases in the concentration of glycerol significantly reduced the glycerol consumption. In addition, the inhibition of the partial pressure of hydrogen could be observed from 80 g/L of glycerol and beyond, which explained the lack of significant differences in 100g/L and 120 g/L of glycerol. These post-hoc tests reaffirmed that 40 g/L is the optimal glycerol concentration for fermentation.
Besides, the post-hoc test for the effect of calcium carbonate on the yield of PDO showed that there were significant differences between 30 g/L and 10 g/L of glycerol (p = 0.038), and with 20 g/L of glycerol (p = 0.030). However, the results were inconclusive. A univariate ANOVA was conducted to analyse the optimal calcium carbonate loading for fermentation. PDO was set as the dependent factor since it could
directly explain the quality of fermentation, whereas the glycerol concentration and calcium carbonate loadings were set as fixed factors. The significance level for this analysis was also set at 0.05. With reference to Figure 4, it was deduced that the calcium carbonate loading was optimal when the concentration was at 15 g/L. It was observed that CaCO3 had no synergistic effect on the PDO production at concentrations higher than 15 g/L.
In addition, the univariate ANOVA was used to investigate the correlation between the glycerol concentration and the calcium carbonate loading. As shown in Table 5a, different glycerol concentrations had a significant effect on the titre of PDO (p = 0.040), whereas calcium carbonate alone and the interaction between these two factors showed no significant differences. A univariate ANOVA was also conducted with the same fixed factors, but the yield of PDO was chosen as the dependent variable. The results in Table 5b show that only calcium carbonate had a significant effect on the yield of PDO (p = 0.019), whereas the glycerol concentration and the interaction between these two factors showed no significant differences.
The results from the statistical analyses suggested that 15 g/L of calcium carbonate be added in place of potassium hydroxide in the fermentation of 40g/L glycerol. The evaluation of the stated condition was conducted in a 3.6-litre bioreactor. As observed from Figure 5, there was a lag phase of 2 hours before the glycerol was converted into PDO due to the high initial pH (pHo=8.85) of fermentation. The effect of calcium carbonate as a buffer was clearly observed after 4 hours of fermentation, where a constant pH of 6 was maintained until the fermentation ended. It was observed that
the yield of PDO was 0.60 mol/mol, but with lower productivity of 1.96 gL-1h-1. From this experiment, it was concluded that calcium carbonate can act as a pH neutralizer in place of a pH controller in the production of PDO.
Conclusion Clostridium butyricum JKT37 was isolated as a highly potential 1,3-propanediol producer in the present study, whereby its productivity surpassed most of the previously reported natural producers by 150%. Unlike the common pH controllers in bioreactors, calcium carbonate was used as a pH neutraliser to accommodate the effect of the acetate and butyrate produced during glycerol fermentation. An optimal concentration of 15g/L of calcium carbonate was statistically proven to enhance the fermentation, without affecting the yield of 1,3-propanediol. The evaluation a bioreactor fermentation showed that a similar yield for 1,3-propanediol was achieved compared with the addition of bases during fermentation.
Acknowledgements The authors would like to thank the Ministry of Higher Education for its financial support (FRGS/1/2014/SG05/UKM/01/2). We would also like to forward our gratitude to Sime Darby East Oil Mill for providing the effluent for isolation purposes.
Competing financial interests The authors declare no competing financial interests.
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24. Selembo, P. A., Perez, J. M., Lloyd, W. A. & Logan, B. E. 2009. Enhanced hydrogen and 1,3-propanediol production from glycerol by fermentation using mixed cultures. Biotechnol Bioeng 104: 1098-106. 25. Szymanowska-Powałowska, D. 2015. The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of Clostridium butyricum DSP1. Electron. J. Biotechnol., 18, 128-133. 26. Szymanowska-Powałowska, D., Białas, W. 2014. Scale-up of anaerobic 1,3propanediol production by Clostridium butyricum DSP1 from crude glycerol. BMC Microbiol., 14. 27. Szymanowska-Powałowska, D., Drożdżyńska, A., Remszel, N. 2013. Isolation of new strains of bacteria able to synthesize 1, 3-propanediol from glycerol. Adv. Microbiol., 3, 171. 28. Van Ginkel, S., Logan, B.E. 2005. Inhibition of Biohydrogen Production by Undissociated Acetic and Butyric Acids. Environ. Sci. Technol, 39, 9351-9356. 29. Wischral, D., Zhang, J., Cheng, C., Lin, M., De Souza, L.M.G., Pessoa, F.L.P., Pereira Jr, N., Yang, S.-T. 2016. Production of 1,3-propanediol by Clostridium beijerinckii DSM 791 from crude glycerol and corn steep liquor: Process optimization and metabolic engineering. Bioresour. Techno., 212, 100-110. 30. Wu, K. J., Saratale, G. D., Lo, Y. C., Chen, W. M., Tseng, Z. J., Chang, M. C., Tsai, B. C., Su, A. & Chang, J. S. 2008. Simultaneous production of 2,3-butanediol, ethanol and hydrogen with a Klebsiella sp. strain isolated from sewage sludge. Bioresour Technol 99(17): 7966-70. 31. Xin, B., Wang, Y., Tao, F., Li, L., Ma, C., Xu, P. 2016. Co-utilization of glycerol and lignocellulosic hydrolysates enhances anaerobic 1,3-propanediol production by Clostridium diolis. Sci. Rep., 6, 19044. 32. Yang, X., Kim, D. S., Choi, H. S., Kim, C. K., Thapa, L. P., Park, C. & Kim, S. W. 2017. Repeated batch production of 1,3-propanediol from biodiesel derived waste glycerol by Klebsiella pneumoniae. Chem. Eng. J. 314: 660-669. 33. Zeng, A.P. 1995. A new balance equation of reducing equivalents for data consistency check and bioprocess calculation. J. Biotechnol, 43, 111-24. 34. Zeng, A.P. 1996. Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum. Bioprocess Eng., 14, 169-175. 35. Zhang, C., Yang, H., Yang, F., Ma, Y. 2009. Current Progress on Butyric Acid Production by Fermentation. Curr. Microbiol., 59, 656-663.
36. Zhang, G., Ma, B., Xu, X., Li, C. & Wang, L. 2007. Fast conversion of glycerol to 1,3-propanediol by a new strain of Klebsiella pneumoniae. Biochem. Eng. J. 37: 256260. 37. Zhu, C., Fang, B., Wang, S. 2016. Effects of culture conditions on the kinetic behavior of 1,3-propanediol fermentation by Clostridium butyricum with a kinetic model. Bioresour. Techno., 212, 130-137.
