Research Article | Volume: 8, Issue: 6, Nov-Dec, 2020

Media optimization studies and production of adenosylcobalamin (Vitamin B12) by environment friendly organism Rhizobium spp

Neha Nohwar Rahul V. Khandare Neetin S. Desai   

Open Access   

Published:  Nov 25, 2020

DOI: 10.7324/JABB.2020.80607
Abstract

Production of Vitamin B12 from microbial sources has many advantages over conventional chemical synthesis. In the present investigation, an attempt was made to isolate and characterize the environment friendly symbiotic Rhizobium species from its natural host, Sesbania sesban (L) root nodules, as source of adenosylcobalamin producer. A total of 75 isolates of Rhizobium were obtained and characterized by morphological, biochemical, and molecular methods. All the isolates obtained, produced the compound of interest in the range of 0.5–7 ppm. Two isolates, namely, AMB and PMT4 showed higher production of Adenosylcobalamin than the others. These isolates, on optimization showed increased production (28±0.26 ppm and 19±0.26 ppm). Beet Molasses, Cobalt Nitrate, and 5,6 DMB were found to be essential components for adenosylcobalamin production. Further, although betaine and Choline Chloride were revealed to affect the cell growth, they could elicit Adenosylcobalamin production. Thus, Rhizobium species has dual advantage as Vitamin B12 producer and as nitrogen fixing environment friendly organism. Further studies are warranted for genetic improvement to enhance Vitamin B12 production without affecting its nitrogen fixing ability.


Keyword:     Rhizobium sp. Sesbania sesban Adenosylcobalamin Media optimization Submerged fermentation.


Citation:

Nohwar N, Khandare RV, Desai NS. Media optimization studies and production of adenosylcobalamin (Vitamin B12) by environment friendly organism Rhizobium spp. J App Biol Biotech. 2020;8(6):38-47. https://dx.doi.org/10.7324/JABB.2020.80607

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. INTRODUCTION

There is an increasing consciousness about nutrition and health all over the globe. However, the developing world is still burdened with the problem of under and malnutrition. This directly relates to vitamin deficiencies [1]. Vitamin B12 is highly essential and one of the most fascinating molecule in the world of nutrition. It is crucial in the production pathways of fatty acids and thus the bioenergetics. It was initially discovered as a treatment of pernicious anemia[2]. Prolonged deficiency of Vitamin B12 leads to irreversible neurological damage. Clams, eggs, oysters, fishes, meat, and milk are known sources of Vitamin B12. However, under specific conditions, intestinal microbes produce Vitamin B12, anaerobically [3,4].

Hydroxycobalamin (OH-Cbl), 1,5–deoxyadenosylcobalamin (Ado-cbl), and methylcobalamin (Me-Cbl) are the natural forms of Vitamin B12 which are produced by microbes. Cyanocobalamin (CN-Cbl) is not the natural form of Vitamin B12 but is commercially synthesized because of its stable structure. Physiologically, Vitamin B12 is essential to maintain myelin sheath of the nerve cells. In addition, it is an essential nutrient for fat and carbohydrate metabolism, and synthesis of DNA in the bone marrow during the formation of red blood cells (RBCs). The deficiency of Vitamin B12 affects growth and development of RBCs leading to megaloblastic anemia [5]. Ado-Cbl and Me-Cbl act as cofactors for the enzymes methyl malonyl coenzyme A (CoA) mutase and methionine synthase [2]. The production of Vitamin B12 from microbial sources involves, approximately, 30 enzymatic steps either through anaerobic pathway as observed in Lactobacillus reuteri, Propionibacterium shermanii, Salmonella typhimurium, and Bacillus megaterium or through aerobic pathway as apparent in Pseudomonas denitrificans [6]. Chemical synthesis of Vitamin B12, in contrast, is a 60 step extensive process [7]. The production of Vitamin B12 from microbial sources is considered as an alternative method because of its simple process. The production of Vitamin B12 has been commercially achieved using bacterial strains such as Pseudomonas, Nocardia and Propionibacterium [8,9] and a higher productivity has been reported from Cobalt-resistant strain of Propionibacteria [4]. Selection of natural Vitamin B12 producers is an endorsed strategy as it does not have any legislative hurdles [10,11]. Various metabolic engineering strategies have been reported for enhanced production of Vitamin B12 in P. freudenreichii [12]. A genetically modified P. Freudenreichii strain harboring a plasmid comprising hemA, from Rhodobacter sphaeroides, and homolog of hemB and cobA, showed 2.2-fold increase in Vitamin B12 [9,13]. These studies have shown that multigene expression systems increase the Vitamin B12 production in Propionibacteria. However, other strategies of enhanced supply of precursors such as aminolevulinic acid and allied intermediates were found to be beneficial [14].

