ON THE METABOLISM OF THE
PURPLE SULPHUR BACTERIA IN
ORGANIC MEDIA
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ON THE METABOLISM OF THE
PURPLE SULPHUR BACTERIA IN
ORGANIC MEDIA
TER VERKRIJGING VAN DEN GRAAD VAN
DOCTOR IN DE WIS- EN NATUURKUNDE
AAN DE RIJKSUNIVERSITEIT TE UTRECHT
OP GEZAG VAN DEN RECTOR MAGNIFICUS,
Dr. C. G. N. DE VOOyS, HOOGLEBRAAR IN
DE FACULTEIT DER LETTEREN EN WIJS-
BEGEERTE, VOLGENS BESLUIT VAN DEN
SENAAT DER UNIVERSITEIT TEGEN DE
BEDENKINGEN VAN DE FACULTEIT DER
WIS- EN NATUURKUNDE TE VERDEDIGEN
OP MAANDAG 30 JANUARI 1933 DES NA-
MIDDAGS TE 4 UUR
door
GEBOREN TE AMSTERDAM
VERLAGSBUCHHANDLUNG JULIUS SPRINGER
BIBLIOTHEEK DER
RIJKSUNIVERSITEIT
f. . •
1-7 ••
M .
-ocr page 7-AAN MlIN OUDBRS
AAN MljN GROOTOUDERS
Vquot;
maar mij ook, zoo veel gij kondet, behulpzaam zijt geweest bij de ver-
wezenlijking mijner plannen en dat gij thans bereid zijt als mijn promotor
op te treden, is iets, waarvoor ik U niet erkentelijk genoeg kan zijn.
Vooral gedurende de jaren, dat ik Uw a.s.sistent mocht zijn, heb ik
veel van U geleerd, dat ik, ook bij mijn latere M^erk, op hoogen prijs
heb leeren stellen en nog steeds gevoel ik mij door talrijke banden met
Uw laboratorium verbonden. Ook de mij door U en Mevrouw Went
betoonde gastvTijheid zal ik steeds in dankbare herinnering houden.
Hooggeleerde Kluyver, het valt mij moeilijk onder woorden te
brengen, hetgeen gij gedurende de afgeloopen drie jaren voor mij
gewee.st zijt. Gij hebt mij de richting getoond, die voor mijn aard en
aanleg de beste was. Uw denkbeelden omtrent de eenheid in de zoo
uiteenloopende chemische verrichtingen der levende organismen hebben
een diepen indruk op mij gemaakt. Ik hoop, dat het mij gegeven zal
zijn, op deze denkbeelden voort te bouwen. Uw hulp bij het ver-
vaardigen van het manuscript van mijn proefwchrift wa» voor mij van
onschatbare waarde. Dat gij hieraan zoo veel van Uw kostbaren tijd
hebt willen besteden, vervult mij met groote dankbaarheid. Ook wil
ik niet nalaten U en Mevrouw Klui/ver voor de groote gastvrijheid mij
bewezen, mijn hartlt;'lijkcn dank te lgt;etuigen.
Veel dank ben ik verschiddigd aan de Nederland»rh-Amt'riknanHche
Fundatie, die mij door de verleening van eon »tudieln-urs instaat gestohl
hoeft, mijn studiën in de Vereenigde Staten van Noord-Amorika voort
te zetton.
I am very much indcbtcHi to Prof. Dr. IF. K. Fisher, director of
tho Hopkins Marino Station of Stanford Univorsity at Pacific (Jrovo,
Calif., for the kind hospitality which I enjoyed in this institution; my
sincorost thanks aro also duo to the momlKTs of tho staff of tho Hopkins
Marino Station for their manifold assistance and kindnoss during my
stay at Pacific Grovo.
H(M)gge)cgt;ordo Ur Meulen, V(H)r Uw waardovollon stlt;Min inzake do
koolstofl)ojmling Immi ik U zolt;t orkontolijk.
ZtHTgoUn-rdo Kleina, vool dank In'n ik II vorschuldigd voor hot-
goon ik van U gololt;Td hob on voor do hulp mij bowozon. Ook l', wannU'
Kingina Holtje» on ZotTgoloonh- tie ({raaf, dank ik v(M)r don sU'un go-
durondo mijn work in lgt;'lft van U ondorvontion.
Tonslotto iM'tuig ik mijn wolgonKH-ndon dank aan liot jHTsonwl
van hot Lalnjratorium voor Microbiologie tlt;» Dolft voor do gr(M)to In-md-
willighoid, waamioo hot stoods mijn wonsohon lun-ft vorvtU«!.
Dor Kodaktion dos „Archiv für Mikrohiologioquot;, sowio dor Vorlags-
buchhandlung Juliujf Springer bin ioh whr orkonntlioh für ihr fnMind-
lichcH Entgogónkommon iK'i dor V'oröffontlichung moinor Doktorarln'it,
(•«llfoillR.
Introduction ............................
Chapter I. Material and tlt;gt;ichnique...............134
, § 1. Material and methods of culture employed........134
§ 2. Analytical methodn...................13«J
Chapter II. Kxiwriment« on the lt;lovelopment of tho purple milphur
bacteria in or({ani(' media................ 13i)
Chapter III. InveHtiRation of tho metnl)oliHm of the purple aulphur
btictcrin in organic melt;iia................140
§ 1. The nature and the iiuantitioH of the metabolic protluct« formtMi 14«)
S 2. p]xperimentM on the Hiniiifionnre of Uio assimilation in the
niutal)oli8ni.......................145
Chapt^^r IV. I^reliminary Kurvey of the exfx^riinental roHultH obtnined 148
Chapter V. AnalyaiB of the tnetalgt;oIism of the purjAr tntlphur hnrlrrin
in organio media.......................
8uriunary...........................1«5
Literature citlt;Hi........................1(15
Introdiirlion.
Though it ha« long lxgt;en known that the purple sulphur Imctrna
are able to develop atitotrophically in a mineral modium containing
hydrogen Hulphid««, many jMiints concerning their inetaholiHni remained
difficult to explain.
Archiv fttr Mikrobiolofrie. IM. 4.nbsp;10
k.
Winogradaky (43) concluded from his experiments that HjS was
indispensable for the development and was oxidized to HjSO«. but also
that these bacteria had to be considered as strongly anaerobic. Tlie
investigations of Etu/elmann (10), Buder (7) and others showed that radiant
energy was very stimulating, if not necessary for their development. Finally
Bavendamm (3) stated that both H^S and light were indispensable, but
failed to give an explanation of this phenomenon.