Figure captions Figure 1 Phylogenetic tree showing position of Clostridium butyricum JKT37 among top 10 BLAST results
Figure 2 Profiles of products and by-products of glycerol fermentation at concentrations of (A) pure 20 g/L,(B) crude 20 g/L,(C) pure 40 g/L,(D) pure 60 g/L, (E) pure 80 g/L, and (F) pure 120 g/L. E and F were plotted at intervals of 2 and 3 hours respectively, although the samples were taken hourly.
Figure 3 Titre of PDO for fermentation at high glycerol concentrations. Data shown as the average of triplicates
Figure 4 Estimated Marginal Means of PDO at different calcium carbonate loadings
Figure 5 Profile of glycerol fermentation using calcium carbonate as pH neutraliser
Table 1: Glycerol fermentation of JKT37 at different glycerol concentrations Experiment Glycerol (g L-1) Fermentation time (h) Glycerol (g L-1) PDO (g L-1) Acetate, AA (g L-1) Butyrate, BA (g L-1) AA/BA ratio Biomass, X (g L-1) µ max (h-1) Yield (mol/mol) Productivity (g L-1 h-1)
A 20 ± 0.4 5 0 ± 0.0 10.29 ± 0.2 2.21 ± 0.1 1.40 ± 0.1 1.57 1.64 ± 0.1 0.58 0.62 2.06
B 20 ± 0.4 6 0 ± 0.1 10.77 ± 0.4 2.33 ± 0.2 1.18 ± 0.1 1.97 1.64 ± 0.1 0.57 0.65 1.80
C D 40 ± 0.9 60 ± 0.7 7 13 0 ± 0.0 0.85 ± 0.1 19.59 ± 0.3 30.24 ± 0.8 3.41 ± 0.2 4.44 ± 0.5 3.01 ± 0.5 5.42 ± 0.2 1.13 0.82 2.00 ± 0.0 1.95 ± 0.1 0.53 0.56 0.6 0.59 2.80 2.33
E 80 ± 1.0 24 0.51 ± 0.1 42.61 ± 0.7 4.35 ± 0.3 8.5 ± 0.2 0.51 1.78 ± 0.1 0.57 0.59 1.76
Note: The readings are the average values from three independent analyses for the same sample. The range of errors is displayed where necessary.
F 120 ± 1.8 37 4.38 ± 1.8 56.53 ± 1.4 4.46 ± 0.2 15.06 ± 1.3 0.30 1.31 ± 0.2 0.34 0.53 1.52
Table 2 Various batch PDO production from glycerol fermentation in bioreactor Microorganism C. butyricum DSM 5341 C. butyricum VPI 3266 C. butyricum CNCM 1211 C. butyricum F2b C. butyricum VPI 1718 C. butyricum DSP1 C. butyricum DSM 10702 C. beijerinckii DSM 791 C. diolis DSM 15410 C. butyricum M01 C. butyricum JKT37
Glycerol (g/L) 110
Time (h) 29
Titre (g/L) 56
Yield (g/g) 0.51
Productivity (g/L.h) 1.9
References
65
48
35
0.54
0.72
121
39
65.4
0.55
1.67
40
26
22
0.55
1.2
61
54
33.6
0.55
0.65
70
33
37.6
0.53
1.12
70
64
40
0.57
0.63
33
24
17.5
0.55
0.72
20.93
9
11.64
0.56
1.29
Saint-Amans et al. (1994) Himmi et al. (1999) Papanikolaou et al. (2000) Chatzifragkou et al. (2011) SzymanowskaPowałowska and Białas (2014) Loureiro-Pinto et al. (2016) Wischral et al. (2016) Xin et al. (2016)
48.7
10
23.4
0.50
2.49
Zhu et al. (2016)
40
7
19.59
0.49
2.80
This study
Biebl et al. (1992)
Table 3 Experimental data for fermentation at different calcium carbonate loading CaCO Glycer Consumptio ol pH PDO Acetate Butyrate n Yield 3 mol/m g/L g/L g/L g/L g/L % ol 6.6 0.82 ± 2.51 ± 10 20 5 6.28 ± 0.43 0.11 0.24 99.28 0.498 6.1 16.45 ± 1.86 ± 5.25 ± 40 4 1.52 0.34 0.87 95.56 0.473 6.1 21.00 ± 2.94 ± 3.95 ± 60 0 1.18 0.51 0.55 69.53 0.596 6.0 15.11 ± 0.45 ± 2.25 ± 80 1 4.31 0.13 0.30 47.08 0.420 6.0 21.54 ± 1.85 ± 4.47 ± 100 6 1.91 0.44 0.31 40.79 0.596 6.2 16.47 ± 2.18 ± 2.23 ± 120 9 0.75 0.18 0.18 41.03 0.454 6.6 1.31 ± 2.45 ± 15 20 4 7.32 ± 0.26 0.20 0.24 99.07 0.490 6.2 19.07 ± 3.43 ± 4.55 ± 40 3 0.98 0.47 0.27 97.03 0.503 6.2 26.00 ± 4.78 ± 7.69 ± 60 0 2.69 0.64 1.13 98.86 0.482 6.1 26.20 ± 4.01 ± 5.16 ± 80 9 0.32 0.79 0.74 56.50 0.552 6.2 22.52 ± 3.68 ± 3.40 ± 100 1 1.37 0.73 0.31 53.74 0.514 6.2 20.67 ± 2.26 ± 3.29 ± 120 6 2.82 0.15 0.22 41.11 0.415 6.6 1.68 ± 2.71 ± 20 20 4 6.95 ± 0.64 0.25 0.19 99.13 0.472 6.3 18.80 ± 2.35 ± 5.61 ± 40 0 1.17 0.33 0.46 98.92 0.549 6.2 23.66 ± 4.08 ± 3.83 ± 60 6 1.13 0.58 0.37 71.87 0.576 6.1 25.58 ± 4.56 ± 5.21 ± 80 3 0.79 0.28 0.20 68.66 0.513 6.2 22.47 ± 3.69 ± 2.42 ± 100 9 1.90 0.40 0.23 51.32 0.486 6.3 18.50 ± 2.34 ± 2.57 ± 120 5 2.18 0.43 0.19 42.10 0.462 6.3 18.26 ± 3.22 ± 4.73 ± 25 40 3 0.98 0.17 0.28 99.35 0.493 6.2 22.90 ± 4.17 ± 4.47 ± 60 3 1.51 0.31 0.34 80.30 0.469 80 6.1 24.62 ± 3.47 ± 5.02 ± 63.57 0.528
100 120 30
40 60 80 100 120
8 6.1 6 6.3 5 6.4 4 6.1 9 6.3 4 6.3 6 6.3 9
1.82 24.51 ± 1.69 20.07 ± 2.08 17.22 ± 1.19 21.64 ± 1.55 20.25 ± 0.62 19.30 ± 1.30 16.46 ± 1.70
0.27 4.20 ± 0.30 1.85 ± 0.17 2.15 ± 0.21 4.19 ± 0.26 2.97 ± 0.19 2.18 ± 0.37 2.44 ± 0.31
0.44 4.64 ± 0.30 3.38 ± 0.41 5.43 ± 0.67 7.66 ± 0.83 2.91 ± 0.16 3.19 ± 0.37 2.36 ± 0.35
63.56
0.424
46.91
0.434
99.71
0.385
91.43
0.483
55.11
0.441
50.89
0.377
45.22
0.334
Table 4 One-Way ANOVA based on the factors of the glycerol and CaCO3 concentrations Glycerol pH PDO Acetate Butyrate Consumption Yield
Sum of Squares 0.524 632.544 17.445 32.324 13148.077 0.027
df 5 5 5 5 5 5
Mean Square 0.105 126.509 3.489 6.465 2629.615 0.005
F 12.389 20.221 4.106 4.871 47.721 1.479
Sig. 0.000 0.000 0.009 0.004 0.000 0.237
pH PDO Acetate Butyrate Consumption Yield