The genetically engineered strain of P. denitrificans gave around 100–300 mg/l of productivity [8]. Commercial productions mostly rely on the strains which show rapid growth and high productivity and therefore adoption of genetically modified microbes is advocated.

The main focus of this study was to isolate the root nodulating, nitrogen fixing bacterial strains having potential to produce Vitamin B12 and optimization of media for its high recovery.


2. MATERIALS AND METHODS

Sesbania sesban was collected from eight industrial areas around Mumbai city [Table 1]. A total of 120 test isolates were obtained from the root nodules. All isolates were morphologically and biochemically characterized, and 75 isolates were confirmed using 16s RNA sequencing [15]. The confirmed stains were preserved in a glycerol stock at −80oC. These 75 isolates were screened for the production of adenosylcobalamin using Submerged Fermentation Technique. The culture was inoculated into 250 ml Erlenmeyer flasks containing 30 ml of media [Table 2] and was incubated at 30°C on rotary shaker at 200 rpm for 48 h. Then, 10% media of this culture were inoculated in 30 ml of seed medium [Table 3] and incubated at 30°C with 200 rpm for 25 ± 1 h. Then, 10% (v/v) seed culture was transferred to 60 ml of production medium in 500 ml of Erlenmeyer flasks [Table 4].

Table 1: Collection site for test sample (Sesbania sesban) – A total of eight industrial areas in the Mumbai Metropolitan region.

IsolatesIndustrial areasLocations
PMTMaharashtra Industrial Development CorprationPanvel
TBTTC Industrial Area , PawneThane Belapur Road
ANMaharashtra Industrial Development CorprationAnand Nagar, Thane
PGMaharashtra Industrial Development CorprationPatalganga
AMBMaharashtra Industrial Development CorprationAmbernath
BDMaharashtra Industrial Development CorprationBadlapur
ZSJindal steel plantKhalapur
MTMaharashtra Industrial Development CorprationTaloja

Table 2: Composition of inoculum media.

ConstituentConcentration(g/l)
Amonium sulfate0.2
Diammonium hydrogen phosphate2.35
Magnous sulfate0.2
Beet Molasses120
Magnesium sulfate2.5
Sucrose40
Zinc sulfate0.2

Table 3: Composition of seed media.

ConstituentConcentration (g/l)
Beet molasses50
Magnous sulfate0.25
Zinc sulfate0.01
Sodium molybdate0.02
Magnesium sulfate0.05
Glycerol3.5
Nutrient broth4.5

Table 4: Composition of production media.

ConstituentConcentration (g/l)
Beet molasses10, 60, 120, 180
Calcium carbonate2
Glycerol1.5
Sucrose8
Choline chloride2.5
Magnesium sulfate1.5
Diammonium hydrogen phosphate2
Ammonium sulfate1.8
Cobalt nitrate0.1, 0.2, 0.3, 0.4
Ferrous sulfate0.02
Magnous sulfate0.02
Monosodium glutamate3
Zinc sulfate0.02
Potassium dihydrogen phosphate0.25
5,6 Dimethyl benzemidiazole0.02, 0.05, 0.1, 0.15
Betaine monohydrate0, 1, 2, 5

After screening, eight (one from each industrial area) out of these 75 isolates were selected as high producers of adenosylcobalamin. Two of these highest adenosylcobalamin producing strains, namely, AMB and PMT4 were selected for further studies.

