Random mutagenesis is a generally an accepted and widely used approach for increasing tolerance of a bacterial strain towards an unknown inhibitor. EMS and N-methyl-N-nitro-N-nitrosoguanidine (MNNG), which both act by direct mutagenesis inducing base substitutions or deletions, have been used with success in the closely related C. acetobutylicum
([Annous and Blaschek 1991
]; Elkanouni et al. [1989
]). Recently, Malaviya et al. ([2012
]) succeeded in developing an effective butanol producing mutant strain of C. pasteurianum
by the use of MNNG. However, both [Lemmel (1985
]) and Syed et al. ([2008
]) obtained C. acetobutylicum
mutants with higher tolerance and productivity using EMS and reported that MNNG was a less effective mutagenic compound. Our success with increased tolerance towards crude glycerol and increased butanol productivity by EMS mutagenesis confirmed the efficiency of EMS for development of C. pasteurianum
mutants. As the mutant strain was developed by chemical mutagenesis it will not be classified as a Gene Modified Organism (GMO) reducing the operating costs and safety precautions necessary to run a GMO-based production.
We have previously examined yields and fermentation of stored crude glycerol supplemented with activated stone carbon by C. pasteurianum
wild type (Jensen et al. [2012
]). The fermentations by MNO6, performed in this study was done with the same crude glycerol as substrate and under almost similar conditions, and benchmarking of MNO6 against data from the wild type strain study, is therefore relevant (Table
Comparison of fermentation data obtained in this study with a related study
The maximum glycerol utilization rate attained by MNO6 was 7.59
g/l/h whereas the wild type strain reached rates of 4.08
g/l/h and 4.94
g/l/h utilizing stored crude glycerol and technical grade glycerol, respectively. This corresponds to an increased rate of 86% and 55% compared to the wild type on crude and technical grade glycerol. The production rates were similarly increased by 33% for 1,3-PDO and 46% for butanol compared to the wild type grown on technical grade glycerol. When the rates achieved with MNO6 were compared to rates of the wild type grown on similar glycerol, maximum 1,3-PDO productivity was increased by 89% and maximum butanol productivity was increased by 49%. Malaviya et al. ([2012
]) demonstrated significantly increased production rates in a high cell density continuous bioreactor using a so-called hyper producing C. pasteurianum
mutant strain. Under batch conditions the mutant strain had a 14% higher butanol productivity compared to the wild type. This is lower than the increase in productivity by MNO6 demonstrated in this study.
In a glucose fed batch bioreactor with gas-stripping, Ezeji et al. ([2004
]) achieved a maximum butanol productivity of 1.81
g/l/h (65% of total solvent productivity) utilizing the well known ABE producing strain C. beijerinckii
BA101. This is similar to the maximum butanol productivity achieved by MNO6. However, Ezeji et al. reached the productivity by utilizing pure glucose where MNO6 utilized stored crude glycerol.
In the mass balance the carbon recovery was 87% (Table
). A similar carbon recovery was observed in the fermentation by the wild type utilizing stored crude glycerol supplemented with activated stone carbon (Table
). We assume that the resulting carbon was non-condensed butanol in the gas-phase. Also, activated stone carbon has been considered as an adsorbent for butanol in downstream processing (Qureshi et al. [2005
]; [Groot and Luyben 1986
]). When fermentations were carried out without activated stone carbon (on technical grade glycerol) a higher carbon recovery was observed (Table
After adjusting the mass balance, the yield of butanol from stored crude glycerol by MNO6 was 0.296
mol/mol compared to 0.264
mol/mol by the wild type utilizing technical grade glycerol. Venkataramanan et al. ([2012
]) reported a butanol yield of 0.347
mol/mol by C. pasteurianum
utilizing purified crude glycerol and 0.322
mol/mol when technical grade glycerol was used. Both yields are higher than those achieved by MNO6 and the wild type. Low initial glycerol concentration, low utilization of glycerol as well as other dissimilarities in growth conditions in the two studies, could cause this difference.
Strain MNO6 had a 1,3-PDO yield of 0.249
mol/mol, which is 47% higher than the yield by the wild type (0.169
mol/mol) grown on stored crude glycerol. Even though the 1,3-PDO yield was lower than the theoretical maximum of 0.720
mol/mol ([Zeng 1996
]) MNO6 has an interesting potential, as 1,3-PDO is produced simultaneously with butanol. Productivity of 1,3-PDO in fed batch has been reported to 0.9-3.0
g/l/h by the dedicated 1,3-PDO producer C. butyricum
([Willke and Vorlop 2008
]; Chatzifragkou et al. [2011
]; Wilkens et al. [2012
]; [Reimann and Biebl 1996
]). The achieved maximum 1,3-PDO production rate of 1.21
g/l/h by MNO6 is comparable to C. butyricum
MNO6 produced ethanol in very small amounts (0.018
mol/mol). Compared to the fermentation by the wild type, the ethanol yield was reduced by 68% and 89% from stored crude glycerol and technical grade glycerol, respectively. The biomass yield of MNO6 was also significantly reduced, constituting only 90% of the wild type. The reduced amount of biomass diminishes the need for ATP. By extrapolating the ATP yield from the mass balances (both in this study and from Jensen et al. [2012
] (data not shown)) it is clear that also the ATP yield is reduced by 10% in MNO6. The reduced ATP requirement/production may be causing the shift in product pattern, as observed in this study.
In a fed-batch fermentation with MNO6 and in situ
removal of butanol, 1,3-PDO would accumulate in the reactor, reaching high titers critical for downstream processing. Recently, the discovery of a bacterial strain producing both 1,3-PDO and ethanol from crude glycerol has been published (Rossi et al. [2012
]). In order to reach high 1,3-PDO titers, the concentration of the second product, ethanol will also increase and possibly inhibit the organism. The fermentation by MNO6 only leads to small amounts of ethanol but high concentrations of butanol, which is even more toxic than ethanol. However, as butanol can be removed simultaneously by gas-stripping, it is possible to achieve high 1,3-PDO titers.
In this study, we have demonstrated that our mutant strain of C. pasteurianum can tolerate high concentrations of crude glycerol, has a high glycerol utilization rate, and high productivity of butanol and 1,3-PDO. Based on these results and on the results on non-treated crude glycerol, we consider MNO6 a more robust and more efficient strain than the wild type and, therefore, also better suited for industrial applications.