Sum of Squares 0.070 104.754 10.948 4.808 270.769 0.040
df 4 4 4 4 4 4
Mean Square 0.018 26.189 2.737 1.202 67.692 0.010
F 0.629 0.905 2.499 0.487 0.111 3.323
Sig. 0.647 0.477 0.071 0.745 0.978 0.027
CaCO3
Table 5a Univariate ANOVA based on glycerol concentrations and calcium carbonate loadings on PDO titre
Source Type III Sum of Squares Corrected Model 227.648 Intercept 8.536 CaCO3 57.341 Glycerol 106.415 CaCO3 * Glycerol 42.045 Error 542.535 Total 11177.527 Corrected Total 770.183
df 3 1 1 1 1 24 28 27
Mean Square 75.883 8.536 57.341 106.415 42.045 22.606
F 3.357 0.378 2.537 4.707 1.860
Sig. 0.035 0.545 0.124 0.040 0.185
Table 5b Univariate ANOVA based on glycerol concentrations and calcium carbonate loadings on PDO yield Source Type III Sum of Squares Corrected Model 0.070 Intercept 0.826 CaCO3 0.017 Glycerol 0.016 CaCO3 * Glycerol 0.015 Error 0.039 Total 6.540 Corrected Total 0.109
df 11 1 1 5 5 16 28 27
Mean Square 0.006 0.826 0.017 0.003 0.003 0.002
F 2.600 339.285 6.795 1.321 1.268
Sig. 0.040 0.000 0.019 0.305 0.325
Highlights • A novel strain for PDO production was isolated from palm oil mill effluent. •
Batch productivity in bioreactor can achieve as high as 2.80 g/L.h.
•
Proposed the use of calcium carbonate to maintain pH range during fermentation.
•
A control fermentation volume was established without the addition of acid or base.
S0960-8524(17)30239-0 http://dx.doi.org/10.1016/j.biortech.2017.02.110 BITE 17682
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
7 December 2016 21 February 2017 22 February 2017
Please cite this article as: Tee, Z.K., Jahim, J.M., Tan, J.P., Kim, B.H., Preeminent Productivity of 1,3-Propanediol by Clostridium butyricum JKT37 and The Role of Using Calcium Carbonate as pH Neutraliser in Glycerol Fermentation, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.02.110
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Preeminent Productivity of 1,3-Propanediol by Clostridium butyricum JKT37 and The Role of Using Calcium Carbonate as pH Neutraliser in Glycerol Fermentation
Zhao Kang Tee1, Jamaliah Md Jahim1 *, Jian Ping Tan1, Byung Hong Kim2
1
Department of Chemical and Process Engineering, Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor, Malaysia. 2
Fuel Cell Institute, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,
Malaysia.
*Corresponding Author: [email protected]
Abstract Calcium carbonate was evaluated as a replacement for the base during the fermentation of glycerol by a highly productive strain of 1,3-propanediol (PDO), viz., Clostridium butyricum JKT37. Due to its high specific growth rate (µ max = 0.53 h-1), 40 g/L of glycerol was completely converted into 19.6 g/L of PDO in merely 7 hours of batch fermentation, leaving only acetate and butyrate as the by-products. The accumulation of these volatile fatty acids was circumvented with the addition of calcium carbonate as the pH neutraliser before the fermentation was inoculated. An optimal amount of 15 g/L of calcium carbonate was statistically determined from screening with various glycerol concentrations (20 – 120 g/L). By substituting potassium hydroxide with calcium carbonate as the pH neutraliser for fermentation in a bioreactor, a similar yield
(YPDO/glycerol = 0.6 mol/mol) with a constant pH was achieved at the end of the fermentation.
Keywords: 1,3-propanediol, isolation, fermentation, calcium carbonate, pH neutraliser
1.0
Introduction
Over the last two decades, many researchers have been performing the bioconversion of glycerol into 1,3-propanediol. The most common microorganisms used are Clostridium sp. and Klebsiella sp. Under batch fermentation, Clostridium sp. can produce a yield ranging from 0.62 to 0.66 mol of PDO /mol glycerol, while the yield from Klebsiella sp.ranges from 0.53 to 0.77 mol/mol (Liu et al., 2010; Saxena et al., 2009; Yang et al., 2017; Zhang et al., 2007). However, Clostridium sp. with its lower yield, has the advantage over Klebsiella sp. due to the non-pathogenicity and independent characteristic of vitamin B-12 on the enzyme glycerol dehydratase. Biebl et al. (1992) started their work by isolating Clostridium diolis DSM 15410 (previously known as C. butyricum DSM 5431) from soil samples. They were able to obtain a yield of 0.62 mol/mol of PDO. Papanikolaou et al. (2000) attained a yield of 0.66 mol/mol of PDO from isolated C. butyricum F2b. Furthermore, C. butyricum AKR102a was screened out as the best isolate from the soil sample from a palm oil refinery, with an efficiency of 0.404 mol/mol in 100 g/L of glycerol fermentation (Ringel et al., 2012). Recently, C. butyricum DSP1 was found to have achieved a yield of 0.66 mol/mol in a 5-litre bioreactor fermentation (Szymanowska-Powałowska et al., 2013).
