Appendix l-D— The Impact of Genetics on Ethanol— A Case Study • 301 
the separation cost of increased ethanol tolerance is 
smaller once ethanol concentrations ha\e reached 
approximately 6 percent. Howexer, the importance 
of increased ethanol concentration to fermenter pro- 
ductix ity remains. 
It is likely that the most important inhihitorv ac- 
tion ot ethanol takes place at the cell membrane. 
Strategies for manipulating the cell membrane com- 
position and properties, and understanding in this 
area, are increasing rapidly. 
i Genetics and ethanol tolerance 
I 
The study of ethanol tolerance by micro-orga- 
nisms has been approached using strains with 
altered genetic makeup. Sex eral kinds of Escherichia 
coli mutants hax e been isolated hax ing different 
tolerances to ethyl alcohol.'- Solxent resistant strains 
either had larger amounts of total phospholipid (type 
III) or had an altered phospholipid and membrane- 
hound protein composition (type II). On the other 
hand, mutants with a lesion mapping close to pss 
gene (which codes for phosphotidylserine syn- 
thetase) were either solx ent sensitixe or resistant.'^ 
The physiologx- of an E. coli ethanol resistant mu- 
tant has been characterized similarly.'^ This strain 
had pleiotropic groxx th defects including abnormal 
cell dix ision and morphologx'. It also had an altered 
tac permease that x\ as not due to a mutation in the V 
gene. It xxas concluded that altered membrane com- 
position xx as responsible for this abnormal behax ior. 
More recently, ethanol tolerant mutants hax e been 
isolated from C. thermocellum.^^ Indirect exidence 
lead to the conclusion that strain S-4 xx as defectix e in 
hydrogenase, since this strain produced loxx^er 
amounts of acetic acid.'® A different ethanol resistant 
isolate of the same bacterium, strain C9, proved to 
hax e a loxver actu ation energx- for groxx th than the 
xvild type, a property that has been related to mem- 
brane composition. 
There are three categories of changes that could 
influence the fermentation process: 
1. Manipulate the existing controls on metabolism. 
Consider an example. In many organisms the 
'-D. P. Clark and J. P. Beard. ".Altered Phospholipid Composition in .Mutants 
of Escherichia Coli Sensitive or Resistant to Organic Solvents." J. Gen. 
Microbiol. 113:267-274, 1979. 
'^.A. Ohta and I. Shibuva, 'Membrane Phospholipid Synthesis and Pheno- 
typic Correlation of an E. Coli pss -Mutant," J. Bacteriol. 132:434M43, 1977. 
“X . .A. Fried and A. Xovick, "Organic Solvents as Probes for the Structure 
and Function of the Bacterial Membrane: Effects of Ethanol on the XX ild 
T\pe and as Ethanol Resistant -Mutant of Escherichia Coli," J. Bacteriol. 
114:239-248. 1973. 
•®S. D. XX ang, "Production of Ethanol From Cellulose by Clostridium Ther- 
mocellum, .M S. Thesis, Department of -Nutrition and Food Science, Massa- 
chusetts Institute of Technology, 1979. 
'Mbid. 
energy' level of the cell, expressed through 
adenosine monophosphate (AMP), adenosine di- 
phosphate (ADP), and adenosine triphosphate 
(,ATP) levels, partially controls the rate of gly- 
colysis. A defective cell membrane xvould pro- 
xide an energy sink, to keep glycolysis at its 
maximum rate. Strategies such as this could be 
attempted noxv. 
2. Increase the amount of each transport and cata- 
bolic enzyme in the fermentation pathway. This 
requires the ability to isolate the genes of in- 
terest and to amplify them xvith in vivo or in 
x itro recombinant techniques in the microbe of 
interest. This is not an immediate prospect. 
3. Accomplish complete deregulation of the fer- 
mentation pathxvay in the microbe of interest. 
Essential catabolic enzymes are difficult to 
manipulate, and this is also not an immediate 
prospect. 
Genetic manipulation of the microbe can influence 
fermentation processes in other^ ways as well. These 
are less important than improvements in yield, final 
ethanol concentration, and productivity, but they 
also affect the cost. Examples are: 
• tx'pe of fermenter used; 
• nonsubstrate nutrients; 
• strain stability; 
• cell separations for byproducts, recycle, or eth- 
anol recovery (i.e., increased size for recovery); 
• operating conditions, i.e., higher groxvth tem- 
peratures for yeast and mesophilic bacteria; and 
• range and efficiency of substrate utilization (i.e., 
complete utilization of all sugars). 
More detailed examples are: 
• Type of fermenter.— If the organism, whether it 
be a yeast or a bacterium, can be made to grow 
under conditions of pH, ethanol concentration, tem- 
perature, etc., that preclude contamination, inexpen- 
sive lined basins can be used instead of tanks, since 
steam sterilization of the fermenter is not required. 
In this case, some operating and capital costs asso- 
ciated xvith sterilization are avoided as well. 
A type of continuous beer fermenter requires 
groxxth in the form of fast-settling pellets. In other 
fermenters, fast-settling particles (such as mycelia) 
present problems that are best avoided by agglom- 
eration of the cell mass. This type of control over the 
growth form of micro-organisms is amenable to 
genetic manipulations. 
• Nonsubstrate medium costs.— In addition to the 
carbon-energy substrate and water, growing cells 
must be supplied with other nutrients. Some orga- 
nisms can make all of their biochemicals from quite 
simple sources of nitrogen, phosphorus, sulfur, 
magnesium and trace metals. Others require more 
