Fructose metabolism by Hydrogenomas eutropha, auxotrophic mutants which produce polysaccharides, and large batch culture apparatu

Tischer, R. G.

English

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N A S A  
PROGRESS REPORT NUMBER V 
INFLUENCE OF METABOLIC ACCUMULATION OF PRODUCTS 
OF HYDROGENOMONAS CELLS ON "HEIR CONTINUED GROWTH 
NASA Research Grant No. NsG 650 to the Mississippi State University 
For period beginning M a y  1, 1966 and ending October 31, 1966 
Principal Investigator: Dr. Robert G. Tischer 
Professor and Chairman 
Department of Microbiology 
Mississippi State University 
State College Mississippi 39762 
DISTRIBUTION LIST OF N A S A, REPORT V ' *  
Dr. Leonard H. Bongers 
Martin Marietta Company 
Space Systems Division 
Research Department 
Baltimore 3, Maryland 
Dr. Doris H. Calloway 
Department of Nutritional Sciences 
University of California 
Berkeley, California 94720 
Dr. John F. Foster 
Physical Chemistry Department 
Battelle Memorial Institute 
505 King Avenue 
Columbus, Ohio 43201 
, 
Mr. Bernard H. Goldner 
Magna Corporation 
Research and Development Division 
1001 South East Street 
Anaheim, California 
Dr. Dale W. Jenkins 
Bioscience Programs 
Chief, Environmental Biology 
Office of Space Science & Applications 
National Aeronautics and Space Administration 
Washington, D . C. 20546 
Miss Winnie M. Morpn 
Grants and Research Contracts 
Technical Reports Officer 
Office of Space Sciences 
National Aeronautics and Space Administration 
Washington, I). C. 20546 
Dr. Roy Repaske 
Institute of Allergy and Infectious Diseases 
National Institutes of Health 
Bethesda, Maryland 20014 
Dr. Joseph Saunders 
Chief, Environmental Biology 
Bioscience Programs 
Office of Space Science Applications 
National Aeronautics & Space Administration 
Washington, D.C. 20546 
Dr. Henry M. Tsuchiya 
Department of Chemical Engineering 
University of Minnesota 
Minneapolis, Minnesota 
Summary. 
TABLE OF CONTENTS --
. .  . 
I. 
11. 
111. 
IV. 
V. 
VI. 
V I 1  . 
Extracellular Products of Hydrogenomonas eutropha 
Table I 
A High Temperature Hydrogen Oxidizing Organism 
The Isolation of Polyssacharide-Producing Hydrogen-Utiliziw 
Micro-organisms 
Large Batch Cultures 
Future Research 
References and Notes on Publications Forthcoming 
Budget Report 
PahSe 
1 
2 
5 
6 
6 
6 
7 
8 
9s 
. 
Of the sugars tested, 11. e!ium& grew only on fructose. It 
produces several sirnple sugars as metabolic products. 
The first information on the thermophilic hydrogenomonad has 
been accepted for publication. Work will continue in an effort to 
find new thermophilics and to compare these with H. eutrophtl. - ----- 
Mutation studies have not produced useful results in terms of 
auxotrophic mutants which produce polysaccharides and will be both 
continued and expanded. 
Large batch culture techniques will be strengthened if appro- 
priate monitoring equipment is made available. At present the 
spray-nozzle technique is under continuous investigation. 
-1- 
I. Extracel lular  Products of Hydrop;enomonas eutro& 
As previously reported, four sugars have been isolated f r o m  
the  spc?.t medium en. which Hydrogenomonas eutropha had grown. 
were ident i f ied  by paper chromatography as  glucose, ribose, arabi- 
nose and xylose. Skudies indicated t h a t  thebe sugars, along with 
galactose,  lyxose, mannose, rhamnose, sedoheptulose, sorbose, and 
sucrose could not be oxidized by e i t h e r  autotrophically or  hetero- 
t rophical ly  grown c e l l s  of Hydrogenomonas eutropha, nor could these 
sugars support the growth of t h i s  organism. 
sugars  i n  the  medium did not detectably a f f ec t  the autotrophic 
growth of Hydrogenomonas eutropha. 
These 
The presence of these 
(See Table 1. ) 
Fructose which w a s  not detected i n  the  spent medium, was  the  
only sugar tested which could be metabolized by Hydrogenomonas 
eutropha. 
f i l t e r  s t e r i l i z e d  fructose under an atmosphere of air w a s  abundant. 