Although there have been few reports on the use of a mixed culture in glycerol fermentation, one common problem was the difficulty of maintaining microbial communities in a mixed culture. In 2013, Liu et al. studied the effect of fresh soil and old soil as the inoculum for the production of PDO. It was found that after 6 months of soil storage, the microbial community had changed, where the yield of PDO was reduced from 0.49 mol/mol to 0.44 mol/mol. Besides, Selembo et al. (2009) reported that a different substrate can significantly affect which bacteria become predominant, and the yield of products. As glycerol waste from biodiesel plants varies according to its composition, the use of crude glycerol in a mixed culture fermentation has to be studied in detail. The effects of impurities and pre-treatments on crude glycerol vary with the microorganism sources used (Samul et al., 2014). Recently, the study of the regulation of the pH in glycerol fermentation through the use of a mixed culture was carried out (Moscoviz et al., 2016). However, the experiment was conducted with only pure glycerol in order to reduce other variabilities that resulted from a mixed culture fermentation.
Due to the formation of acetate and butyrate as the bacteria grow, glycerol assimilation by Clostridium sp. resulted in low pH after the fermentation. During the glycerol fermentation without any pH control, the pH of the extracellular medium dropped below the dissociation constants of acetate (pKa = 4.76) and butyrate (pKa = 4.8) (Monot et al., 1984), which resulted in the undissociated form of the acids in equilibrium. Consequently, the undissociated acetic and butyric acids were diffused across the bacterial membrane into the cell, and were dissociated at the more alkaline region (Russell & Diez-Gonzalez, 1997). However, the anionic species of the acids
were unable to pass across the membrane, thus causing them to remain in the intracellular fluids. These anionic species acted as an uncoupler, where the proton motive force was not dissipated in a cyclic manner as more energy was utilized for the maintenance of the pH in the cell (Van Ginkel & Logan, 2005). As a result, the fermentation was inhibited and the PDO production was halted. A research conducted by Colin et al. (2001) showed that the undissociated acetate and butyrate acids caused a reduction in the PDO and biomass, respectively.
It is more preferable to use a bioreactor approach in conducting fermentation due to the automatic adjustment of crucial parameters, such as the pH of the media (Wu et al., 2008). Nevertheless, the addition of diluted acids or bases to control the pH can dilute the fermentation media, where the dilution factor has to be considered. The use of a pH-buffer in glycerol fermentation serves as an alternative in the pH control, particularly in shake flasks or serum vials. Calcium carbonate is widely used as a pHbuffer due to its low cost and non-hazardous properties (Ai et al., 2014). During the screening of Clostridium diolis 15410, 1 g/L of calcium carbonate was added as the pH buffer (Otte et al., 2009). However, Ringel and co-workers used 30 g/L of CaCO3 in their screening work to select the best PDO producer (Ringel et al., 2012). The addition of CaCO3 maintained the pH of the media within the range of 5 – 9 during the fermentation, even when added in excess. As calcium carbonate is insoluble, a higher concentration of the pH buffer in the media provided a larger buffer capacity to neutralise the volatile fatty acids (Ai et al., 2014). A proteomic analysis showed that the addition 4 g/L of CaCO3 had higher positive effects than a concentration of 2 g/L in
acetone-butanol-ethanol (ABE) fermentation, but there was no further improvement with more than 4 g/L of calcium carbonate (Han et al., 2013).
The aim of this study was to isolate a pure culture of a local1,3-propanediol producer that was able to utilise glycerol as the sole carbon source. It was then evaluated for its glycerol consumption, PDO yield and productivity by using a bioreactor for batch fermentation. Calcium carbonate was added to the fermentation medium, which was then screened for optimal buffer loading with different glycerol concentrations. A statistical analysis was used to further investigate the effect of calcium carbonate on the PDO production. Finally, batch fermentation was performed in a bioreactor by using the glycerol concentration with the highest productivity and optimal buffer loading, which was regarded to replace potassium hydroxide as the pH neutraliser.
2.0
Materials and Methods
2.1
Source of Microorganisms
The bacteria were isolated from palm oil mill effluent (POME), which was collected from Sime Darby East Oil Mill, Selangor, Malaysia.
2.2
Isolation of Local Producer
The bacteria were isolated by using a synthetic agar medium containing the following components per litre of distilled water: K2HPO4, 3.4 g; KH2PO4, 1.3 g; (NH4)2SO4, 2.0 g; MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.02 g; yeast extract, 1.0 g; and agar powder, 20 g. Pure glycerol at a concentration of 20g/L was solely used as the carbon source for the
isolation. The enrichment was conducted by inoculating the isolate with Reinforced Clostridium Medium, RCM (BD Difco), with the following composition in one litre of distilled water: peptone, 10g; beef extract, 10 g; yeast extract, 3 g; dextrose, 5 g; sodium chloride, 5 g; soluble starch, 1 g; L-Cysteine HCl, 0.5 g, sodium acetate, 3 g; and agar. 0.5 g. All the media were autoclaved at 121ºC for 20 minutes prior to being dispensed onto petri plates. The pH of the autoclaved media was 6.8 ± 0.2.
A sample of 0.2 mL of non-sterilized POME was cultured in Hungate tubes containing 5 ml of a glycerol-based culture medium. The tubes were heat-shocked at 80ºC for 10 minutes to remove any methanogens in the samples. The tubes were then incubated for 48 hours at 37ºC with nitrogen gassing for 10 minutes. Next, 1 mL of the suspension was then re-inoculated into a Hungate tube containing 9 mL of melted synthetic medium agar. Roll tube method was used for the cultivation, where the inoculum was solidified on the surface of the tube. The colonies that were found on the surface of tube after three days of incubation were then transferred to an agar plate containing RCM for enrichment. All the agar plates were incubated in an anaerobic jar for three days. A purified Clostridium colony was then further identified using the 16S rRNA coding sequence.