2.1. Media Optimization for Adenosylcobalamin Production

Medium optimization was carried out by the traditional method of taking one variable at a time and keeping other variables fixed, i.e., varying one factor while keeping all others constant. Using this approach, the important media components were identified, and they were further used to optimize the fermentation media. The effect of varying concentrations of Betaine, Cobalt Nitrate, Beet Molasses, Choline Chloride, and 5,6 DMB on cell growth and adenosylcobalamin biosynthesis, was tested by designing set of experiments using Taguchi’s method [Table 5]. The flasks were incubated at 200 rpm for 7 days at 30°C. 10% (v/v) of 50 % Sucrose feeding was done in a production flask from log 48 to 120 h at 24 h interval.

Table 5: Composition of different sets of Production medium designed using Taguch’s method.

Set of experimentsMedium constituent (g/l)

Betaine monohydrateCobalt nitrate5,6 DMBCholine chlorideBeet molasses
100.10.022.510
200.10.055.560
300.20.16.5120
400.30.157.5180
510.40.056.5180
610.20.027.5120
710.30.152.560
810.40.15.510
920.10.17.560
1020.20.156.510
1120.30.025.5180
1220.40.052.5120
1350.10.155.5120
1450.20.12.5180
1550.30.057.510
1650.40.026.560

2.2. HPLC Analysis

The samples were analyzed using HPLC as per Singh et al., from 120 h onward up to 168 h, respectively [16].

2.3. LC MS Analysis

The samples were analyzed using LC MS method, executive Plus – Orbitrap MS, column details – Hypersil gold 3 micron 100 × 2.1 mm, solvent used: Solvent A – 0.1% food acid in milliq water, solvent B – 100% acetonitrile, run time – 30.000 [min], syringe type – Hamilton, flow rate – 3.000 mL/min, inner diameter – 2.303 mm, volume – 250 mL, polarity – Positive, In-source CID – 0.0 eV, microscans 1, resolution – 70,000, AGC target – 3e6, Maximum IT – 200 ms, number of scan ranges – 1, scan range – 133.4 to 1800m/z.

2.4. Determination of Dry Cell Weight (DCW)

The liquid culture was centrifuged at 5000 rpm for 10 min after fermentation, followed by three washes with distilled water and pellet was obtained. The biomass was then dried to a persistent weight at 100°C.

2.5. Statistical Analysis

The analysis of the data was done with the help of SPSS 24.0 software (SPSS Inc., Chicago, IL, USA). Primary and secondary variables under study were analyzed and statistical such as like percentages; standard deviation and mean were calculated. Logistic regression was applied considering amount of adenosylcobalamin produced by the isolates as the dependent variable and medium components as an independent variable. P < 0.05 was statistically significant.


3. RESULTS AND DISCUSSION

3.1. Screening of Isolates and Media Optimization for Adenosylcobalamin Production

A total of 75 isolates were screened for adenosylcobalamin production [Figure 1]. Out of these, eight significant producers were selected based on their performance and were further taken for characterization and optimization studies [Figure 2]. Finally, two highest producing strains were used for further studies.

Figure 1: Adenosylcobalamin production efficiency of different isolates.



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Figure 2: Media optimization for high producers of adenosylcobalamin.