It was not before van Niel's important contribution to the subject (28)
appeared, that we got a clear insight into the metabolism of these bacteria
£is it occurs in a mineral meditun containing HjS and CO,^. From his
experiments van Niel concluded that, imder uj^take of radiant energy, the
COj is reduced by the HjS according to the elt;|uation:
COj ^ 2 HjS ^ CHjO 2 S - HjO.
The sulphur appears as an intermediate product inside or outside the
cells and in its turn reduces COj, thereby being oxidized to HjSO,. so that
we get the final equation:
2 CO, HiS 2H,0 -gt;2CH,0« H,K()«.
Van Niel further pointed out that, in agreement with tlie theory of
Kluyver and Donker (Vd) about metabolic proce-sses in general, we may
consider this process as a dehydrogenation of H,S in 8ub8elt;iuent steiw»
to HjSO« with COji as the hydrogen acceptor. Comimring the eijuations
given above with that of the photosynthesis of the green plants:
rO, ^ 2 HaOnbsp; O, H,0
he arnvelt;l at the conclusion that the metabolism of the purple sulphur
bacteria in a mineral medium containing H,S and CO, has to ho ronsideroii
as a photosynthotic jMocess, representing a sjiecial case of the general
equation of photosynthesis:
CO, 2 n,A C,H () 2 A H,0.
Van Niel also showed that not only H,S is deliyilrogi'natlt;Hl to MjSO,
(with sulphur as an intlt;^rmediate i)r«)durt), but that, in the nbsenre of H,.S.
sidphur, sulphite and thiosulphat« can serve as hydrogen donators in the
I)hot08ynthotic process, thereby likewise Ix^ing converted into HjRO,.
Moreover van Nid carrielt;i out some experiments with niedia containing
organic suljstances, wlgt;ich settled an old controversy, lis will apinmr from
the following survey of the lit^irature.
Winoffradtiky addlt;Hl to his culture ngt;«Hlium 0.(Ktrgt;-().(gt;l % Ca-
butyratlt;\ Ca-formiate or Nquot;a-acetate and oblnined no l)ett4?r nwult^» with
very lt;lilut.o iwptono or meat extract. On account of the fact tliat 0gt;e iMUit-i'ria
in his cultures did not develop without H,S, lie considered thoni as quot;An-
My own work is a direct continuation of that of van Nirl. In thes««
jiages 1 liave given a brief review of Ills most imixjrtant rwiilts and con-
clusions. but for a goolt;l understanding of my own work tlio rwuler is re-
commendfHl first to make liimself familiar witli m»» Nu-Vh i»ii|)i»r.
« In those ocpiations Cll,0 «lonotw the primary pHwIuct of photo-
«yntJiosis from which all organic cell constituents are derived.
» H,S to S. S.H,0 to SO. H,SO,to SO,. n,SO,to SO,.
-ocr page 11-orgoxydantenquot;, i. e. a.s organisms which derive their energy from the
oxidation of inorganic comjwunds (in ca.su H2S) and empha-sized that they
needed very little organic matter. Since at that time he had not yet got
the idea of the carbon autotrophy of the pfirple sulphur bacteria, he naturally
considered these small amount,s of organic matter a.s neces.sary for their
development.
Molisch (24) held the opinion that organic matter in relatively large
quantities was absolutely indispensable, but based his conclusion chiefly
upon experiments with Athiorhodaceae. which can not oxidize HjS and
therefore can not live aiitotrophically. He mentioned however that a
typical, sulphur storing Chrot»atiuvi developed very well in a peptone-
dextrine mf^ium without H,S.
In connection with the favorable influence of light on the development
Molisch expre.s.srd the ojiinion that (I.e. p. i)l) „zwischen der Assimilation
der organischen Substanz, dem I.ichte und den Farbstoffen irgendein
Zusammenliang lgt;e.stehtquot;, and (]). 02) ..daß die Ern»ihnmgsversuehe mit
Purimrhnktericn uns mit einer neuen Art von Photos^aithese Ix'kannt
gemacht hal)en. Ix'i der organische SuKstanz im Lichte a-ssimiliort wirdquot;
Jhtdrr (7) considered the purple siilphtir bacteria a« obligatelj' auto-
trophic organisms, which do not n(Mgt;d any organic matter for their develop-
ment. btit biiild up their cell material exclusively from (quot;O,. He refuted the
idea that the typical jmrple svlphvr fmrtrria will Im» able to grow in melt;lia
containing organic substances in the a)»sonce of H,S. He mentionetl however
the possibility that intermelt;liatlt;» forms In^twwn Thiorhodaccae ami
Athiorhodaceac may exist which are able to develoj) both autotrojilucnlly
and helerotr()i)hicnlly. Harntdmmu (3) shared Jivdrr'n opinions.
Tan Nifl however u\ade the remarkable observation that Iiis strains, most
of which \uidoubtlt;gt;dly l)elong to tlie tgt;-pical jmrph miJphvrhavtrria. are able
to grow in n medium rfrfwW of oxidizablr nilphur roinpoi/vds, but containiiig
leucomethylene blue or reduced indigo carmine. In the rnurs«gt; of the meta-
bolism these leuco-lt;lyo8 wen' dehydrogenated to the corresigt;onding dyes
which fiirther roinnined intact. Moreover inv Nirl fouiul that a good
developnient of all his purjgt;le strains took place in mon« nlt;»nnnl «)rg»iiic
melt;lin. in the alMWMico of H,S. Oood nwilts were obtained with media
containing jH^ptone, yeast extract. Xa-lactate or Na-pynivate, whereas
glucose plt;'nnittelt;l only a scanty development. As wiult; the case in mineral
melt;lia containing oxidizablo siilplmr complt;nuuls. growth only oeciirnMl
under anaerobic conditions in the light, whereas in the .lark or in contact
with the air no devek)pmeiit was lt;»lgt;s,.rvelt;l,
I.Actio and pyruvic acids in contrast witli jM'ptone, yenst lt;«xt.rart
anlt;l leuco-dyfH — nr«« sinipl«- organic Huhstance». Then-fon« it sivmed
poKsibh' to carry out 0 (piantitAtive clu-mionl investigaticm of the
metjilmlism of tlie ;rtlt;f;)/f Hulphvr Imrlcria in media free from HjS, but
contHininR thow« or other simple organic compounds.