2.3
Bacteria Identification
DNA from the extracted isolate was amplified using the reverse primer, 1492R (5-GGT TACCTTGTTACGACTT-3’), and the forward primer, 27F (3’AGAGTTTGATCMTGGCTCAG-5’). A polymerase chain reaction (PCR) was carried out with a reaction volume of 25 µl containing crude bacterial lysate DNA, 10 pmol of
each primer, deoxynucleotides triphosphates (dNTPs, 400 µM each), 0.75 U Taq DNA polymerase, and the supplied buffer. The PCR was performed as follows: one cycle for the initial denaturation at 95ºC for 5 minutes, 30 cycles for annealing and extension (95ºC for 45 seconds; 51ºC for 15 seconds; 72ºC for 2 minutes), and one cycle for the final extension of the amplified DNA at 72ºC for 10 minutes. The products were purified and sequenced with primers 518F and 800R using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).
The sequences encoding the 16S rRNA of the isolate were identified using NCBI Nucleotide BLAST programme. A phylogenetic tree was constructed using the Top 10 Hit Blast results against the NCBI 16S rRNA sequences (bacteria only) database, excluding the uncultured bacterium (tax id: 77133). The tree was inferred using the neighbour-joining method, with the percentage of replicated trees, in which the associated taxa clustered together in the bootstrap test (500 replicates), being shown next to the branches. The evolutionary distances were computed using the Jukes-Cantor method.
Microbial observations were made using a field emission scanning electron microscope (FESEM) (SUPRA 55VP, Carl Zeiss AG). The isolate was harvested by the microcentrifugation of the culture at 2,000 rpm for 5 minutes. It was then mixed with 2 % glutaraldehyde for 20 hours and refrigerated at 4ºC. Next, 0.1 M phosphate buffer solution was used to wash the pellets three times for 10 minutes each time. Dehydration was carried out by consequent immersion in 30, 50, 70, 80, and 90% ethanol, and finally three times in 100% (v/v) ethanol, for 10 minutes each time. The dehydrated samples
were then immediately dried in a critical point dryer (Leica EM CPD 300) for 1.5 hours. The dried samples were sputter-coated with platinum, and subsequently, analysed using FESEM.
2.4
Batch Fermentation in Bioreactor
The fermentation media used in this study contained (per litre of water) K2HPO4, 3.4 g; KH2PO4, 1.3 g; (NH4)2SO4, 2.0 g; MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.02 g; yeast extract, 1.0 g; L-Cysteine HCl, 0.5 g; 5 g/L resazurin solution, 1 ml; Fe solution, 1 ml; and trace elements solution, 2 ml (Biebl, 1991; Günzel et al., 1991). The Fe solution consisted of FeSO4·5H2O, 5 g/L; and 37% hydrochloric acid, 4 ml/L. The composition of the trace solution (per one litre of water) included ZnCl2, 70 mg; MnCl2.4H2O, 100 mg; H3BO3, 60 mg; CoCl2·2H2O, 200 mg; CuCl2·2H2O, 20 mg; NiCl2·6H2O, 20 mg; and Na2MoO4·2H2O, 40 mg. Pure glycerol with a concentration ranging from 20-120 g/L was added to the medium as the sole carbon source. In addition, crude glycerol obtained from a biodiesel plant was also evaluated on its performance in PDO production. 20 g/L of crude glycerol was used for comparison with pure glycerol.The fermentations were carried out in a 3.6-litre Labfors 5 bioreactor (Infors HT), with a working volume of 1 litre. The pH was set at 7.0, and it was controlled by adding 2M potassium hydroxide automatically. Samplings of the fermentation were taken at hourly intervals.
2.5
Effect of Calcium Carbonate as pH Neutraliser
The pH-buffered fermentations were run with identical fermentation media, but with additional 10 to 30 g/L of calcium carbonate (CaCO3). The 10% v/v inoculum used in
the experiment was transferred into 130-mL serum vials with a working volume of 50 mL by using sterile syringes. Anaerobic conditions were maintained by capping the vials in a Bactron I-2 anaerobic chamber (Shel Lab), with a gas mixture (80% N2, 10% CO2, and 10% H2) and using resazurin solution as an indicator. The fermentation was carried out at 35ºC with an agitation speed of 200 rpm for 24 hours. The experiments were conducted in triplicate, where independent samples were taken at the initial stage (T= 0 h) and at the end of the fermentation process (T=24 h).
A statistical analysis of the data was performed using the IBM SPSS Statistics 21.0. One-way ANOVA was used to investigate the effects of glycerol and the buffer loading on the pH, the PDO titre, acetate and butyrate, glycerol consumption, and yield of PDO. Univariate ANOVA was used to investigate the relationship between these two effects. A significance level of P < 0.05 was used (Saratale et al., 2016). Turkey’s HSD Post-hoc test was conducted right after the ANOVA test to confirm the significant difference between the levels in calcium carbonate loading and glycerol concentration, respectively. As a final verification, the optimal calcium carbonate and glycerol loadings were used in the batch fermentation using a bioreactor.
2.6
Analytical Methods
Glycerol, 1,3-propanediol, acetic acid and butyric acid were analysed using high performance liquid chromatography (Thermo Scientific UltiMate 3000), equipped with a Phenomenex RoA 300 mm × 7.8 mm column and a refractive index detector (RID). In addition, 5 mN of H2SO4 was used as the mobile phase, which was eluted isocratically at a flow rate of 0.6 ml/min. The column temperature was set at 60ºC while it was
detected by using the refractive index at 40ºC. The retention time for each component was as follows: glycerol, 11.94; acetic acid, 16.67; 1,3-propanediol, 20.53; and butyric acid, 25.27 minutes, respectively.
3.0
Results and Discussion
3.1
Phylogenetic Analysis
The partial 16S rRNA genetic sequence of the local isolates was compared with the sequence in GenBank. Figure 1 shows the optimal tree with the sum of the branch lengths being 0.0569. According to the phylogenetic tree, Clostridium butyricum VPI3266, Clostridium butyricum JCM1391 and Clostridium butyricum ATCC 19398 also produce 1,3-propanediol from glycerol fermentation. The isolate was named as Clostridium butyricum JKT37 (GenBank No.: KU513553).
A microbial observation was done on the JKT37 isolate under field emission scanning electron microscopy (FESEM). The images were taken after the culture had been incubated for 3 days, where the growth of the isolate was at the stationary phase. The rod-shaped isolates could be identified as pure cultures, where no contaminants were found on the images. The average size of the isolates was 0.62 × 0.62 µm, with lengths ranging from 3.1 to 5.9 µm. The elongation and division of cells could be seen clearly in the images (see Supplementary Materials), thereby indicating that healthy growth was taking place in the culture.
