Paper No, L.5.

2
Working Group V
May 16, 1968

COMMITTEE ON SPACE RESEARCH (COSPAR)

Eleventh Annual Meeting
Tokyo, Japan
May 9-21, 1968

CURRENT RESEARCH ON REGENERATIVE SYSTEMS

Jacob Shapira, Adrian D . Mandel, Phillip D. Quattrone, and Nancie L. Bell

Biotechnology Division
Ames Research Center, NASA

Moffett Field, California, USA

(ACCESSION NUMBER)

94035

CURRENT RESEARCH ON REGENERATIVE FOOD SYSTEMS

SHAPIRA, Jacob, MANDEL, Adrian D.,

QUATTRONE, P h i l l i p D.,

and BELL, Nancie L.

Ames Research Center, NASA

Moffett Field , California, USA

94035

SUMMARY
Multiple s t u d i e s d i r e c t e d toward t h e development of a regenerative

l i f e support system have shown t h a t e a s i l y synthesized organic compounds

and microbiological materials a r e p o t e n t i a l l y capable of being used as
foods f o r long duration space missions.
ported t h e s e views.

Animal feeding s t u d i e s have supThe organic compounds p r e s e n t l y believed t o o f f e r t h e

greatest p o t e n t i a l are glycerol, simple glycerol d e r i v a t i v e s such as

t r i a c e t i n , and formose sugars.

Laboratory s t u d i e s i n d i c a t e t h a t glycerol

can be synthesized from formaldehyde which i n t u r n i s obtained by t h e

d i r e c t c a t a l y t i c oxidation of methane, a by-product of t h e S a b a t i e r react i o n used i n t h e spacecraft atmosphere control system.
derived from t h e self condensation of formaldehyde.

Formose sugars are

Mixtures of glycerol

and t r i a c e t i n have been shown t o be s u i t a b l e as a major component of d i e t s

fed t o weanling rats f o r prolonged periods.

These compounds do not e x i s t

as stereoisomers and therefore o f f e r advantages over t h e formose sugars.

Hydrogenomonas eutropha i s t h e microbiological system under i n v e s t i g a t i o n .
An automated system f o r t h e continuous autotrophic production of Hydrogenomonas b a c t e r i a i s i n operation, and t h e n u t r i t i o n a l requirements f o r
growth i n t h e system using urea as a nitrogen source a r e being studied.
Nutritional evaluation of Hydrogenomonas b a c t e r i a has shown they a r e capable of supplying t h e t o t a l p r o t e i n requirement of growing r a t s f o r prolonged periods.

The p o t e n t i a l and problems of t h e s e regenerative systems

and t h e prospects f o r t h e accomplishment of a t o t a l l y regenerative food

system w i l l be discussed.

41ntroduction. Development of a system capable of regenerating at

l e a s t p a r t of t h e crew's food requirements during missions o f very long
duration has been shown t o be d e s i r a b l e (1,2).

A v a r i e t y of schemes t o

u t i l i z e metabolic waste as a source o f food have been proposed and two

of t h e approaches are under i n v e s t i g a t i o n here.
Regenerative systems can be of two types.

E i t h e r some b i o l o g i c a l

system i s involved o r else only physicochemical reactions are used.

Examples of t h e former are systems u t i l i z i n g algae, b a c t e r i a , duckweed,
o r some o t h e r l i v i n g organism.
r e l a t i v e l y unexplored.

The physicochemical approach has been

Its usefulness w i l l depend upon finding organic

compounds which can be synthesized r e l a t i v e l y e a s i l y from metabolic products o r o t h e r surplus material on t h e spacecraft and which can be s a f e l y
consumed a s food f o r extended periods.
A search has been conducted t o f i n d organic compounds which can be

t o l e r a t e d as a major component of t h e d i e t ( 3 ) .

Currently, investiga-

t i o n s a r e i n progress t o develop d i e t s which contain v i r t u a l l y a l l t h e

c a l o r i e s as p u r i f i e d organic compounds.