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For media optimization important components such as Betaine, Cobalt Nitrate, 5,6 DMB, Choline Chloride, and Beet molasses were evaluated at various concentrations [Table 6]. It was observed that betaine was a necessary compound for adenosylcobalamin production, although it had a negative impact on cell growth. A maximum DCW of 22 (± 0.01) g/l only was obtained in presence of betaine with the highest adenosylcobalamin titer (5g/L). With the increased concentration of Choline Chloride in fermentation medium, the DCW decreased gradually, confirming that Choline also has an undesirable effect on cell growth. The low yield after addition of Choline Chloride may be due to change in pH during fermentation. This perhaps had a negative effect on activities of enzymes involved in biosynthesis. For the formation of Methionine, Choline Chloride, and betaine are essential and this amino acid is then converted to S-adenosylmethionine by the action of methionine adenosyltransferase enzyme. Betaine, although, has undesirable effect on cell growth, its feeding during fermentation was found effective to enhance production of adenosylcobalamin. The higher synthesis of Vitamin B12 precursors such as methionine, glutamate, and glycine was reported only after addition of betaine to the production media [9,17]. Choline and betaine increased the formation of adenosylcobalamin in Agrobacterium species by, as much as five to six folds [8,18]. As reduction of production cost is an essential aspect, a low cost carbon source, namely, Beet Molasses was screened. The addition of precursors such as DMB, Cobalt ions, or compatible solutes such as Choline and betaine for adenosylcobalamin was found to be beneficial. Under optimal fermentation conditions approximately 28.57 ± 0.26 mg/l of adenosylcobalamin were accumulated in the fermentation medium during 7 days run [Table 6] while, Rhizobium cobalaminogenum was reported as the most active producers of Cyanocobalamin (16.5 mg/l) [2,14]. Margaret et al. screened 70 strains representing six species of Rhizobium, namely, R. trifoli, Rhizobium meliloti, R. japonicum, R. leguminosarum, R. phaseoli, and R. lupine for Vitamin B12 production in which, R. meliloti showed the highest production (1000 mg/ml) of Vitamin B12 under the experimental conditions. Addition of 1 mg/L of Cobalt Chloride in media for R. meliloti and Bradyrhizobium japonicum was found essential for maximum production of Vitamin B12 [13,19].

Table 6: Media optimization for high producers of adenosylcobalamin (AMB and PMT4) and DCW (at 168 h) under four various concentrations of betaine, DMB, beet molasses, choline chloride, and cobalt nitrate.

Set of experimentsAmount of adenosylcobalamin produced by AMB isolateDCW g/l of AMBAmount of adenosylcobalamin produced by PMT4 isolateDCW g/l of PMT4
Set 12.20±0.1122±0.011.16±0.0320.26±0.19
Set 25.07±0.6021.49±0.153.15±0.0319.54±0.10
Set 35.21±0.3720.50±0.163.66±0.2118.36±0.14
Set 48.37±0.2518.28±0.186.25±0.1715.38±0.33
Set 515.30±0.4015.56±0.1112.29±0.1913.36±0.40
Set 610.37±0.2614.45±0.248.59±0.1812.52±0.41
Set 79.2±0.2915.33±0.198.93±0.0613.29±0.29
Set 88.07±0.4418.57±0.227.06±0.0515.39±0.31
Set 918.37±0.2615.48±0.1114.44±0.2113.64±0.21
Set 1010.36±0.1514.19±0.179.40±0.2412.42±0.44
Set 1115.51±0.2213.82±0.1512.5±0.1911.5±0.17
Set 1219.27±0.4214.45±0.3516.47±0.2112.40±0.48
Set 1328.62±0.2610.43±0.4219.29±0.268.61±0.33
Set 1420.1±0.2510.43±0.3917.72±0.118.03±0.04
Set 1512.37±0.2612.46±0.2910.45±0.2110.5±0.42
Set 1615.48±0.1712.69±0.2913.62±0.1610.51±0.42

The results were means ± SD (standard deviation) of triplicate determinations.

The adenosylcobalamin produced in the media was evaluated using HPLC and MS. The UV-vis spectra data retention time (11 min) obtained by HPLC analysis showed the peak matching to adenosylcobalamin [Figures 3 and 4]. The LC MS analysis of the isolated compound produced by Rhizobium isolate was carried out. The mass of the ionized peak confirmed the presence of adenosylcobalamin in isolated compound produced by Rhizobium isolate. The mass of adenosylcobalamin is 1579.58m/z and its spectra show that it is doubly charged. Mass of 790 m/z was observed instead of their singly charged mass of 1581 m/z. The mass spectrometry data [Figures 5 and 6] showed that the spectra of the compound produced by Rhizobium isolate matched the values of Vitamin B12 analog, adenosylcobalamin.

Figure 3: HPLC spectrum of the adenosylcobalamin standard.



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Figure 4: HPLC spectrum of the extract from Rhizobium isolates AMB.



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Figure 5: Mass spectrometry spectrum of the standard adenosylcobalamin.



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Figure 6: Mass spectrometry spectrum of the extract from Rhizobium isolate AMB.



