Coenzyme Q 10 (CoQ 10 ), an important antioxidant molecule playing a major role in electron transport chain, has been commercially produced by fermentation process for the use in oral nutraceutical formulations. Constructing the high-yielding CoQ 10 producing strains is a pre-requisite for cost-effective production. A superior mutant strain P-87 generated from Paracoccus denitrificans ATCC 19367, which showed 1.25-fold improvement in specific CoQ 10 content higher than the wild type strain at shake flask level, was selected to carry out the studies on CoQ 10 yield improvement through fermenter process optimization. In the course of study, initially the cane-molasses-based medium and fed-batch fermentation strategy using pHBA in combination with sucrose were standardized in shake flask using wild type strain. This strategy was subsequently translated at 2 L laboratory fermenter while optimizing the fermentation process parameters using improved mutant strain P-87. Under optimized fermentation condition, mutant strain P-87 produced 49.85 mg/L of CoQ 10 having specific content of 1.63 mg/g of DCW, which was 1.36 folds higher than the specific CoQ 10 content of wild-type strain under similar optimized condition. The temperature and DO were found to be critical parameters for CoQ 10 production by mutant strain P-87. The optimum temperature was found to be 32 °C and the optimum DO concentration to be maintained throughout the fermentation cycle was found to be 30% of air saturation. Overall, a new cost-effective process has been established for the production of CoQ10 using the cheaper substrate “cane molasses” and higher CoQ10 producing mutant strain P-87.
CoQ10 is 2,3-dimethoxy-5-methylbenzoquinone with 10 units of isoprenoid side chain at the 6-position of the quinine ring [
A genetically engineered microorganism synthesizing CoQ10 has also been constructed [
As reported previously, one strain of Rhizobium sp. was elected as a potent CoQ10 producer. Along with the progress of strain improvement, many fermentation condition experiments had been carried out and it has been observed that oxygen supply is one of the important environmental factors in CoQ10 production or other secondary metabolite production by microorganisms [
In our previous study mutant strain P-87 generated through mutagenesis and selection process, showed 1.25- fold improvement in specific CoQ10 content higher than Paracoccus denitrificans ATCC 19367 under shake flask cultivation. In the present study, efforts were made to optimize the CoQ10 fermentation process for mutant strain P-87 in 2 L laboratory fermenter. Initially the media modification and dosing strategy were employed at shake-flask level using Paracoccus denitrificans ATCC 19367 and subsequently the process was transferred to the laboratory fermenter for optimizing fermentation parameters for improved mutant strain P-87. The research work is focused on improving the CoQ10 fermentation process in order to make it cost-effective.
The bacterial strain Paracoccus denitrificans ATCC 19367 and its induced mutant strain P-87 were maintained at 4˚C - 8˚C on Tryptic Soy Agar (TSA) slants. All dehydrated media and media components were procured from Hi-Media, India. All solvents (AR grade) were procured from Merck [
The seed medium contained 60 g of sucrose, 15 g of yeast extract, 15 g of peptone, 5 g of NaCl in 1 L demineralized water and pH 7.2. The 50 ml seed medium in 500 ml conical flask was inoculated with loopful of wild type strain culture on slant and incubated at 30˚C with shaking at 220 rpm. The 10% of grown seed was transferred to 50 ml of different production media in 500 ml conical flask. The production medium PM-A contained 25 g of sucrose, 10 g of (NH4)2·SO4, 0.5 g of K2HPO4, 0.5 g of KH2PO4, 0.25 g of MgSO4·7H2O, 20 g of corn steep liquor (CSL), 20 g of CaCO3, trace element solution 1 ml/L in 1 L demineralized water and pH 7.0. PM-A medium was further modified by changing the concentration of other