I t i s postulated t h a t t h e essen-

t i a l compounds o f t h e d i e t such as t h e vitamins, e s s e n t i a l amino and f a t t y

acids, and trace elements which together comprise only a minor proportion
by weight of t h e t o t a l food requirements w i l l be c a r r i e d along during t h e
very long missions and physicochemically regenerated compounds w i l l comp r i s e t h e bulk of t h e d i e t .

The compounds which appear t o have t h e g r e a t -

e s t promise are t h e formose sugars, glycerol, propylene glycol and some

of t h e i r d e r i v a t i v e s .
An a l t e r n a t i v e and/or complementary system involves t h e p o s s i b l e

u t i l i z a t i o n of Hydrogenomonas b a c t e r i a .

These b a c t e r i a which can u t i l i z e

metabolic waste f o r autotrophic growth have been shown (4,s)

high q u a l i t y p r o t e i n .

t o contain

I f an apparatus could be designed t o produce t h i s

material during space f l i g h t , an even g r e a t e r reduction i n t h e amount of

food which would otherwise have t o be c a r r i e d along would r e s u l t .

It is

unlikely t h a t Hydrogenomonas can be made a major component of t h e d i e t

s i n c e it apparently contains too large an amount of nucleic acids t o be
t o l e r a t e d by humans ( 5 ) .

However, as w i l l be shown subsequently, it may

serve as a very useful food supplement.

N u t r i t i o n a l Studies.

The n u t r i t i o n a l q u a l i t i e s o f t h e formose sugars

In t h e only previously reported

study, Akerloff and Mitchell found ( 6 ) t h a t t h e i r product w a s t o x i c t o
rats. The d i s t r i b u t i o n of sugars present i n t h e f i n a l product a f t e r t h e
self condensation of formaldehyde is dependent upon t h e conditions o f t h e
reaction (7). Further, many of t h e sugars may be present as t h e formal

have not been i n v e s t i g a t e d extensively.

d e r i v a t i v e and l i b e r a t e formaldehyde when exposed t o t h e a c i d i t y of t h e

stomach.

We have removed t h e r e s i d u a l formaldehyde from a product produced

continuously i n a plug-flow r e a c t o r and, by means of gas chromatography of

t h e s i l y l d e r i v a t i v e s , have shown t h a t our product contained predominantly
pentoses and hexoses (8).

I t w a s p o s s i b l e t o incorporate up t o 20% by

weight of t h i s material i n t o r a t d i e t s without major detrimental effect

(3).

A backmix r e a c t o r is now being used t o b e t t e r control t h e r e a c t i o n

and hopefully t o y i e l d a product which .does not contain t h e materials which

have previously l i m i t e d i t s p o t e n t i a l usefulness (8).
Compounds such as glycerol and t r i a c e t i n o f f e r advantages over t h e
formose sugars.

They do not e x i s t as stereoisomers and therefore can be

t o t a l l y metabolized whereas formose sugars contain L-isomers which i n most

p a r t w i l l not be u t i l i z e d by t h e body.
There have been a number of human feeding s t u d i e s with glycerol and
i t has been shown t o have medicinal value (9,lO).

Here, an e f f o r t has

been made t o determine t h e maximum amount of materials such as glycerol

and t r i a c e t i n which can be t o l e r a t e d by growing rats.

In growing rats

(which must c l e a r l y be d i f f e r e n t i a t e d from t h e s i t u a t i o n which obtains i n

mature animals), we have observed t h e following during a number of t h r e e
month feeding s t u d i e s :
(1) There is n e g l i g i b l e reduction i n weight gain when 40% glycerol
replaces conventional carbohydrate i n t h e d i e t .
(2) The animals can t o l e r a t e up t o 20% t r i a c e t i n well.

Greater

amounts cause a decrease i n weight gain.

(3) A l a r g e loss i n weight and considerable mortality is associated

with d i e t s containing e i t h e r glycerol o r t r i a c e t i n as 60% of t h e d i e t .