Growth of t h i s  organism i n  basal medium containing 0.5% 
In  manometric experiments, autotrophically grown c e l l s  required 
about a twenty minute adaptive period before they could oxidize 
fructose,  whereas fructose grown c e l l s  u t i l i z e d  fructose without 
1%- 
When fructose grown c e l l s  were offered fructose,  they ex- 
h ib i ted  a respiratory quotient of near the  theore t ica l  value of 
one indicating t h a t  fructose was converted almost quant i ta t ively 
t o  carbon dioxide. 
to ry  quotient remained at zero u n t i l  the  c e l l s  became adapted t o  
f ructose,  then the RQ value rose t o  s l i g h t l y  l e s s  than one half  of 
t he  theo re t i ca l  value. 
With autotrophically grown c e l l s ,  t h e  respira- 
This may have indicated t h a t  the  autotro- 
-2- 
. phical ly  grown c e l l s  were assimilating about half  of the  carbon 
dioxide produced. 
Although Hydrogenomonas eutropha was unable t o  u t i l i z e  glucose, 
it was found t h a t  t h i s  organism would grow..abundantly i n  a medium 
containing glucose prepared by autoclaving the  glucose i n  the buffer 
before the addition of the  other components of the  basal  medium. 
Cells grown on the  autoclaved glucose medium were shown t o  u t i l i z e  
f ructose without l ag  i n  manometric experiments. 
s tudies  of t he  autoclaved buffered glucose solut ion revealed t h a t  
it contained fructose. Apparently t h e  glucose had been changed 
during autoclaving i n  a reaction similar t o  the  Lobry-.de Bruyn- 
Alberda van Ekenstein transformation t o  y ie ld  fructose. 
explain why other workers have been able t o  grow Hydrogenomonas 
eutropha on glucose. 
Chromatographic 
This may 
Autotrophically growing ce l l s  were shown t o  adapt t o  and t o  
u t i l i z e  fructose under a H2-02-C02 emosphere. 
zation of fructose did not affect  the  quantity of gas used o r  t he  
r a t e  a t  which the  gas was used by t h i s  organism. 
However, the u t i l i -  
Hydrogen i n  the  absence of carbon dioxide was found t o  inh ib i t  
the  a b i l i t y  of autotrophically grown c e l l s  t o  adapt t o  fructose.  
This inhibi t ion could be removed by replacing the  hydrogen-oxygen 
atmosphere with air. 
Manometric experiments indicated t h a t  t he  t r icarboxyl ic  acid 
cycle was operative i n  Hydrogenomonas eutropha because most of the 
intermediates of t h i s  cycle were u t i l i z e d  by both autotrophically 
and fructose grown res t ing  ce l l s  without l a g .  
-3- 
. 
The presence of the enzymes associated with the Entner-Dou- 
doroff metabolic pathway were demonstrated in fructose grown c e l l s  
of yydrogenomonas eutropha. 
trophically grown c e l l s ,  but t o  a lesser extent. 
peared that the Entner-Doudoroff pathway was responsible (wholly 
or in  part) for the metabolism of fructose by Hydrogenomonas eutropha. 
These enzymes were also found in  auto- 
Therefore, it ap- 
-4- 
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;alactose 
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11. - A High Temperature Hydrogen Oxidizing Organism 
One publication resul t ing from t h i s  discovery has been accepted 
by NATURE and w i l l  appear soon. (See Reference a t  end. ) 
Work w i l l  continue t o  foreward the  comparison of t h i s  thermo- 
ph i l e  w i t h  E. eutropha as a possible consti tuent of t h e  space ecology 
sys tem . 
The thermophile w i l l  a l so  be included i n  the  work on mutation 
t o  attempt t o  i so l a t e  a polysaccharide producing mutant o r  t o  estab- 
l i s h  t h e  s t a b i l i t y  of the new s t ra in .  
111. Isolat ion of Polysaccharide-Producing Hydrogen-Utilizing 
M i  cro-organ i sms 
Considerable e f fo r t s  t o  apply ultra v io le t  i r rad ia t ion  have cost  
a considerable amount of time, hundresof p e t r i  p l a t e s  and medium. 
The net results which were sent t o  Dr. DeCicco were two unimpressive 