ingredients to suit CoQ10 fermentation. These media are as followed: PM-B consisting of 50 g of cane molasses, 10 g of (NH4 )2·SO4, 0.5 g of K2HPO4, 0.5 g of KH2PO4, 0.25 g of MgSO4·7H2O, 20 g of CSL, 20 g of CaCO3 in 1 L demineralized water and pH 7.0, PM-C consisting of 50 g of sucrose, 10 g of (NH4 )2·SO4, 0.5 g of K2HPO4, 0.5 g of KH2PO4, 0.25 g of MgSO4·7H2O, 40 g of CSL, 20 g of CaCO3 in 1 L demineralized water and pH 7.0 and PM-D consisting of 80 g of cane molasses, 13 g of (NH4 )2·SO4, 0.5 g of K2HPO4, 0.5 g of KH2PO4, 0.25 g of MgSO4·7H2O, 40 g of CSL, 20 g of CaCO3 in 1 L demineralized water and pH 7.2. The production flasks were incubated at 30˚C with shaking at 220 rpm for 120 h. The best production medium was dosed intermittently with different concentrations of para- hydroxy benzoic acid (pHBA) (5, 10, 20, 25, 40 and 50 mg/L) at 24 h [
The 20 ml of broth was centrifuged at 12,000 rpm for 20 min to get biomass pellet, which was extracted with 20 ml ethanol by heating in shaking water bath at 60˚C for 3 h. The cells were removed by centrifugation and ethanol layer was re-extracted with 20 ml of hexane. The hexane layer was separated, concentrated till dryness and finally reconstituted with 1 ml of hexane. The titer was estimated by comparing the area of sample and standard of known concentration and expressed as mg of CoQ10/L of broth (mg/L). The titer value was divided with DCW to get specific CoQ10 content (mg/g of DCW) [
The CoQ10 extracted from cell biomass was quantified on HPLC (Agilent 1100) using normal phase Kromasil silica column (250 mm × 4.6 mm, 5 μ particle size) and hexane:isopropyl alcohol (95:5) as mobile phase with a flow rate of 1 ml/min. Detection was carried out at 273 nm [
The 10 ml of broth was centrifuged at 12,000 rpm for 20 min in a pre-weighed centrifuge tube. The cell mass was quantified by drying at 60˚C until a constant mass was obtained.
The total sugar was estimated by Anthrone method [
The fermentation process was optimized for mutant strain P-87 using PM-D medium in 2 L Applicon fermenter. A 10% (v/v) seed culture was inoculated into a 2 L fermenter with a working volume of 1 L. The fermentation was carried out by altering the parameters i.e. temperature (25˚C, 28˚C, 30˚C, 32˚C, 35˚C), agitation (300 rpm, 500 rpm, 700 rpm and 900 rpm), aeration (0.3 vvm, 0.5 vvm, 0.7 vvm and 1 vvm) and DO (10%, 20%, 30%, 40% and 50%) of appropriate air saturation [
For analyzing differences between two groups, student’s t-test was used based on PRISM-5 software. P values below 0.05 were considered statistically significant. The values in all graphs are an average of 3 trials. All error bars represent standard error of mean.
The wild type strain of Paracoccus denitrificans ATCC 19367 was found to produce considerably less amount of CoQ10 than the other few bacterial strains [
Being a primary metabolite, a longer cell growing stage would tend to accumulate more biomass and lead to a higher CoQ10 concentration being produced. Hence for CoQ10 fermentation, most researchers have made an effort to increase the biomass by substrate feeding or maintaining high substrate concentration in medium [
other two media namely PM-B and PM-C showed relatively less titer as well as specific CoQ10 content. Based on the media studies, medium PM-D containing 80 g/L of cane molasses and 40 g/L of CSL was found to be best suited medium for screening of mutants in shake flasks.
pHBA is a precursor of aromatic ring of CoQ10 biosynthesis and hence has been used in fed batch fermentation for CoQ10 production. It was observed that significant improvement in CoQ10 content of Sporidiobolus johnsonii was achieved by feeding pHBA precursors, resulted in achieving the maximum content of 10.5 mg/g DCW [
The concentration of sucrose in the fermentation medium is one of the major factors for CoQ10 production using microorganisms [
The best pHBA and sucrose in combination were used for feeding purpose in the next trials of shake flask.