3.2
Fermentation in Bioreactor
The bioconversions of pure glycerol with concentrations ranging from 20 – 120 g/L are summarised in Table 1A, 1C, 1D, 1E and 1F. Table 1B represents the fermentation of crude glycerol at a concentration of 20 g/L as a comparison. Generally, glycerol fermentation by Clostridium butyricum JKT37 produces PDO as the main product, while co-producing acetate and butyrate throughout the fermentation. The fermentation profile for each parameter is shown in Figure 2.
In all the experiments, it was observed that the glycerol was almost completely utilized, with the average yield being 0.6 mol/mol. The fast-growing JKT37 isolate showed an outstanding productivity with regard to PDO, with the maximum productivity being 2.8 g/L.h during batch fermentation. However, the productivity of PDO decreased with an increase in the glycerol concentration, coupled with a decrease in the AA/BA ratio. The accumulation of butyrate during the fermentation inhibited the growth of JKT37, resulting in a lower biomass production and lower glycerol uptake. It was observed in Figure 2F that there was a slow down after 18 hours of fermentation, where the glycerol was not fully consumed at the end of the fermentation. A low AA/BA ratio of 0.30 at the stated experiment indicated a lower ATP yield and low biomass yield (X=1.31 g/L) (Zhang et al., 2009).
Glycerol fermentation in Clostridium butyricum JKT37 consists of oxidoreductive pathways, where glycerol is oxidized to dihydroxyacetone, and progressively to pyruvate (Xin et al., 2016). At the same time, glycerol is also converted to 3hydroxypropioaldehyde, and then reduced to 1,3-propanediol (Deckwer, 1995). The possible products and by-products formed by Clostridium sp. from glycerol metabolism can be mainly represented by biochemical reactions as follows:
(1) PDO production + 2 → +
(2) Acetate production → + 6 + 2 + −
(3) Butyrate production 2 → + 8 + 3 + 2
(4) Biomass generation 4 + 3 + 35 → 3 + 8 + 6
It was assumed that the molecular formula of the biomass was C4 H7O2N, with a yield of 8.6 g/mol of ATP (Zeng, 1996). The maximum theoretical yield for PDO production is 0.72 mol/mol due to the reducing cofactors in the reductive pathways that have to be regenerated by converting glycerol into pyruvate via the oxidative pathway, thus producing acetate as a by-product (Zeng, 1995; Zeng, 1996). Nevertheless, most of the experimental works on glycerol fermentation produced acetate and butyrate as byproducts. For the equal utilization of cofactors in the fermentation, the maximum theoretical yield for PDO production decreased to 0.69 mol/mol. In the current study, the experimental yield by JKT37 reached 0.60 mol/mol, which was equivalent to 87% of the maximum theoretical yield. Table 2 gives a summary of the PDO production from batch fermentation in a bioreactor scale. Surprisingly, the microorganism isolated
from this study was considered to have among the highest productivity compared to previous studies. One reason for this promising result was the rapid growth of JKT37 (µ max= 0.53 h-1), where no lag phase was observed during the fermentation. This result was comparable to the estimated µ max of 0.527 h-1 when the Contois model was used in a modelling study (Papanikolaou & Aggelis, 2003).
3.3
Effect of Calcium Carbonate as pH Neutraliser
Calcium carbonate was added to serve as an acid buffer pool to neutralise the volatile fatty acids produced by the bacteria. An increase in buffer concentration enabled the accommodation of more acetate and butyrate, thereby, allowing a higher conversion of glycerol. The experiments were carried out with calcium carbonate loadings ranging from 10 to 30 g/L. The experimental data are shown in Table 3. Note that for glycerol fermentation at a concentration of 20 g/L, the carbonate loading was tested only between 10 – 20 g/L due to the complete conversion of glycerol under the abovementioned conditions, where further additions of calcium carbonate did not show any additional positive effect on the fermentation (data not shown). Therefore, the investigation of the calcium carbonate loadings focused on the higher initial glycerol concentrations, i.e. ranging from 40 g/L to 120 g/L. Generally, the final pH for all the samples was slightly acidic due to the presence of a buffer in the fermentation medium. An obvious improvement was observed when the carbonate loading was from 10 to 15 g/L, as shown in Figure 3. Further increments in the carbonate loading did not have much effect on the fermentation. The best PDO result recorded was a concentration of 19 g/L that was obtained from 40 g/L of glycerol with a carbonate loading of 15 g/L.
Nearly all the glycerol at concentrations of 20, 40 and 60 g/L was consumed within 24 hours by the fermentation process. In contrast, the consumption of glycerol declined to half at concentrations higher than 80 g/L. Note that there was not much change in the final pH after 24 hours of fermentation, and therefore, the effect of the dissociated butyrate and acetate could be neglected. It was presumed that at high glycerol concentrations, the osmotic and hydrogen gas pressure may have inhibited bacterial growth (Szymanowska-Powałowska, 2015).
In order to investigate the effect of glycerol and calcium carbonate on the effectiveness of the fermentations, the data in Table 3 were used for the one-way ANOVA calculations. The response parameters, including the pH, PDO titre, acetate and butyrate, glycerol consumption and PDO yield, were set as dependent variables, whereas the glycerol and calcium carbonate concentrations were set as factors in each analysis, as shown in Table 4. The glycerol concentration acted as a direct factor on the pH, PDO, acetate and butyrate, and glycerol consumption with a p-value of less than the significance level at 0.05. In contrast, the calcium carbonate loading did have a significant effect on the yield of PDO production (p = 0.027). However, the one-way ANOVA tests did not show the specified levels of glycerol or calcium carbonate concentrations that were significant.
As such, Turkey’s HSD post-hoc tests were conducted to further investigate the matter through multiple comparison tables (see Supplementary Materials). The results showed that fermentation of 20 g/L of glycerol had a significant effect on the pH compared to glycerol concentrations at 40 g/L and above. Additionally, there was no
significant difference at 40 g/L and higher concentrations of glycerol. In this case, 40 g/L was the optimal glycerol concentration in the batch fermentation. On the other hand, there was a significant difference in the PDO titre when 20g/L of glycerol was used in the fermentation compared to higher concentrations of glycerol. However, by using the same comparison method, 60 g/L of glycerol was found to be the optimal concentration for the highest PDO titre. While acetate and butyrate are dependent on the consumption of glycerol, the results of the glycerol consumption in the post-hoc tests could be used to confirm the optimal concentration of glycerol. The results showed that 20 g/L of glycerol consumption had no significant effect as compared to 40 – 120 g/L of glycerol. However, there were significant differences between 40 g/L and 60, 80, 100, and 120 g/L of glycerol. It was noted that at a concentration of 80 g/L of glycerol, there were no significant differences in the consumption with 100 and 120 g/L. As 20 and 40 g/L of glycerol fermentation can achieve almost complete consumption, further increases in the concentration of glycerol significantly reduced the glycerol consumption. In addition, the inhibition of the partial pressure of hydrogen could be observed from 80 g/L of glycerol and beyond, which explained the lack of significant differences in 100g/L and 120 g/L of glycerol. These post-hoc tests reaffirmed that 40 g/L is the optimal glycerol concentration for fermentation.