(4) A d i e t containing a mixture of 40% glycerol and 20% t r i a c e t i n i s

t o l e r a t e d f a i r l y well (also see Table I ) .

two compounds is not a d d i t i v e .
2

Thus, t h e t o x i c i t y of these

. (5)

The type and q u a n t i t y of t h e p r o t e i n present i n t h e d i e t has an

influence upon t h e weight gain i n t h e presence of glycerol and t r i a c e t i n .

(6) Excess vitamins are of b e n e f i t t o t h e animals consuming t h e s e

diets.
The r e s u l t s of a feeding study t o determine whether a mixture of

H.

eutropha, glycerol and t r i a c e t i n could be made a major proportion of

t h e d i e t of weanling rats is shown i n Table I .

The H. eutropha was

obtained as a s i n g l e uniform l o t (Grain Processing Corporation, Muscatine,

Iowa) which had been grown h e t e r o t r o p h i c a l l y f o r 24 hours on a medium cont a i n i n g salts, casein hydrolysate, protopeptone, y e a s t e x t r a c t and sucrose.
The cells were k i l l e d by heating a t 90C f o r 15 min., washed t h r e e times

with d i s t i l l e d water and used as a frozen p a s t e containing 21.6% by weight

dry cells and contained 144 mg N/gm dry weight,

The casein used f o r t h e

control d i e t s contained 140 mg N / g m dry weight,

I n addition t o t h e com-

ponents shown i n Table I , a l l d i e t s contained 5% LISP XIV s a l t mixture

(plus 16.5 mg ZnS04/100 gm d i e t ) , 5% safflower o i l , 2% a - c e l l u l o s e , 1%
vitamins, 1%agar and a t o t a l o f 2 p a r t s water t o each p a r t dry weight.
After formulation, t h e d i e t s were a thick p a s t e which w a s s t o r e d a t 4'C

and fed f r e s h d a i l y .

There were e i g h t male Sprague-Dawley r a t s i n each

group; t h e animals were caged as p a i r s and weighed d a i l y .

I t can be seen from Table I t h a t 1 2 % p r o t e i n i n t h e form of e i t h e r

casein o r H. eutropha i s not adequate f o r maximum growth of t h e animals.

Substitution of 60% of t h e s t a r c h i n t h e d i e t with a mixture of glycerol
and t r i a c e t i n reduced t h e growth of t h e animals.

When t h e concentration

of p r o t e i n was increased t o 24%, maximum growth occurred and i n t h i s case,

s u b s t i t u t i o n of t h e s t a r c h with g l y c e r o l - t r i a c e t i n i n t h e d i e t containing

H.

eutropha did not r e s u l t i n decreased growth.

However, when casein was

t h e p r o t e i n , t h e growth was s i g n i f i c a n t l y (P < 0.05, t

t e s t ) lower.

Growth of t h e animals was e s s e n t i a l l y t h e same with d i e t s containing 48%

H . eutropha, whether or not t h e
p r o t e i n i n t h e form of e i t h e r casein or -

carbohydrate component of t h e d i e t was s t a r c h or t h e g l y c e r o l - t r i a c e t i n

mixture.

The differences shown are not s t a t i s t i c a l l y significant.

These d a t a show t h a t over 90% of t h e c a l o r i e s of t h e d i e t of growing

r a t s can be composed o f a mixture of H . eutropha, glycerol and t r i a c e t i n

without d e t r i m e n t a l effect on t h e growth o f t h e animals,

Rats excrete

allantoin as t h e c a t a b o l i c product of nucleic a c i d purines r a t h e r than

u r i c a c i d which is t h e e x c r e t i o n product of man. Consequently, one would
not expect t o observe i n rats t h e undesirable effects which are a s s o c i a t e d
with excess u r i c a c i d i n man.
Systems Development. I t i s of no value t o demonstrate t h e p o t e n t i a l
usefulness of materials such as H. eutropha, glycerol and t r i a c e t i n as
food unless s a t i s f a c t o r y systems can be developed f o r t h e i r production
from metabolic waste during missions of long duration. Accordingly, w e

have been conducting research t o determine t h e weight, volume and power

requirements of regenerative systems.
Synthesis of formose sugars and glycerol w i l l r e q u i r e t h e i n t e r mediary s y n t h e s i s of formaldehyde.

