The effect of sulfur globules on the buoyant density of Chromatium vinosum and Beggiatoa alba was examined. The potential use of sulfur as a terminal electron acceptor in the anaerobic metabolism of Beggiatoa alba is also examined. The effect of the reduction of intracellular sulfur was investigated during dark metabolism on the buoyant density of C. vinosum. It is hypothesized from the results that the sulfur reduction to sulfide is part of an anaerobic energy operating system. Carbon stored as PHB can be oxidized with the concomitant reduction of sulfur to sulfide

Schmidt, T. M.

English

NASA Technical Reports Server

N85 = 32 1 3  Q 
T.M. Schmidt 
Introduc t i on 
Chrorastiun u i n o ~ u m ,  a f l age l l a ted  photosynthetic rod 
bacterium, and Beggzatoa alba, a g l i d i n g  f i lamentous 
bacterium, both ox id ize  s u l f i d e  t o  sulfur, which i s  stored ins ide  
t h e i r  c e l l s  i n  the form of su l fu r  globules. Although these 
bacter ia  are morphologically and phys io log ica l l y  d i s t i n c t ,  t h e y  
both belong t o  the purp le bac te r ia l  group as determined by t h e i r  
56 rRN4 sequences (Krieg, 1984). 
Filaments of Beggiatoa g l i d e  through sediments so t h a t  
they are s i tuated at tha oxic/anoxic i n te r face  where s u l f i d e  and 
oxygen coexist. fit this. iq ter facc,  sulfur i s  accumulated by the 
ce l l s .  Since th i s  i n te r face  r i s e s  a t  n igh t  and f a l l s  dur ing the 
day, Baygistoa f i laments m u s t  be able t o  generate energy under 
both ox ic  and anoxic condi t ions as they g l i d e  towards the 
interface. The aerobic axidat ion of acetate by Beggiatoa has 
been wel l  documented and shown t o  be responsible f o r  energy 
generation (Strohl,  1981). However, there i s  no known mechanism by 
which Bsggiatoa can generate enerqy i n  the absence of oxygen. 
Chromatiurn, i s  found i n  the anoxic layers  o f  lakes. 
Electrons released from the ox idat ion of  sui-?id@ are used i n  
anoxygenic photosynthesis. The sulfur t h a t  accumulates from t h i s  
react ion increases the buoyant densi ty of t. 2 ce l l s .  A s  s u l f u r  
globules accumulate, the c e l l s  sink out o f  the phot ic  zone if the 
bac ter ia l  layer  i s  concentrated enough t o  l i m i t  the l i g h t  
avai lab le for  photosynthetic energy production. Once below the 
phot ic zone  Chromatiurn, gains maintenance energy + r o m  the 
oxidat ion of polyglucose t o  poly- -hydroxybutyric ac id  (PHE) and 
the reduct ion o f  sulfur t o  s u l f i d e  (van Gemerden, 1968?. Decrease 
i n  the amount of  i n t race l l u l i i t -  su l fu r  globules reduces the buoyant 
density of the c e l l  and may permit c e l l 5  t o  r e t u r n  to the phot ic  
zone. 
T h i s  p ro jec t  examined: 
1) The e f f e c t  o f  sulfur globules on the buoyant densiry of 
Chromatiur v z n o s u ~  and Beggiatoa alba, 
2) The po ten t i a l  use of su l fu r  as a terminal e:ectron acceptor 
i n  the anaerobic my:tabolism o f  Beggzacoa a l b a ,  and 
3) The e f f e c t  of the reduction of i n t r a c e l l u l a r  sulfur during 
dark metabolism on the buoyant densi ty of C, vinosus. 
10 8 
https://ntrs.nasa.gov/search.jsp?R=19850024426 2020-03-20T18:19:56+00:00Z
Culturing Freshwater Strains of B e g g i e t o s  
(Type s t ra in :  B .  a lba  B18LD) 
STOCK SOLUTIONS BASIC GROWTH MEDIUM 