The remarkable point 1« again that growth in these media only
occurB in tl»o light. It ih evident that this noceHsity of radiant energy
for organismH thriving on organic suhstancoH ronstitut^'H a most int^T-
esting problem. Moreover one mipht oxin-ct that a more profound
10*
-ocr page 12-knowledge of this remarkable metabolism would contribute to our
insight into the metabolism of the A thiorhodaceae. W hen connected with
vanNieVs data regarding the metaboUsm of the purple sulphur bacteria
in mineral media, such knowledge might even deepen our insight into
photosynthesis in general [cf. van Niel and Muller (29)].
f'hai)ter I.
gt;Iatorial and technique.
§ 1. Material and methods of culture emploi/ed.
All my experiments were carried out with pure cultures, which
were obtained by the method described by wn iVteZ (28, p. 22—24).
I used the following strains of van Nielnrs. 1, 4, 7, 0, 12 and 19 (28,
p. 27) and three strains, named a, b and c, which 1 had brought from
Holland. Strain a was obtained by Dr. J. B. van, der I^k from an
enrichment culture started with mud from the Zuiderzee near the island
of Wieringen; strains b and c were obtained by myself as infections
from the air in bottles with a NaHCOg—NajS mineral medium in tho
laboratory for microbiology of the Technical University at igt;ltt.
Strains a, b and c aro very similar tt) each other and to strains U
and 19; thoy all l)olong to van NieVa Chromatium typo. Strains 4 and 12
reprosont his Thiocystis typo and strains 1 and 7 his PfteudomonnJi typ««.
For the groatlt;'r majority of my oxp-riments I havo takon ono ropro8lt;ui-
tativo of each of the throo types.
The pure cultures woro kopt in gloHS-Btopix-rod bottlos of .'Jd com
with a layer of paraffine oil on top of the modium, which filled up the
remaining part of the bottle complotoly. Contaminations which might
fall on tho bottle can not ontor thrtmgh tho paraffino oil Iwtweon nock
and 8toi)por and aro killed boforo ojH'ning the bottle by boating tho
nock in a burner flame.
mineral Na^S NaHCOg modium as woll as yoast lt;gt;xtraot and
Na-pyruvatc media woro usod. Tho niinoral modium had tho following
com|kgt;Hition:
n'hci............'-i';.,
(Xll4)f^lt;»4.........quot;•'%
k,hi'(),..........(•.0.quot;.%
with tho addition of 0.1 % No^S. 9 a«}, for strains n, h, c, 1 and 7 and
().(gt;.')% NajS . 9 aq. for strains 9, 19. 4 and 12.
NrtllCO, and Xii,S havo to Iw luldixl «ift«r st^^riliwition, »XM-auw thoy
low* iMvrt of thoir CO, and H,K whoii hout^nl in sohition. on arrount of hydro-
lysis. Tho XiiUCO, was dissolv«««! jimi st4gt;iriliHlt;Ml Hojwriit^'ly by filtration
throiifih a Seitz filter; the XajS was added from a stock solution of 10%
XajS . 9aq. which can be sterilised in an autoclave without loss of H,S.
The PH was adjusted to 8.0 for .strains a, h, c, 9 and 19; to 8.5 for 4 and 12
and to 8.7 for 1 and 7, with drops of sterile 10% solutions of HsP04 and
Xa,C03.
The yeast extract medium contained 2%Xa{quot;l and had by itself a ph
of about 7.2, which was .suitable for strains a, h, c, lt;1 and I Jl; for the other
strains adjustments to pn's 8.5 and 8.7 were made.
The pyruvate medium contained 0,25% N'a-pyruvate and the same
inorganic salts as the mineral medium, with the omission of XaHCOj and
was adju-sted to the same Ph's of 8.0. 8.5 and 8.7 for the different strains.
In order to exclude the oxygen as much as possible, the media
were either used immediately after oi)enirg the autoclave or after
l)oiling, followed by a rapid cooling; then the NaHCOg was ad«led and
in the case of the organic media one drop of a stlt;'rile solution of 10%
Na,S.9aq. for each KM) com of culture medium, in order to remove
the last trace of oxygen. This quantity of Na^S by its(gt;lf is too small
to cause an a])preciable development of the bacteria.
For the analytical ex{H^rimentKnbsp;125 ccm-bottles were used,
which were completely filled with the medium, without adding ])araffine
oil. Neck and stojqx'r were covered with paraffine after inoculati{)n.
The media employ«'»! contained the sanu^ base of inorganic salts a«
the pyruvaU' i)ure cultures, to which 0.25- 1% of the organic suh-
stance which was to be investigated and, if necessary, 0.25 or 0.5%
Na H COa were added. A little Na^S was added and the pn was adjusted
in the same way as with the jiure culturea.
Together with each cidture a blatik was jirepared.
The bottles were kept under continuous illumination in a light
cabinet as deHcrihed by »viw A'»'^ P-
Ah the p s. h.^ grow rather slowly, eultiires atul blanks often had
to Ik« kept in the light cabinet for thn-e weeks or longer. It sometimeK
happ«'nelt;l that during this long stny in the light p. .s. h. develoiied in
the blanks.
In all prolmbility this infection will have originated fnmi the air.
When pouring large (puintilies of medium from a flask into the ctdtun*
bottles, much air is drawn into the flask and in a room wh(gt;re intieh
work is done with p.n.h., thewgt; organisms will be present also in the
air«. The development of ƒgt;.nbsp;the blanks lH)wever eould Im'
prevented «.asily I.V ndlt;ii»R a little mercuric iodide.
' The tern\ '•jrtirpk tulphur Ixirtrrui'' will l)e H»gt;llt;reviHtelt;l from now
* Apiwrently th«w strongly antien)hic hact^TiH can cx-cur ni the air
in a form whieli is resistant tfgt; oxyK«'»«.
-ocr page 14-§ 2. Analytical methods.
The following substrates were selected for analytical experiment«:
lactic, pyruvic, succinic, malic, acetic and butyric acids, all used in
the form of the sodium salts.
The procedure of the analysis of the \ g l-cultures was as follows:
Immediately after opening the bottle two portions of 25 ccm were
taken out for the determination of COg, which was carried out in the
same way as described by van Niel (28, p. 86). P'ollowing this the pn
was estimated with Clark's and La Moite's indicators.