Besides, the post-hoc test for the effect of calcium carbonate on the yield of PDO showed that there were significant differences between 30 g/L and 10 g/L of glycerol (p = 0.038), and with 20 g/L of glycerol (p = 0.030). However, the results were inconclusive. A univariate ANOVA was conducted to analyse the optimal calcium carbonate loading for fermentation. PDO was set as the dependent factor since it could
directly explain the quality of fermentation, whereas the glycerol concentration and calcium carbonate loadings were set as fixed factors. The significance level for this analysis was also set at 0.05. With reference to Figure 4, it was deduced that the calcium carbonate loading was optimal when the concentration was at 15 g/L. It was observed that CaCO3 had no synergistic effect on the PDO production at concentrations higher than 15 g/L.
In addition, the univariate ANOVA was used to investigate the correlation between the glycerol concentration and the calcium carbonate loading. As shown in Table 5a, different glycerol concentrations had a significant effect on the titre of PDO (p = 0.040), whereas calcium carbonate alone and the interaction between these two factors showed no significant differences. A univariate ANOVA was also conducted with the same fixed factors, but the yield of PDO was chosen as the dependent variable. The results in Table 5b show that only calcium carbonate had a significant effect on the yield of PDO (p = 0.019), whereas the glycerol concentration and the interaction between these two factors showed no significant differences.
The results from the statistical analyses suggested that 15 g/L of calcium carbonate be added in place of potassium hydroxide in the fermentation of 40g/L glycerol. The evaluation of the stated condition was conducted in a 3.6-litre bioreactor. As observed from Figure 5, there was a lag phase of 2 hours before the glycerol was converted into PDO due to the high initial pH (pHo=8.85) of fermentation. The effect of calcium carbonate as a buffer was clearly observed after 4 hours of fermentation, where a constant pH of 6 was maintained until the fermentation ended. It was observed that
the yield of PDO was 0.60 mol/mol, but with lower productivity of 1.96 gL-1h-1. From this experiment, it was concluded that calcium carbonate can act as a pH neutralizer in place of a pH controller in the production of PDO.
Conclusion Clostridium butyricum JKT37 was isolated as a highly potential 1,3-propanediol producer in the present study, whereby its productivity surpassed most of the previously reported natural producers by 150%. Unlike the common pH controllers in bioreactors, calcium carbonate was used as a pH neutraliser to accommodate the effect of the acetate and butyrate produced during glycerol fermentation. An optimal concentration of 15g/L of calcium carbonate was statistically proven to enhance the fermentation, without affecting the yield of 1,3-propanediol. The evaluation a bioreactor fermentation showed that a similar yield for 1,3-propanediol was achieved compared with the addition of bases during fermentation.
Acknowledgements The authors would like to thank the Ministry of Higher Education for its financial support (FRGS/1/2014/SG05/UKM/01/2). We would also like to forward our gratitude to Sime Darby East Oil Mill for providing the effluent for isolation purposes.
Competing financial interests The authors declare no competing financial interests.
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Figure captions Figure 1 Phylogenetic tree showing position of Clostridium butyricum JKT37 among top 10 BLAST results
Figure 2 Profiles of products and by-products of glycerol fermentation at concentrations of (A) pure 20 g/L,(B) crude 20 g/L,(C) pure 40 g/L,(D) pure 60 g/L, (E) pure 80 g/L, and (F) pure 120 g/L. E and F were plotted at intervals of 2 and 3 hours respectively, although the samples were taken hourly.
Figure 3 Titre of PDO for fermentation at high glycerol concentrations. Data shown as the average of triplicates
Figure 4 Estimated Marginal Means of PDO at different calcium carbonate loadings
Figure 5 Profile of glycerol fermentation using calcium carbonate as pH neutraliser
Table 1: Glycerol fermentation of JKT37 at different glycerol concentrations Experiment Glycerol (g L-1) Fermentation time (h) Glycerol (g L-1) PDO (g L-1) Acetate, AA (g L-1) Butyrate, BA (g L-1) AA/BA ratio Biomass, X (g L-1) µ max (h-1) Yield (mol/mol) Productivity (g L-1 h-1)
A 20 ± 0.4 5 0 ± 0.0 10.29 ± 0.2 2.21 ± 0.1 1.40 ± 0.1 1.57 1.64 ± 0.1 0.58 0.62 2.06
B 20 ± 0.4 6 0 ± 0.1 10.77 ± 0.4 2.33 ± 0.2 1.18 ± 0.1 1.97 1.64 ± 0.1 0.57 0.65 1.80
C D 40 ± 0.9 60 ± 0.7 7 13 0 ± 0.0 0.85 ± 0.1 19.59 ± 0.3 30.24 ± 0.8 3.41 ± 0.2 4.44 ± 0.5 3.01 ± 0.5 5.42 ± 0.2 1.13 0.82 2.00 ± 0.0 1.95 ± 0.1 0.53 0.56 0.6 0.59 2.80 2.33
E 80 ± 1.0 24 0.51 ± 0.1 42.61 ± 0.7 4.35 ± 0.3 8.5 ± 0.2 0.51 1.78 ± 0.1 0.57 0.59 1.76
Note: The readings are the average values from three independent analyses for the same sample. The range of errors is displayed where necessary.