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Vitamin B12 is a highly essential and indispensable component for normal functioning of human body. Different producer-strains synthesis pathways for production of Vitamin B12 are under consideration by research institutions. New Technologies and strategies are being developed to increase the potential of the microbes for its production.

In the recent years, a lot of interest on this subject has been shown by the researchers in various parts of the world. In one such metagenomic analysis study, it was observed that less than 10% of soil archaea and bacteria (Predominantly Nitrospirae, Proteobacteria, Thaumarchaeota, Actinobacteria, and Firmicutes) could initiate the coding process of the genetic makeup required for de novo production of the enzyme cofactor, which is required for manufacturing adenosylcobalamin. In the same study, the enrichment of DMB and corresponding DMB synthesis genes, relative to corrin ring synthesis genes, suggests an important role for cobalamin remodelers in terrestrial habitats [20].

In another study, the corrinoid compounds production in Lactobacillus (L.) strains (such as L. reuteri CRL 1098 and L. coryniformis CRL 1001) was increased by adding 5,6-dimethylbenzimidazole and Co2+. Whereas, when L-threonine was added, it only increased the production of corrinoid compounds by CRL 1001 strain [21]. As stated above, the biosynthesis of Vitamin B12 is limited to only few bacteria and archaea and depends as such on microbial fermentation. Current innovations in metabolic engineering and synthetic biology are being involved to efficiently construct many microbial chemical factories [22].

In one more study, genes responsible for biosynthesis of adenosylcobinamide phosphate from Rhodobacter capsulatus were studied in vitro and/or in vivo. The analysis suggests that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are same in both the anaerobic and aerobic pathways. The yield of Vitamin B12 from a genetically engineered, recombinant E. coli strain could be increased by more than ∼250-fold to 307.00?mg?g−1 DCW by metabolic engineering and optimizing the favorable conditions required for fermentation [23].

3.2. Statistical Analysis

Selection of media components plays a key role in adenosylcobalamin production. Sixteen sets of experiments were performed using different combinations of variables – Beet Molasses, 5,6 DMB, Choline Chloride, and Cobalt Nitrate as per Taguchi’s method. Independent T-test was performed and P-value represents that the model was significant [refer in supplementary file Table S1]. Correlation analysis was performed and the relationship between the significant variables and the response was determined. It was observed that the Pearson Correlation was positive and the variable under study had beneficial impact on the adenosylcobalamin production. Negative Pearson Correlation for the variable was also observed which had its beneficial effect at the lower concentrations. The above-mentioned relationship between the variables and the response for adenosylcobalamin production was considered in the next stage for regression analysis. Multiple regression analysis was used to analyze the data for adenosylcobalamin production [refer in supplementary file Table S2 - S5]. The regression model’s goodness of fit was checked by multiple correlation coefficients (R2). The model proved to have accuracy, precision and reliability as the R2 value is close to 1. The P-value of the model revealed that the model was significant.

Table S1

Independent t-test

Set of experimentsGroupnMean Standard deviationP value
Set 1AMB isolate32.20330.119300.0001
PMT4 isolate31.16000.03000
Set 2AMB isolate35.07670.601780.005
PMT4 isolate33.15000.03606
Set 3AMB isolate35.21670.375810.003
PMT4 isolate33.66670.21032
Set 4AMB isolate38.37330.254230.0001
PMT4 isolate36.25000.17349
Set 5AMB isolate315.30330.400790.0001
PMT4 isolate312.29330.19140
Set 6AMB isolate310.37000.266270.001
PMT4 isolate38.59670.18877
Set 7AMB isolate39.20000.296140.207
PMT4 isolate38.93670.06658
Set 8AMB isolate38.07330.447360.018
PMT4 isolate37.06000.05292
Set 9AMB isolate318.37000.266270.0001
PMT4 isolate314.44330.21008
Set 10AMB isolate310.36330.159480.005
PMT4 isolate39.40330.24583
Set 11AMB isolate315.51000.226050.0001
PMT4 isolate312.50000.19157
Set 12AMB isolate319.27000.425320.001
PMT4 isolate316.47000.21633
Set 13AMB isolate328.62670.267640.0001
PMT4 isolate319.29330.26083
Set 14AMB isolate320.10000.250600.0001
PMT4 isolate317.72330.11930
Set 15AMB isolate312.37330.261600.001
PMT4 isolate310.45330.21548
Set 16AMB isolate315.48000.176920.0001
PMT4 isolate313.62330.16503
Table S2