The culture liquid was then filtered in order to get rid of the larger
agglomerations of bacteria and brought to a pu of about 9.6. Two
thirds of the original volume were destilled off, the destillate was
acidified and 2/3 of its volume were destilled off. This was repeated, till
the volume of the destillate was less than 1(K( ccm. The de8tillatlt;.' from
the alcaline culture liquid contained the neutral volatile prcxlucts
eventually formed, and was tested for ethyl alcohol in the following
manner: 10 ccm were mixed with the same volume of concentrated
sulphuric acid and a few drops of a 0.2 n solution of potassium di-
chromate were added. When ethyl alcohol is present, the characteristic
smell of acetaldehyde is easily noticed. Control experiments proved
this test to be very sensitive. Of the de.stillatos from the lactate and
pyruvate cultures the refractive index was determined by means of
a Bausch and Ijomb refractomct^-r. The dcstillates from the succinat(gt;,
malatlt;gt;, acetate and butyrate cultures were also teHtlt;'d for acetone
with a 1% solution of 2, 4-dinitrophenylhydrazine in dihitlt;' HCl [cf.
ran (U r Uk (23)].
The residue of the alcaline destination was filtered from a prccii)itate
originating from the bacteria and acidified with HjS(gt;4 to a pn of
about 2.5. Nine tenths of the original volume wore dpHtillod off and
tho destillate was neutralis«'d with o.l n baryta. In the ease of the
Jicctate and butyrate cidtures twice or thrice • of the original volume
were destilled off, in order to collect all the remaining Hubstrat«- from
the medium. The combined neutraliwd destillat'CH were conoontrated
and acidified with an amount of HgSC)^ equivah-nt to tho anunint of
baryta used for the neutralisation. Following this a few «Irojw of a
saturaU'd Ag2S()4-Holution were alt;l(h'd in order to preripitate tho H('l
originating fnmi tho NaCl in the culture medium. Ksix-cially when
tho volume of tin» liquilt;l in tho doHtillation flask Iwcomos small, somo
HCl will go ovor together with th«- fatty acids,
Tho liquid was then brought t(» a vohmio of 1(Mgt; ccm anlt;l three
times JM) ccm wore destilled off. With butyric as woll as with aootic
acid three destinations of jq of the original volume are sufficient to
collect all the acid present. In the.sc three destillates the acid could
be titrated with sufficient accuracy.
In order to check whether the volatile acid remaining in the medium
was pure acetic or butyric acid, the titrated dcstillatos were concentrated
and after addition of the calculated amount of HjSO^, a Duclanx-
destination was carried out in the manner described by van der Lek (23) :
From a volume of 110 ccm 5 times 20 ccm are destilled off and collected
in volumetric flasks of 20 ccm. The figures so obtained were compared
with those from destinations of the pure acids.
During the acid destination another precipitate of bacterial origin
was formed. The residue was neutralised and treated with magnesia
mixture to remove the phosphate^. After filtration and evajioration
tlt;gt; dryness, 2 ccm of 50% HgSO^ were added and enough anhydrous
sodium sulphate to get a dry mass, which was extracted with ether in
a Sorhlet apparatus during 8 hours. The ether extract, which contained
the non-volatile acids, was drilt;gt;d over night with anhydrous NajSO^.
filtered and the ether was destilled off. After dissolving the residue
in watlt;'r and filtering, the aqiieous solution was neutralised M-ith 0.1 n
baryta. The haritim salts of succinic and mahc acids were then pre-
cipitated with the 14-fold volume of {gt;')% ethyl alcohol, the ])recipit«te
iH'ing allowj'd to settle over night. The next day it wa« conectlt;'d on
a .Jena glass filter nr. 4 and thoroughly washed with hot alcohol of the
!lt;ame concentration as that in which the ])recii)itate had lu'en forme«l.
.\fter drving and weighing the filter tlie Ba-salt was dissolved in HOI
and washetl out with hot water. 'I'he filter was then dried and weigiied
again in order to ctjrn'ct for small qiugt;ntities of liaSO^, which may
originatlt;' from a little H-jSO^ in spite of the treatment of the ether
extrart with anhydrous NugSO,. The amount of unattaeked succinate
or malate in th«' succinat«« and nialate cultur(gt;s can Im- detlt;'nnined in
this way with sufficient accuracy, if only in the case of malat4gt; the
»lentnilisation of th«« acid is carried out at boiling temiM-rature, in (mhgt;r
tlt;) d«'eomiM)se the malic anhydride.
The natun- of the alcohol-insolultle Ba-»alt (succinate or n»alHt4')
ean Ik- estahlislied by determining its Imriimi content. This was done
by adding 1 ccm of Ao^o MjSO^ U) the H(M-filtrate and collecting the
precipitate of liaSO^ on a .lena glass filter nr. 4, which was waslied,
' Phosphorie acilt;l is solulilo in ««ther to .Hotn«» extent and. when tlio
extra«! is neutraliwMl with Il.i(()n)f |'r«HM|»it«te(l as H«H1M)lt;. Siiu-e
ihiK «»lit is Hohd)le in HCU it interferes with tlte »letenninHtion of the non-
vohitile iicicls «s ilewriNnl l»el«)w and therefore it is iiiH-«»iM»gt;ry to riMuovo
the pluwphate iK^foro the oxtnution.
dried and weighed. In some cases the non-volatile acid in the filtrate
from the BaS04-precipitate was further identified by the vacuum
subHmation method of Klein and Wemer (16). In the sublimate succinic
acid was identified microchemicaUy as Pb-succinate, malic acid as Ag-
malate.
The alcoholic filtrate was tested for lactic acid after removal of
the alcohol, in the manner indicated l)elow and in some cases by the
microchemical reaction with yttrium nitrate.
When lactate was used as a substrate the amount remaining in
the medium was determined directly in the culture liquid by the method
of Ulzer and Seidd, following the prescription of Smit (35). The lactic
acid is oxidized to oxalic acid with permanganate in an alcaline medium
and the excess permanganate removed with Na^SOg. After filtration
from the precipitate of MnOg the liquid is acidified with IIjSO^ and
boiled to drive out the SOg. The oxalic acid is then titrated with
standard KMnO^. It is advisable to avoid a large excess of NajSOg,
since this substance may react with MnOa to form non-volatile sulphur
compounds (e. g. dithionic acid) which also reduce permanganate in an
acid medium. The lactic acid determinations were carried out in duplo
or in triplo.