A c o n t r a c t o r of t h e NASA (General

American Transportation Corp., Niles, I l l i n o i s ) has been s u c c e s s f u l i n

developing a method f o r t h e d i r e c t c a t a l y t i c oxidation of methane t o formaldehyde,

The methane i s produced as a by-product of t h e S a b a t i e r process

i n t h e s p a c e c r a f t atmosphere c o n t r o l system.

The recycle system c u r r e n t l y

being i n v e s t i g a t e d uses a r e a c t o r composed of Berl saddles coated with

sodium t e t r a b o r a t e h e l d a t 675OC.

Conversions of about 30% a r e obtained

when t h e feed is composed o f 40% methane, 36% oxygen, 23% nitrogen and
0.5% n i t r i c oxide.

Addition of r e a c t o r s t o convert by-product carbon

monoxide and carbon dioxide back i n t o methane w i l l r e s u l t i n a system

capable of e f f e c t i n g t h e q u a n t i t a t i v e conversion of carbon dioxide t o
formaldehyde.

The product i s obtained as i t s s o l i d polymer and can be

v o l a t i l i z e d by g e n t l e h e a t i n g f o r use i n subsequent s t e p s .
The conversion o f formaldehyde t o glycerol i s being i n v e s t i g a t e d f o r

us by t h e Esso Research and Engineering Corporation, Linden, New J e r s e y .

They have shown t h a t it i s p o s s i b l e t o c o n t r o l t h e self condensation of
formaldehyde t o y i e l d predominantly t h e t h r e e carbon sugars, glyceraldehyde and dihydroxyacetone.
of t h e s e compounds

Our feeding s t u d i e s have shown t h a t n e i t h e r

per se is s u i t a b l e as food. However, t h e mixture can

be c a t a l y t i c a l l y reduced with hydrogen t o produce glycerol.

The bacterium, Hydrogenomonas eutropha, when grown i n t h e autotrophic
mode requires salts, hydrogen, oxygen, carbon dioxide and a nitrogen
source such as urea o r ammonia.

An experimental laboratory model of an

4

-%

automated system s u i t a b l e f o r s p a c e c r a f t a p p l i c a t i o n was developed by

Battelle Memorial I n s t i t u t e f o r NASA (11). Using t h e same sensors and

c o n t r o l s , t h e c u l t u r e volume can be e i t h e r two liters o r twenty l i t e r s .
The automatic oxygen sensor c o n t r o l u t i l i z e s a Beckman polarographic
d e t e c t o r (Clark type e l e c t r o d e ) i n e i t h e r t h e l i q u i d o r gas phase.

Carbon

dioxide t e n s i o n i s regulated by a Beckman carbon dioxide s e n s o r (a modif i e d pH e l e c t r o d e ) .

Hydrogen c o n s t i t u t e s t h e remainder of t h e gas phase

and is c o n t r o l l e d by a t o t a l pressure r e g u l a t o r .

The hydrogen concentra-

t i o n is always g r e a t e r than 80%. In a d d i t i o n , a thermistor temperature

c o n t r o l l e r and a pH r e c o r d e r - c o n t r o l l e r system connected t o r e s e r v o i r s of
a c i d and base are present i n t h e system,

A modified autoanalyzer arrange-

ment i s used t o monitor and c o n t r o l t h e c e l l density ( t u r b i d i t y ) and t h e

urea concentration i n t h e c u l t u r e .
The primary goal of i n v e s t i g a t o r s working with continuous c u l t u r e

H.

eutropha systems has been t o optimize c u l t u r a l conditions s o t h a t

maximum growth rates a t high population d e n s i t i e s can be maintained r e l i a b l y

f o r an i n d e f i n i t e period.