Correlations (PMT4)

AMB isolateBetaine monohydrateCobalt nitrate5,6 DMBCholine chlorideBeet molasses
Pearson Correlation0.736**0.0880.157−0.0350.394
P value0.0010.7470.5610.8960.131
n1616161616

Correlation is significant at the 0.01 level (2-tailed).

Table S3

Correlations (PMT4)

PMT4 isolateBetaine monohydrateCobalt nitrate5,6 DMBCholine chlorideBeet molasses
Pearson correlation0.767**0.2200.124−0.0940.375
P value0.0010.4130.6460.7300.152
n1616161616

Correlation is significant at the 0.01 level (2-tailed)

Table S4

Linear regression (For dependent variable: PMT4 isolate)

Unstandardized coefficientstP valueR square change95% confidence interval for B


BStandard errorLower boundUpper bound
Constant6.1021.2904.7320.00013.3368.868
Betaine Monohydrate2.1060.4714.4730.0010.7821.0963.116
Table S5

Linear regression (For dependent variable: AMB isolate)

Unstandardized coefficientstP valueR square change95% confidence interval for B


BStandard errorLower boundUpper bound
Constant7.5641.7444.3360.0013.82311.305
Betaine Monohydrate2.5900.6374.0670.0010.8521.2243.956

4. CONCLUSION

A total of 75 Rhizobium species were screened for the production of adenosylcobalamin using submerged fermentation technique. All the isolates were capable of producing in the range of 2–28 ppm. In comparison to others, isolates AMB and PMT showed high yield (28 ± 0.26 ppm and 19±0.26 ppm). In this study, we reported higher yield of adenosylcobalamin from Rhizobium isolate than that of earlier research work. Betaine and Choline Chloride were found to affect the cell growth; however, they could stimulate the adenosylcobalamin production. It was observed that the Beet Molasses, Cobalt Nitrate, and 5,6 DMB are essential components in production media. Media optimization is necessary for each fermentation process. Thus, Rhizobium species has dual advantage as Vitamin B12 producer and as nitrogen fixing environment friendly organism. Further studies will be warranted for genetic improvement to enhance Vitamin B12 production without affecting its nitrogen fixing ability.


5. AUTHORS’ CONTRIBUTIONS STATEMENT

Neha Nohwar have made substantive intellectual contributions to the content of this manuscript in the areas of concept and design, data acquisition, data analysis/interpretation, drafting manuscript, critical revision of manuscript, statistical analysis, funding, admin, technical or material support, and final approval.

Rahul V. Khandare have made substantive intellectual contributions to the content of this manuscript in the areas of concept and design, data acquisition, data analysis/interpretation, drafting manuscript, critical revision of manuscript, admin, technical or material support, supervision, and final approval.

Neetin S Desai have made substantive intellectual contributions to the content of this manuscript in the areas of concept and design, data acquisition, data analysis/interpretation, drafting manuscript, critical revision of manuscript, admin, technical or material support, supervision, and final approval.


6. ACKNOWLEDGMENT

We would like to thank “Indian Institute of Technology Bombay (IIT B) Maharashtra, India,” for providing the facilities to carry out HPLC and LC MS studies for our research work.


7. Conflict of interest

Authors declared that there are no conflicts of interest.


8. Financial support and sponsorship

None.

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Reference

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2. Martens JH, Barb H, Warren MJ, Jahn D. Microbial production of Vitamin B12. Appl Microbiol Biotechnol 2002;58:275-85.https://doi.org/10.1007/s00253-001-0902-7

3. Kang Z, Zhang J, Zhou J, Qi Q, Du G, Chen J. Recent advances in microbial production of δ-aminolevulinic acid and Vitamin B12. Biotechnol Adv 2012;30:1533-42.https://doi.org/10.1016/j.biotechadv.2012.04.003

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