The pyruvic acid remaining in the pyruvate cidtures was determined
by the method of Kayser, as described by van Niel (27). This method
is based on the reaction of CO-groups with hydroxylamine. When
NH^OH . HCl is used, HCl is set free which can be titrated with
standard alcali. Unfortunately it appeared that in my case this
method yielded results which were not quite satisfactory (cf. Cha|)ter III,
§ 1, h).
No indications of the presence of iu)n-volatilo neutral jjroducts
(e. g. glycerol) were found, cxcept traces of acetylniethylcarhinol in
the pyruvate cultures which substance was determined by the well-
known method of Ijemoigne in the maimer descrilM'd by mn Niel (2»)).
The 125 ccm-culturea were analytwd only for COj and for the
H'maining substrate in the manners indicated ttlM)vo. Kurthermonquot;
the amount of carbon present in the bacteria was detlt;Tmined by the
method developed by Ileslinga (12) for the elementary analysis of
organic HubstancoH. After the 2 samples of 25 cem for the COj-ilet.«^.
mination had been taken oiit of the bottle, the remaining culture lilt;|uid
was ccntrifuged in order to collect the bacteria. The suiK-rnoting lifpiid
was still turbid with breakdown products of the bactt^rin, hut afUT
atlding a few drops of 5()% sulfuric acid (up to a p» of alwmt 2.5), u
pn-eipitttU' was formed which couhl Ik» eentrifuged, leaving a clear
supernatant liquid which was then used for the determination of the
remaining substrate.
The liquid used for the C Og-determinations was also centrifuged.
so that the bacteria from the whole bottle were collected. The combined
precipitates were washed twice on the centrifuge with destilled water.
The bacteria were then rinsed from the centrifuge tubes into a porcelain
boat, dried at 45quot; C and burned in a quartz tube in a stream of oxygen,
using a mixture of equal amounts of manganese dioxide and lead per-
oxide as a catalyst. The amount of COj resulting from the combustion
was determined in the usual way.
By running analyses of pure succinic acid I convinced myself
that I had this method well in hand.
(quot;hapter II.
Kxpprlment« on the devplopmeiit of the purple sulphur hnctoria in orcanic
media.
As has Ix-en mentioned in the introduction, van Niel had already
obtained a good growth of the p. s. b. in yeast extract, jH'jit^jne, Na-
lactate and Na-pyruvate media, in the absence of HjS, which results 1
could fully confirm for all strains. In order to get a notion which other
simple organic compoinids were suitable substratea for the p. s. /gt;.,
I prepared cidtures in 30 ccm-bottlcs (in the same way as described in
Chapter I, § 1 for the pure cultures), containing the following substances
in quantities of 0.25, 0.5 or 1 % witli the addition of 0.25 or 0.5% biear-
Iwnate :
Nitrogeti-free organic acid« (as Na-salts): formic, acetic, |)roi)ionic,
butyric, succinie, fumaric, glycolic, malic and tartaric acids.
Alcohols: ethyl alcohol, glycerol.
Sugars: glucose.
J want to emphasize that, with the cxcei)tion of a siH'cial series of
control exiK-riment« (see Ik'Iow), all exixTinjcnts were carried out under
anaerobic conlt;litionH in the light.
A good growth of all three tyjx's of p. s. b. occiirred with acetaU-,
proi)ionate, succinate, fumarate and ngt;alat4'. With butyratlt;' only the
Chrfmiatiuni and I'srudowmms tyiM's showed a good develoj»mlt;'nt,
when«as glucoHlt;gt; was a suitable substrate for the Thiofi/stis and I'snido-
mma^ tyin-s only. Th(« higher p» to which the media for the last nu«ntio-
ned tyiH'H are adj»»st«(l (S.5 anlt;l H.7 resiwctively) as eomi)ared with
that of the media for the Chrnwatiuw tyjH- (S.O), may 1m« favorable to
an attack of the ghicose nu)locule, as sugars an» known to unlt;lergo
pungt;ly oheniical change« even in a slightly alcaline medium. Finther
investigation will l)c neceSHary to tlt;-st this assumption.
That the cultures I employed were really pure was shown by
inoculating yeast extract and peptone agar plates from them, which
remained sterile after prolonged incubation at 30quot; C^.
As already stated in the introduction, van Niel (28, p. 106) found
that also in organic media no development occurred in the dark. I have
had the same experience with yeast extract, peptone, pjTuvate and
glucose cultures (other substrates were not tested). The cultures with
the two last mentioned substrates were brought into the light after a
stay of a week or more in the dark at 25quot; C, whereupon they developed
readiljquot; into normal cultures.
That development with an organic substrate can occur in the
complete absence of HgS, is shown by the following experiment: a
pjTuvate medium was cooled immediately after sterilisation in a stream
of oxygen-free nitrogen. Cultures with this medium and with a pyruvate
medium prepared in the ordinary way with a small amount of Na^S,
developed equally well, whereas in cultures in a medium prepared
without either of these precautions to remove the last trace of oxygen,
development was either very much retarded or did not occur at all.
Van Niel (1. c. p. 103) also tackled the question whether prolonged
cultivation in organic media might render the p. s. b. incapable of
growing in a mineral medium with oxidizable sulphur compotinds and
ro|K)rted that four months of cultivation in yeast extract had no effect
in this direction. When I transferred my strains to a mineral Na^S-
medinm after cultivation in yeast extract for nine months, they all
develo{K'd well, showing the characteristic sulphur droplets inside or
outside tho cells. Even afU'r cultivation in yoast extract for a year and
a half, strain a, when transferred to a mineral Nji^S-modium, showed
a distinct growth ami all the colls stuffed with sulphur th«- noxt day!
Tho results of thoso oxjK'rimonts make it highly improbable that tho
p. 8. b. will ovor loose their ability to grow autotrophicaIly afU'r culti-
vation in organic modia.
Chapter III.
InvoNfiirutlon of llip nifgt;(Hho|iHm of Ihr purple sulpiiiir buc-tpriH In organic
modiu.
§ 1, The mtlurf ami the ifiiatUitie/i of the niHaholic prwIvcUi formed.