Such a c u l t u r e would reduce t h e volume and weight

requirements of t h e system t o a minimum.

With our apparatus, it i s p o s s i b l e t o a t t a i n s t a b l e c e l l population
d e n s i t i e s i n t h e range of 4 . 1 gm dry weight/L a t growth rates of 0.25/hr

( i . e . , population p e r l i t e r increases by 25% each h o u r ) ,

The same high

growth rate i n c u l t u r e s containing 6 . 1 gm/L has been reported by L . Bongers

Growth r a t e s as high as 0,35/hr have been observed occasionally as well as

population d e n s i t i e s of 10 gm/L.

Thus, t h e genetic l i m i t a t i o n s on growth

and c e l l density have not y e t been reached.

A t present s t a b l e production

rates, it i s estimated t h a t 12 l i t e r s of c u l t u r e is s u f f i c i e n t t o convert

the e n t i r e inventory of one man's carbon dioxide output t o c e l l mass.

However, it is u n l i k e l y t h a t H . eutropha w i l l comprise a l l of t h e a s t r o n a u t ' s

d i e t and a smaller c u l t u r e volume w i l l s u f f i c e t o supply h i s p r o t e i n
requirements.
Unlike t h e s i t u a t i o n with carbon dioxide conversion, t h e e f f i c i e n c y of
t h e organism i n converting u r i n a r y nitrogen t o p r o t e i n nitrogen has not
been s t u d i e d i n any d e t a i l .
urea.

Our s t u d i e s are being performed with p u r i f i e d

I n batch c u l t u r e H. eutropha can u t i l i z e n e a r l y a l l t h e major

nitrogenous components of urine when supplied individually and the intermediates o f degradation appear to be ammonia and/or urea.

The effect that

human urine w i l l have on the growth rate and carbon dioxide assimilation
i n continuous culture remains t o be determined.

-"
"p.

s4

!J"

1.

!'Conference on Nutrition i n Space and Related Waste Problems," NASA

SP-70 (1964).

2.

"The Closed Life Support System,*1NASA SP-134 (1967).

3.

Shapira, J.

%pace Feeding:

Approaches t o t h e Chemical Synthesis of

Food," Cereal Science Today 13, 58-63 (1968).

4.

Shapira, J. and Mandel, A. D.

"Nutritional Evaluation of Bacterigl

Diets i n Rats , I 1 Nature 217, 1061-2 (1968).

5.

"Hydrogenomonas eutropha as

Calloway, D, H. and Waslien, C. I .

Human Food," Second Conference on Global Impact of Applied Microbiology,

Addis Ababa, Ethiopia (1967).
6.

Ackerloff, G. C. and Mitchell, P. W. D.

"A Study of t h e F e a s i b i l i t y

of t h e Regeneration of Carbohydrates i n a Closed Circuit Respiratory

System," J. Spacecraft 1, 303-10 (1964).

7.

Gabel, N. W. and Ponnamperuma, C.

Wodel f o r Origin of Monosaccharides,"

Nature 216, 453-5 (1967).

8.

Weiss, A. H. and Shapira, J.

'The Kinetics of t h e Formose Reaction,"

Ind. Eng. Chem. (In Press).

9. Johnson, V.

, Carlson,

A. J . and Johnson, A.

"Studies on t h e Physio-

logical Action of Glycerol,'1 Am. J. Physiol. 103, 517-34 (1933).

10.

Freund, G.

"Metabolic Effects of Glycerol Administered t o Diabetic

Patients," Arch. I n t . Med. 121, 123-29 (1968).

7

11.

Foster, J . F. and Litchfield, J. H.

"A Continuous Culture Apparatus

f o r t h e Microbial U t i l i z a t i o n of Hydrogen Produced by E l e c t r o l y s i s

of Water i n Closed-Cycle Space Systems," Biotech. and Bioeng. 6,

441-56 (1964).

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