mutants. The sum of these occurrences s w g e s t s  t h a t  g. eutropha i s  
a f a i r l y  s tab le  microorganism. 
The e f f o r t s  t o  produce a polysaccharide-producing strain are 
continuing and plans are t o  use several -types of mutagens and t o  
devise, if possible,  better methods of detecting auxotrophs which 
produce polysaccharides. 
I V . .  Large Batch Cultures 
S’tudies wi th  t he  large batch cul ture  apparatus were con8inued. 
During the experiments, t h e  gas mixtures, l i qu id  volume, and growth 
medium const i tutents  were varied i n  an e f f o r t  t o  increase the  growth 
rate and to ta l  growth of Hydrogenomonas eutropha. 
The results of t h e  experhents  are as follows: 
-6- 
The volume of growth medium used i n  the  apparatus was 6 l i ters.  
"his volume w a s  used throughout the  experiments. 
The gas mixtures which proved best  f o r  growth i n  the  large 
batch apparatus consisted of 6756-701 hydrogen, 20%-22% oqygen, and 
lO$-11% carbon dioxide. 
The simplified medium as described by Bongers (1965) supported 
t h e  growth of E .  eutropha bu t  yielded somewhat l e s s  growth than did 
the  normal growth medium. 
Increased concentrations of urea i n  the  medium changed the  
growth rate o r  t o t a l  growth suf f ic ien t ly  to merit t he  addition of 
urea i n  quant i t ies  above 1.0 gram per l i t e r .  
The growth experiments normally ran f o r  a 48 hour period. 
The results show t h a t  when using the  growth medium described 
by Bongers (1963) and the 67% hydrogen, 22% oxygen, and 11% carbon 
dioxide mixture, t he  large batch cul ture  apparatus w i l l  y ie ld  an 
average harvest of 2.0 t o  2.5 grams dry weight of c e l l s  per l i t e r  of 
medium i n  a 48 hour growth period. 
From the r e su l t s  of an extended growth experiment i n  which 
portions of t he  c e l l s  were harvested and fresh medium was  added at  . 
in te rva ls ,  it seems possible tha t  t h i s  system might be modified f o r  
t he  continuous cul ture  of H. eutropha. 
V. Future Research 
Attention w i l l  be sh i f ted  from neutral  metabolic products t o  
acid and basic products with special  a t ten t ion  a l so  t o  the  separa- 
t i o n  and idont i f icat ion of proteins and protein break down products. 
New attempts w i l l  be made t o  i s o l a t e  thermophiles and t he  
thermophile i n  hand w i l l  be tested for genetic s t a b i l i t y .  
-7- 
. 
Application of ultra-violet  t o  H. eutropha produced few mutants 
and none which produced polysaccharides but t h i s  search w i l l  be con- 
t inue d . 
Large batch cul tures  w i l l  be continued as i n  the  past ,  i n  an 
effort t o  perfect the  method i n  use. If addi t ional  monitoring 
equipment is made available,  addit:'.onal work w i l l  be undertaken t o  
prove the  course of change of pH, Eh, tu rb id i ty ,  temperature, dry 
weight, e tc .  during closely controlled runs i n  an e f f o r t  t o  f ind  
maximum operating conditions by sequential  i t e ra t ion .  
VI. References and Notes on Publications Forthcoming 
1. Cook, D.W., Brown, L.R., and Tischer, R.G. Metabolic Pathway 
of Fructose i n  Hydrogenomonas eutropha. 
publication. 
S-itted f o r  
2. McGee, J.M., Brown, L.R., and Tischer, R.G. I so la t ion  and 
Characterization of a High Temperature, Hydrogen-Oxidizing 
Bacterium--&drogenomonas thermophilus , n. sp . 
f o r  publication by NATURE. 
Accepted 
3. Cook, David W . ,  Tischer, Robert G . ,  and Brown, Lewis R.  
Carbohydrate Metabolism i n  Hydrogenomonas eutropha. 
Accepted f o r  publication by Canadian J. of Microbiology. 
- 8- 
Equipment 
Supplies 
Travel 
Salaries and Wages 
EXPENDITURES TO DATE 
FISCAL YEAR 1966-4967 
Professional 
Non-professiond 
TOTAL EXPENDITURES: 
$943.40 
1834.52 
18.76 
'1535.21 
280.00 
t 
-9- 


Influence of Metabolic Accumulation of Products of Hydrogenomonas Cells on Their Continued Growth Progress Report, 1 May - 31 Oct. 1966

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