In order to establish tho nature of tho motAlMilic jinnluot«
formed from tho difforont substrates, analyses of oulturos in l.lM)ttlo8
' Of cours«» tills only shows that aerobic contaminations were altsent ;
ordinary anaerobic cuntaminaticms however would have l)oen easily noticinl,
lK«cause they would have develo|MHi much faster Ihan the slow growing
p. a. b.
were carried out. I used the following substrates for theae experiments:
lactate, pyruvate, acetate, succinate, malate and butjTate. I did not
obtain a good growth with other substrates until shortly before the
close of my experimental work. An analysis of e. g. glucose and pro-
pionate cultures certainly would have yielded interesting results, but
the experiments with the above mentioned substrates were so time-
consuming that I could no more analyse cultures with these two sub-
strates.
a) Lactate.
11 cultures were analysed, all with the same result, viz. that,
wherm-8 a luxurious developimtU of the bacteria took flare, no products
other than a small quantity of cnrbon dioxide and a very smaU quantity
of volatile acid could be detccttd. The alcaUne destillate was practically
pure destined water and the ether extract of the residue of the acid
destination contaiiu-d no non-volatile acids other than lactic acid.
Only about V'g of the substrata- (1% Na-lactatlt;gt;) had been used up.
The Ph of the medium had increased markedly, especially in the cultures
without NaHCOg, as a consequence of the formation of NaOH from
the lactate which had l)een consumed. Details of the analyses of five
cultures are given in Table I.
Tahlr 1.
Cultures on 1% Xa-lactatc in '/i Iquot;•»lt;»♦ ♦I«'«-
lAcUtc nupil COiforme«!, Vol. »old,
u.,nbsp;Chanito „„ mllllmoln inlllimoU mllllcnulvnlents
Mr«innbsp;per 100 rem. per 100 rem. per lOOecm.
9 |
81 |
7.8- 9.3 |
7 |
73 |
9.0-10.5 |
h |
88 |
7.8- 9.5 |
lt;) |
6» |
8.0- 8.8 |
7 |
6«J |
8.6-10.2 |
no Nail CO,
id.
id.
0.75% Nh net )s
ill.
0.04
0.06
0.04
0.05
0.46
0.10
0.60
0.79
fl.W
1.17
2.63
1.62
2.40
2.82
b) Pyruvate.
Thlt;- n-sults I obUiin.'d witli this suhstrat^- are similar tlt;gt; tlu»se of
the lactate' cultures in ho far that I lt;lid not find any neutral volatile
pnMluets or n..n.volatile acids oth.-r than pyruvic acid and that only
n.lativelv small nmlt;.unt^ of volatile aei.l wen« formed. The quantity
of CO howevcgt;r was much larger and I could det^-ct traces of ac.lt;tyl-
methylnirbinoP. The ctilture liquid had the oharaet^.ristic smell of
« The r.«.i.hgt;e of the carbinol ,let.gt;nnination was twt.Ml for 2. a-butyleno-
givcol I^^K^hng with bromitie NW (2«)1. but 1 found n.gt; i„d.c«tugt;n
of the prnsc^nce of thin «uJjHtance.
diacetyl, which substance was probably formed in very small traces
from the carbinol by oxidation by the air after the opening of the bottle.
I never noticed this smell in cultures with other substrates.
The Ph as a rule decreased. Table II shows the figures for the six
cultures I analysed.
Table II.
Cultures on 1 % Na-pyruvate in Vs l-bottles.
strain |
Ape in |
Change |
Pj-ruvate used |
COo formed, tnlllfmols |
Vol. acid, |
b |
bl |
7.8-8.0 3.37 |
3.77 |
0.17 | |
9 |
53 |
7.8 8.3 |
3.22 |
2.03 |
0.28 |
7 |
51 |
8.7-8.3 |
3.36 |
2.50 |
0.27 |
h |
50 |
7.5-7.4 |
4.51 |
4.11 |
0 27 |
9 |
59 |
7.5-6.8 |
4.62 |
3.99 |
0.4lt;) |
7 |
58 |
8.4-6.9 |
3.12 |
2.81 |
0.24 |
I used an EaMman Kodak proj)aration of pyruvic acid, which was |
a clear liquid with a slight yellow colour. The Na-salt was prepared by
adding NagCOg. The determination of pyruvic acid in the blanks always
gave much lower figures than expected on the base of the quantity
of pyruvic acid added to the medium, which was calculatlt;gt;d from direct
titration with 0.1 n KOH of a certain volume of the stock preparation.
Therefore not much significance can 1m' attached to the vahugt;s for
quot;pyruvate used upquot; in Table II. The explanation of this discrepancy
probably lies in the great reactivity of the carhonyl gro»q) in the pyruvic
acid molecule, which causes polymerisation, through which pnn'ess the
nnmlxT of free CO-groups is diminished and then-fore lower vahies in
the determination by the Kayner method are obtained. I tried to
purify the acid by fractionat«' destination in vacuo, followed by a pn«-
paration of tho Na-salt, which was precipitated with alcohol in tho
form of pungt;Iy whit«' n«H'«Uos which woro fro«' from wat^-r. However
when I titrated the frolt;gt; CO-groups in a fresh solution of this salt hy
tho Kayser method, I got a value which was 22% lower than th«' oalcu-
laU'd on«'.
Kvon when a really pur«' preparation of pyruvic acid is used, there
still remains tho probalgt;ility that, owing to th«' long durttti«»n of tho
exiK'rinu'nts (tho cultun-s havo to stay slt;mu' days in tho light at 25quot; C),
tho |)yruvatlt;' in tho slightly alcalino culture licpiid will uinlorg«! changes
which will ngt;nlt;lor tho «lotormi»mtions unn'liahlo [cf. rf/ (14)].
iSinco it woidd tak«« consilt;lorahlo time to work oiit this proi)l«'m
in such tt way that reliable results could Im* obtained, 1 havo not uwil
pyruvate as a substrate for «piantitativ«' «'xix-rinvnts as «h'sorilM'd
hi §2.
c) Acetate.
The raetaboHsm in acetate closely resembles that in lactate. I
analysed three cultures of strain 7 ^ after 1, 2 and 3 weeks; the quantity
of COg formed was likewise small and the pa in the oldest culture
hml increased considerably, as is shown in Table III.
Table III.
Cultures on 0.25 Na-acetate with 0.5% XaHCOj in V» l-'nittles.
strain |
Age In (lays |
Chango of pn |
Acetate nselt;i |
CO, formed, milllmols |
7 |
7 |
8.7-9.0 |
1.50 2,68 |
0.24 |
No neutral volatile products or non-volatile acids were formed.
/gt;»/r/flwa:-destillation8 showed that the remaining volatile acid was
practically pure acetic acid.
(1) Succinate and Malate.