Modi+ied Frinqsheim’s 
M i  croEl ement Solut ion 5 m l / l  
N H d l  (5 percent) 5 m l / l  
CaCla (15 percent) 15 m l / l  
MqS047Hdl 1 percent 1 1 m l / l  
KHaP04 ( 1  percent) 1 m l l l  
Sodium acetate 0.5 911 
Sodi u m  sul f i d e  0.24 g / l  
Adjust the pH of the  medium t o  7.4 w i th  1N NaClH and 
autoclave. Add ne.Atralized and s t e r i l i z e d  s u l f i d e  S.O a f i n a l  
concentrat ion of 1 mM. Use a 10 to 15 percent inoculum and 
gent ly  ag i ta te  the c u l t u r e  f o r  best growth. Cultures can be 
grown i n  the  presence or  absence of su l f ide .  
MODIFIED PRINGSHEIM’S MICROELEMENTS 
H3B03 - 8: ) .  0 0 1  percent 
Na~NoO+ 7H20 - 0.0001 percent 
FeSOo 7HZ0 - 0.07 percent 
Mn504 7Hz0 - 0.0002 perhent 
CoCla 6H20 - 0 . 0 O C l l  percent 
ZnS04 7Hz0 - 0.001 percent 
CuS04 -- O.OOCiOOO5 percent 
EDTA - 0.2 percent 
Prepare i n  ddH,O and add 1 m l / l  H C I  to prevent 
p r e c i p i t a t i o n  of 1 ran. 
Maintaining Cultures on Agar Plate% 
Cultures can be maintained f o r  two t o  three weel:s.on aqar 
plztes.  Prepare the growth medium as described abLve except 
lower the acetate concentrat ion t o  0.1 g / l  and add 0.1 q / l  yeast 
extract .  Sulfide should he added to 1 mM a f t e r  autoclaving. 
Transfer- p l a t e  cu l tu res  every t w o  to three weeks by c u t t i n g  
aut. a slab of  agar arid gent lv  s l i d i n g  i t  over the  surface of a 
new plate.  
S ta r t i ng  Suspension Cultures f r o m  Agar  P l a t e r  
f repare 100 m l  of 2 percent agar- i n  a 250 m l  f lask.  
Autoclave, then ad-1 neutra l ized and stei-i l i z e d  s h l f i d e  t o  a f i n a l  
concentration of 1 TIM. U f t v r  t h i s  s o l i d i f i e s ,  add 100 m l  of 
hasic qruwth medium (+ s u l f i d e )  and inoc i l la te  the f l ask  w i t h  a 
sect ion of the agar cu l ture.  h g i t a t e  gen’ 1 .  GroNth should 
occur i n  t w u  days. The agar plug w i l l  provide a continuous 
release o+ s u l f i d e  i n t o  thc  inedium. Beygiataa survives best 
under microaeraphi 1 ic condit ions. 
109 
Hateriala and t4erthods 
Pure cultures of Chroratiur vinosur and Beggiataa 
alba were grown upder conditions where elemental sulfur nas 
accumulated by the cells. The cultures were then transferreo 
to the apparstus diagrammed in Figure 11-17. In the 
experiments with Beggiatoa, the culture was centrifuged and 
washed twice in basal salts (Strohl and Schmidt, 1984) before 
being transferred to the appaTatus. Nitrogen was flushed 
through the system and the outfiowing gas was bubbled through 
two test tubes that each contained 10 tnl of 2 percent zinc 
acetate to precipitate sulfide. The zinc acetate tubes were 
changed every hour duri-rg the course of the experiments. This 
apparatus per-mir.i i-J the continuous removal of sulf idt so that 
sulfide toxicity did not limit metabolic rtion of sulfur 
glohules in the cell (Table 11-3)- C, v z n o s u r  and 8 ,  
alba  reduced the i.itracellular sulfur to sulfide at similar 
rates (Fiq. 11- 18). duggiafoa filaments that lacked sulfur 
inclusions produced no sulfide, suggesting tnat 
sulfur-containing amino acids were not the source of the 
sa1 f i de measured. 
The buoyant density of Chporatiur cills was increased 
to i-lSO g/cm by providing illuminated cells with 1 mH sulfide 
4 hours before ti.e densitv measurement. This ensured that the 