F 120 ± 1.8 37 4.38 ± 1.8 56.53 ± 1.4 4.46 ± 0.2 15.06 ± 1.3 0.30 1.31 ± 0.2 0.34 0.53 1.52
Table 2 Various batch PDO production from glycerol fermentation in bioreactor Microorganism C. butyricum DSM 5341 C. butyricum VPI 3266 C. butyricum CNCM 1211 C. butyricum F2b C. butyricum VPI 1718 C. butyricum DSP1 C. butyricum DSM 10702 C. beijerinckii DSM 791 C. diolis DSM 15410 C. butyricum M01 C. butyricum JKT37
Glycerol (g/L) 110
Time (h) 29
Titre (g/L) 56
Yield (g/g) 0.51
Productivity (g/L.h) 1.9
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65
48
35
0.54
0.72
121
39
65.4
0.55
1.67
40
26
22
0.55
1.2
61
54
33.6
0.55
0.65
70
33
37.6
0.53
1.12
70
64
40
0.57
0.63
33
24
17.5
0.55
0.72
20.93
9
11.64
0.56
1.29
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2.49
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40
7
19.59
0.49
2.80
This study
Biebl et al. (1992)
Table 3 Experimental data for fermentation at different calcium carbonate loading CaCO Glycer Consumptio ol pH PDO Acetate Butyrate n Yield 3 mol/m g/L g/L g/L g/L g/L % ol 6.6 0.82 ± 2.51 ± 10 20 5 6.28 ± 0.43 0.11 0.24 99.28 0.498 6.1 16.45 ± 1.86 ± 5.25 ± 40 4 1.52 0.34 0.87 95.56 0.473 6.1 21.00 ± 2.94 ± 3.95 ± 60 0 1.18 0.51 0.55 69.53 0.596 6.0 15.11 ± 0.45 ± 2.25 ± 80 1 4.31 0.13 0.30 47.08 0.420 6.0 21.54 ± 1.85 ± 4.47 ± 100 6 1.91 0.44 0.31 40.79 0.596 6.2 16.47 ± 2.18 ± 2.23 ± 120 9 0.75 0.18 0.18 41.03 0.454 6.6 1.31 ± 2.45 ± 15 20 4 7.32 ± 0.26 0.20 0.24 99.07 0.490 6.2 19.07 ± 3.43 ± 4.55 ± 40 3 0.98 0.47 0.27 97.03 0.503 6.2 26.00 ± 4.78 ± 7.69 ± 60 0 2.69 0.64 1.13 98.86 0.482 6.1 26.20 ± 4.01 ± 5.16 ± 80 9 0.32 0.79 0.74 56.50 0.552 6.2 22.52 ± 3.68 ± 3.40 ± 100 1 1.37 0.73 0.31 53.74 0.514 6.2 20.67 ± 2.26 ± 3.29 ± 120 6 2.82 0.15 0.22 41.11 0.415 6.6 1.68 ± 2.71 ± 20 20 4 6.95 ± 0.64 0.25 0.19 99.13 0.472 6.3 18.80 ± 2.35 ± 5.61 ± 40 0 1.17 0.33 0.46 98.92 0.549 6.2 23.66 ± 4.08 ± 3.83 ± 60 6 1.13 0.58 0.37 71.87 0.576 6.1 25.58 ± 4.56 ± 5.21 ± 80 3 0.79 0.28 0.20 68.66 0.513 6.2 22.47 ± 3.69 ± 2.42 ± 100 9 1.90 0.40 0.23 51.32 0.486 6.3 18.50 ± 2.34 ± 2.57 ± 120 5 2.18 0.43 0.19 42.10 0.462 6.3 18.26 ± 3.22 ± 4.73 ± 25 40 3 0.98 0.17 0.28 99.35 0.493 6.2 22.90 ± 4.17 ± 4.47 ± 60 3 1.51 0.31 0.34 80.30 0.469 80 6.1 24.62 ± 3.47 ± 5.02 ± 63.57 0.528
100 120 30
40 60 80 100 120
8 6.1 6 6.3 5 6.4 4 6.1 9 6.3 4 6.3 6 6.3 9
1.82 24.51 ± 1.69 20.07 ± 2.08 17.22 ± 1.19 21.64 ± 1.55 20.25 ± 0.62 19.30 ± 1.30 16.46 ± 1.70
0.27 4.20 ± 0.30 1.85 ± 0.17 2.15 ± 0.21 4.19 ± 0.26 2.97 ± 0.19 2.18 ± 0.37 2.44 ± 0.31
0.44 4.64 ± 0.30 3.38 ± 0.41 5.43 ± 0.67 7.66 ± 0.83 2.91 ± 0.16 3.19 ± 0.37 2.36 ± 0.35
63.56
0.424
46.91
0.434
99.71
0.385
91.43
0.483
55.11
0.441
50.89
0.377
45.22
0.334
Table 4 One-Way ANOVA based on the factors of the glycerol and CaCO3 concentrations Glycerol pH PDO Acetate Butyrate Consumption Yield
Sum of Squares 0.524 632.544 17.445 32.324 13148.077 0.027
df 5 5 5 5 5 5
Mean Square 0.105 126.509 3.489 6.465 2629.615 0.005
F 12.389 20.221 4.106 4.871 47.721 1.479
Sig. 0.000 0.000 0.009 0.004 0.000 0.237
pH PDO Acetate Butyrate Consumption Yield
Sum of Squares 0.070 104.754 10.948 4.808 270.769 0.040
df 4 4 4 4 4 4
Mean Square 0.018 26.189 2.737 1.202 67.692 0.010
F 0.629 0.905 2.499 0.487 0.111 3.323
Sig. 0.647 0.477 0.071 0.745 0.978 0.027
CaCO3
Table 5a Univariate ANOVA based on glycerol concentrations and calcium carbonate loadings on PDO titre
Source Type III Sum of Squares Corrected Model 227.648 Intercept 8.536 CaCO3 57.341 Glycerol 106.415 CaCO3 * Glycerol 42.045 Error 542.535 Total 11177.527 Corrected Total 770.183
df 3 1 1 1 1 24 28 27
Mean Square 75.883 8.536 57.341 106.415 42.045 22.606
F 3.357 0.378 2.537 4.707 1.860
Sig. 0.035 0.545 0.124 0.040 0.185
Table 5b Univariate ANOVA based on glycerol concentrations and calcium carbonate loadings on PDO yield Source Type III Sum of Squares Corrected Model 0.070 Intercept 0.826 CaCO3 0.017 Glycerol 0.016 CaCO3 * Glycerol 0.015 Error 0.039 Total 6.540 Corrected Total 0.109
df 11 1 1 5 5 16 28 27
Mean Square 0.006 0.826 0.017 0.003 0.003 0.002
F 2.600 339.285 6.795 1.321 1.268
Sig. 0.040 0.000 0.019 0.305 0.325
Highlights • A novel strain for PDO production was isolated from palm oil mill effluent. •
Batch productivity in bioreactor can achieve as high as 2.80 g/L.h.
•
Proposed the use of calcium carbonate to maintain pH range during fermentation.
•
A control fermentation volume was established without the addition of acid or base.