The amounts of COj formelt;l from these substrates are larger than
those formed from lactate and acetate; more COj is formed from malate
Struin Arc In (l»y»
Tabic IV.
Culturivs on 0.25'',, Xa-succinate with 0.5% Nail CO, and on 0.25%
Xa-mahite in '/, 1-botties.
Subntrmo unpd t; O. formod, j Vol. nclil,
ChanKO nf itii ' up, nilllimolii nillllnioiR nilltl(gt;lt;)uivnl(gt;iitti
' por UK) com. per UK) pcin. I per loo com.
Succiiinte cultures.
8.0-9.0nbsp;0.Ü1
0.81
0.77
0.45
0.02
0.0()
0.04
7
21
25
I,
1
12
0.92
0.()3
H.7-y.2
H.6-8.8
MnUt« culluro«.
1.(58
0.70
0.75
2.09
0.95
0.78
0.05
0.04
0.07
«.0-8.H
8.7-9.0
8.5- H.9
20
24
82
a
1
12
' My firMt attempts «gt; grow tho other strams in acetnto were un-
surcm-ful «nlt;l it was n.gt;t bu( shortly Ix^for«. tho close of tlio ox,Hgt;ri,ne.ital
work (hat I obtained well develoixnl aeetnte n.ltun-s of lugt;m. Vor this
manon I l.ave n..t earri.nl ou( any an.dymv, of ace(a(« euKun« of strains
other than nr. 7.
than from succinate. Therefore the p« does not become as high as in
the lactate and acetate cultures. The malate medium did not contain
any NaHCOg; sufficient poising action was obtained from the COg
produced. No neutral volatile products and only very small quantities
of volatile acid are formed; besides the remaining substrates no non-
volatile acids could be detected. The Ba-content of the alcohol-insoluble
Ba-salts prepared from the substrates remaining in the medium agreed
fairly well with that calculated for succinate, resp. malate; in the
succinate and malate cultures of strain 12 the acids were moreover
identified as succinic, resp. malic acid by sublimation in vacuo. The
figures for these analyses are collected in Table IV.
e) Butyrate^
\\'ith this substrate a very remarkable phenomenon was observeil:
COg, instead of being formed, was taken up fro?» the medium. Again no
volatile neutral products or non-volatile acids were formed. The figures
for the Xgt;«cZatta:-de8tillation8 of the volatile acid remaining in the medium
agreed closely with those obtained from pure butyric acid. The results
obtained with butyratc are given in Table V.
Table V.
Cultures on 0.25% Na-butyrat« with (».5% NaHCOa in Vi l-bottlos.
strain |
Ar« in day» |
ChanKP of pn |
Bntyrate used | |
CO, takon np, millimol» |
b |
21 |
1 8.0- ^ 9.5 |
1 1.38 |
1.71 |
f) ('onolusions.
From the results of the analyses of the '/j l-culturos it appears
that the metabolism of tho p. 8. b. in organic mo«lia is not a fomu^ntation
process, such as occurs with other analt;'robic microorganisms (o. g.
alcoholic and butyric acid formontations and sulphate reduction),
which convert thoir 8uhstratlt;'8 for 5Mgt;% or mon» into motalM)litlt;gt;8, under
lil)oration of considerable amounts of energy and only for 10%
into coll material. With tho oxco])tion of COj in relatively small quan-
tities, no waste products of tho motalwilism have Imhmi founlt;l®. It slt;«omH
gt; Since dovolopmont of strains 4ami 12 (Thioctjatia typo) in a butyralo
medium was very irregular and insufficient for an aceuratlt;gt; analysis, no
data from culturw of these strains are available.
« The very small e|uantitio« of volatile acid found in the cultures with
non-volatile acids as sulwtrates are prolwibly pHwlucts of autolysis.
therefore that the substrata- is almost complet^ely converU-d into cell
material and CO2, i. o. w. that the assimilation predominates in the
metahohsm. In order to establish this more firmly, I have carried out
analyses of cultures in 125 ccm-liottles in which, besides the amount of
substrate consumed and of COj formed (or taken up), the amount of
carbon present in the bacteria was determined, so that it was possilile
to draw up a carbon balance of the cultures. The results of these analyses
are given in the next paragraph.
§ 2. Experiwevts 011 the significance oj the assimilniimi
in the metabolism.
Before giving the detailed results obtained with the 125 ccm-
cultures, I want to point out that as a rule a perfect carbon balance
(in so far that 1(gt;0% of the carbon of the consumed substrate can be
accounted for as carbon in bacteria and in Gi\) was not obtained. This
may be due to the fact that a certain portion of the bacteria had under-
gone autolysis, which means that the cell constituents had been broken
down to either soluble products or to fragments so small that they
were not carried down by centrifuging, not even aftlt;'r acidification,
which always gave a slight précipitât*». It Ih true that in very
young cultures but little aut^.lvsis had occtnred, but then only small
amounts of substrate had been used up- T»quot;« iniigt;aired the accu-
racy of the analy«^«. es]H'cially of the C(Vdetermiiuvtion, since the
quantity of CO, formed was often not larger than a few mdhgrams
per 25 ccm.
As was alreadv mcntion.nl, no 125 ccm-cultures with i)yrtivatc«
wen. atuvlysed. The n'sults obUincnl with the other five substrat*'«
follow iH'low. In the tables the amounts of «ubstratc- used up of CO,
form(Hi and of carbon in bact.Tia an' given in „ulligramatoms of carbon
tM'r KK) ccm of medium; the figun's in the last cohnnn n'pn-m'nt the
IMTcent^ige of the carlM.n of the substniU' accounted for ivs hacU'ria
carlM)n in bact. earlgt;on in (M), ^ ^^^^nbsp;analym-s of
and CO,:nbsp;carbim in substrat*-
. .nbsp;w il » ..f till. CO which ha^lt; m'en tlt;tken up,
the butyrat«'cultun'8 the carlMUi of thenbsp;, i •nbsp;A
,nbsp;.1 ♦ . fjmin. is obtained which is dinn'tlv
18 countlt;.d negative, so that a figun is odum
comparal.le with the figun's for the other suhstnitlt;.s: •
carbon in bact. - carbon in CO, ^ ^^^^
carlM)!» in 8ul)«trftU'
a) Lactate and Malate.
Table VI shows the n'sults obtained with these substratrs.
-ocr page 24-TabU VI.