lfur to sulfile during sic hours did not make a significant 
change in the buoyant density of the cells. Although sulfur 
redurtion to sul+ide with corelated decrease in buoyant density 
is rot a mechanism by which Chromatiurn returns to the 
photic zone after 6 hourz. it may be important to cell 
maintenance as the sulfur continues to decrease for several 
dcys. 
Since Bbggiataa can also reduce sulfur tc sulfide, we 
h./pothesized that sulfur to sulfide is part of an anaerobic 
energy-generating system. This pathway was previously 
suggested to exist in Feggzatoa (Nelson apd Castenholz, 
1981). CLr work provides evidence to con+irm such a 
suqqestiun. Carbon stored as PHB m a ,  b e  oxidized with the 
concomitant reduction of sulfur to sblf Ide. Additional 
research 1-3 requirod to determine whether the oxidation of PHB 
usir,g sulfur as a tee-minal electron source provides enerqy in 
6 e g g i a t c a  and whether t h i s  is sufficient to maintain calls 
and prc. i ide energy for .novement back into the oxic =on€. 
C r a d f o r d ,  ?4.?4,, 1976. P rapr-;' .nd sensiti*:e method for the 
quantification of  microg. a m  quantities of protein utilizing 
t h e  prirsciples of proteus dye binding, Anal. Biochem., 
72: 248-254. 
110 
Figure 11-17. CIpparatus for continuous removal and mtasurcement of 
sulfide. 
culture 
, m e d i u m  
i 
I 
I 
I 
Trap 1 Trap 2 / 
/ Zn Acetate traps 
magnet i c st i rrer 
I11 
4 - 
+ 
+4 
rftw 3 hwrr  
dark incubrrtion ++ 
a f t u  6 hours 
dark incubation 4+ 
(I U 
1.115 
1.095 
1.087 
1.130 
1.130 
1.13G 
Table 11-3. Buoyant d e n s i t i e s  of B ,  uIba &..a C. V i n o s w  
nit). varying -ts of i n t r a c e l l u l a r  sulfur 
1.6 I 
1.4 
1.2 
1.0 
0. e 
0. L 
0.4 
0.2 
Fiqure  11-18- Reduction of i n t r a c e l l u l a r  w l f u r  to s u l f i d e -  
112 
Cl ine,  J. ,  1969. Spectrophotometric determination of hydroaen 
sulfide in natural envirmaretrts. Limnol. Oceanoq.. 
I 14: 454--459. 
Guwrero, R a y  Has, J.,  and Pedros-cllio, C., 1984. Euoyant 
density chanyes due to intracellular content of sulfur in 
Chrorat - iur  wsrrinjJi and Chroraiiur V ~ ~ O S U S ,  flrch. 
fli crob i 01 . . 137: 350-556. 
Krieg, N-R. end Holt, J.6.. 1904. In G e r g e y ' s  Manual o f  
S y s t e - r s t i c  Defcrrinative Bacteriology. Vo1. 1, Williams and 
Wi 1 kins. Baltimore. Md. 
bison, D.C. and Cestmholz, R.' , 1Y81.  Use of reduced 
sulfur ccj'npound by Beggiat,a sp.. .- Eracteriol.. 
~ 147: 140-154. 
Strohl, W. R., Cannon, 6. C,, Shivley, 3. H., &de, H., tbok, L- 
A- ,  Lane, C, H., and Larkin, 3. N., 1981. Heterotrophic 
carbon metabolism by Bepgiatoa alba .  J. bacte.- iol .  
- 148:572-83. 
Strohl, W. R. and Larkin, J, tl. 1378. Enumeration. isolation 
and characterization O C  Fegyiat .oa from freshwater- 
sediments. Appl- Environ, 1.11 c r - o b i o l . ,  &: 755-7ii. 
Strnhl ,  W. R- and Schmidt, T. H., 1'7a4. MixGtrophy of the 
colorless, sulf ide-oxidizinq gliding bacteria Regqzatoa 
and T h i o t h r i s .  in t f icr-ot ial  C t )e ruaut .o$rGpr : , :  i W .  I?. 
Strohl and 0. ti. Tuovlnen, e d s . ; ,  O h i o  S t a t e  U n : v o r s i t y  
Press. Columbus. 
van -den, H.. 19aEi. On the A ; F  generation b y  
Chromatictr 11-1 dat-l.nese,. Fir-ch. Plicrobiul.. 
63: 11u-124. 
113 


Anaerobic reduction of elemental sulfur by Chromatium vinosum and Beggiatoa alba

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