The optimized fed batch process from the shake flask was transferred to the fermenter level with additional optimization studies with respect to fermentation parameters. For this purpose an improved mutant strain P-87 previously generated from Paracoccus denitrificans ATCC 19367 showing improvement in CoQ10 production in shake flask level was used [
During fermentation of Paracoccus denitrificans ATCC 19367 on cane molasses based PM-D medium at shake flask level, dosing was carried out using 25 mg/L of pHBA at 24 h followed by 30% of sucrose solution at 48 h and 72 h respectively. The total sugar concentration dropped from 40 g/L to 17.5 - 18.5 g/L at 46 h and then it was maintained at around 22.5 - 24.5 g/L till end with the help of intermittent dosing at 48 h and 72 h. The same fed batch strategy was adopted for further optimization in fermenter by altering the parameters using mutant strain P-87. During optimization of temperature condition in fermenter, we have tried different temperatures like 25˚C, 28˚C, 30˚C, 32˚C, 35˚C and the results are expressed in
Both agitation and aeration are involved to a different extent in overall mass and oxygen transfer in the process fluid. Agitation controls the nutrient transfer and distribution of air and oxygen. Aeration not only determines the oxygenation of the culture, but also contributes to bulk mixing of the fermentation broth. High agitation promotes good mass transfer but is energy-intensive which increases the production cost. Agitation creates shear forces which cause morphological changes, variation in their growth and product formation, and also damages the cell structure. The combined effect of aeration/agitation and fed batch strategy on CoQ10 production by Pseudomonas diminuta was reported [
The effect of limited supply of air on CoQ10 production by R. sphaeroides was studied. The high aeration decreased the CoQ10 content [
During optimization of agitation condition in fermenter, we have tried different agitations like 300 rpm, 500 rpm, 700 rpm, 900 rpm and the results are expressed in
It was proposed that cell growth and CoQ10 production were affected by various DO concentrations with Agrobacterium sp. and it was also been showed earlier that DO concentration had a great effect on the specific cell growth rate and DCW of Rhizobium radiobacter WSH2601 [
Fermenter operated at temperature 32˚C with appropriate DO concentration of 30% (of air saturation) showed 1.3 folds improvement in titer and specific CoQ10 content than above standardized parameters i.e. agitation 500 rpm, aeration 0.3 vvm and temperature 32˚C as shown in
At identical fermenter condition i.e. 30˚C, 500 rpm, 0.5 vvm, Paracoccus denitrificans ATCC 19367 found to produce 29.23 mg/L with 0.8960 mg/g of DCW specific content whereas at optimized fermenter condition, it produced 34.55 mg/L with 1.1935 mg/g of DCW specific content as shown in
Strain | Condition | Aeration (vvm) | Agitation (rpm) | Temperature (˚C) | Cycle (h) | PCV (%) | Yield (mg/L) | Specific CoQ10 content (mg/g of DCW) |
---|---|---|---|---|---|---|---|---|
Wild type strain | Identical | 0.5 | 500 | 30 | 120 | 8.9 | 29.23 | 0.8960 |
Optimized | Appropriate DO concentration of 30% (of air saturation) | 32 | 120 | 13.3 | 34.55 | 1.1935 | ||
Mutant strain P-87 | Identical | 0.5 | 500 | 30 | 120 | 11.4 | 32.1 | 1.1096 |
Optimized | Appropriate DO concentration of 30% (of air saturation) | 32 | 120 | 17.6 | 49.85 | 1.63 |
in specific CoQ10 content at identical fermentation condition and 1.36 folds improvements in specific CoQ10 content at optimized fermentation condition than Paracoccus denitrificans ATCC 19367 as shown in
At optimized fermenter condition mutant strain P-87 found to produce 49.85 mg/L at 120 h of fermentation cycle with a PCV of 17.6% whereas wild type strain produced 34.55 mg/L with a PCV of 13.3%. It was observed that mutant strain P-87 produced more biomass and titer than wild type strain under two different fermentation conditions as mentioned in
A mutant strain P-87 derived from Paracoccus denitrificans ATCC 19367 was utilized for CoQ10 fermentation optimization studies in 2 L laboratory fermenter. The fed-batch fermentation strategy developed in the shake flask level was transferred to the fermenter with optimization of the fermentation process parameters. The optimized fed-batch condition includes dosing of pHBA at a concentration of 25 mg/L at 24 h followed by 30% of sucrose solution at 48 h and 72 h respectively. The optimized fermentation parameters are at the temperature of 32˚C and at the appropriate DO concentration of 30% (of air saturation). Under optimized condition mutant strain P-87 produced 49.85 mg/L of CoQ10 having specific content of 1.63 mg/g of DCW which is 1.36 folds higher than that produced by wild type strain under optimized fermentation condition. The use of cane molasses in the medium-improved mutant strain and optimized fermentation parameters helped in reducing the cost of production with improved yield. Overall the laboratory scale process for production of CoQ10 using mutant strain P-87 was established.
We acknowledge Dr. Arun Balakrishnan, Senior Vice President, Piramal Enterprises Ltd., Mumbai, India for his support and encouragement towards this project.