Culturamp;s on 0.35% Xa-lactate and 0.25% Xa-malate in 125 ccm-bottles.
strain |
Age in |
Change of p^ |
Substrate |
COj |
Carbon in |
Percentage |
Lactate cultures. | ||||||
b |
30 |
8.0-9.5 |
3.29 |
0.41 |
2.95 |
102 |
12 |
30 |
8.5-9.2 |
1.04 |
0.12 |
0.82 |
90 |
1 |
30 |
8.7-10.0 |
3.70 |
0.25 |
3.15 |
92 |
Malate cultures. |
19
7
12
25
28
30
8.0-8.8
8.7--
8.5-9.2
4.48
5.41
2.92
1.25
1.77
0.86
2.72
2.94
1.93
89
87
96
We may consider these results as highly satisfactory, when we
bear in mind that the cultures were quite old at the time they were
analysed and that the pn had increased markedly in all cases both
factors favoring autolysis. The high yield in tho first ex|x'rimcnt is
probably due to a somewhat too high value for the lactate remaining
in the medium. With a view to the limitations of the analytical methods,
a percentage of recovery of about (M) % may be considered as a strong
indication that indeed the substrate is converted quantitatively into
cell material and CO,.
b) Acetate, Succinate and Hiityrate.
With these substrates serious difficulties were oncounten'd. Auto-
lysis, which was rather limitt'd in lactate and malate cultures, apiK»ared
to be quite strong even in young cultures with acetate, succinate and
butyratt?, as I could tell already from the peculiar smell of the culture
liquid. Another striking difference with lacUito and malate cultureH
was tho nature of tho bactlt;'ria: whon grown in thow two substratos
they were rather slimy and stuck together at tho Iwttom of the contrifugc
tub«!8, 8o that it was oasy to get them into tho jKircelain boat for the
combustioji. When grown in acetate, succinato and butyraU' howovor,
thoy formed a very finely divided precipitate which adhered strongly
to the glass and thoroforo was only difficultly removed from the tuln-B.
Owing to th(^ strong autolysiB tho first analyHcs with tluw huIgt;-
stratos showed [H-rcontagos of recovery of only fM)- •80%, though tho
cultures mostly wore younger than 20 and somotinu's not older than
7 days.
Now wo must lKgt;ar in mirul that in acotato, siiccinato and butyrato
cultures tho pn will rise strongly froni the start of tho dov«gt;lo|mu'nt:
with the first two clt;miplt;ninds ono equivalent of aloali i8 sot fnn^ for
every two gramatoms of carln)!) that are assimilated, which is only
counterbalanced by the formation of small amounts of CO2 and o
a little HgSO^, originating from (NH^JgSO^ after the a.ssimilation of
NH3. With butyrate one equivalent of alcali is produced for every four
gramatoms of carbon assimilated, but here COg is taken up instead of
being formed. With lactate however one equivalent of alcali is set
free for ever^- three gramatoms of carbon assimilated and with malate
much more COg is produced.
Moreover I observed that in many cases development with
these three substrates was much more rapid than with lactate or malate;
in one acetate culture 0.25% Xa-acetate had disappeared complet(»ly
after a week! It is evident that snch a rapid development will be
accompanied by a very rapid rise in pu, so that the bacteria will be
exposed to a high p» during a longer period than in a slow-growing
culture. I therefore startlt;Hl some more cultures, which I anah'sed after
only a few days, i. e. as soon as I thought that they would have deve-
loped enough to warrant a sufficiently accurate analysis. Indeed I
Tubk Vll.
Cultures with lt;t.25»(, Xa-aeetate. 0.25% Xu-succinate and 0.25% Xa-
butyrate. all with 0.5% XHHCO3. in 125 ccm-l)ottlos.
StrHinnbsp;ChunKe of p„
Substrate
used up
COj
fomiod
Carbon in
bacteria
PcrrentBffc
recoverea
AcetHt« cultures. | ||||||
7 |
10 |
8.7-9.5 |
3.72 |
0.28 |
2.30 |
69 |
7 |
20 |
8.7-9.1 |
3.rgt;i |
0.16 |
2.36 |
70 |
7 |
17 |
8.7- 9.5 |
4.28 |
0.43 |
2.26 |
63 |
7 |
7 |
8.7--- 9.5 |
5.84 |
0.38 |
3.94 |
74 |
7 |
3 |
8.7— -gt; 9.5 |
3.70 |
0.30» |
2.72 |
82 |
7 |
7 |
8.7— gt; 9.5 |
4.72 |
0.49 |
2.69 |
67 |
Suocinato culture». | ||||||
u |
13 |
8.0-9.3 |
4.73 |
0.73 |
2.51 |
69 |
12 |
17 |
8.5-9.3 |
4.01 |
0.63 |
2.10 |
68 |
1 |
12 |
8.7-9 3 |
5.06 |
1.10 |
2.83 |
78 |
u |
7 |
8.0-8.9 |
4.30 |
0.63 |
2,21 |
()6 |
n |
4 |
8.0—8.9 |
3.27 |
0.45 |
1.70 |
6(gt; |
I |
5 |
8.7-9.4 |
3.60 |
0.55 |
2.20 |
76 |
HutymtP culture«. | ||||||
II |
1 28 |
8.0 -8.7 |
2.74 |
-0.35 |
2.38 |
74 |
1 |
! 24 |
8.5 8.7 |
1.38 |
-0.28 1 |
1.35 |
78 |
a |
IR |
8.0- 9.5 |
5.40 |
— 0.84 1 |
4.53 |
66 |
1 |
8 |
8.7— ^ 9.5 |
3.33 |
— 0.45 |
3.53 |
92 |
1 a |
! 7 |
8.0-9 5 |
6.45 |
- 0.79 |
5.90 |
79 |
n i |
3 |
8.0—9.0 |
1.52 |
-0.39 |
1.97 |
104» |
' The OOj-d'termlnailon» In elil» «nalynii wore Io»t; Iho ftRurp in the table ii the imgt;aji
«•»Iciilaiod on the biui» of «he oihe-r «nulyw» (cf. p. I-V)).
* Ai only little baiyratc hu been roniiume«! In ihii rnllure, the orror« in the analytical
prooifdore« have be^n relnilvely larRP, which may account for the hlRh pcrcenURf of rocoverj-.
Archiv fflr Mikrobiologie, ltd. 4.nbsp;11
-ocr page 26-obtained better results with l)utyrate in this way; with succinate and
acetate results were still disappointing, but then it is difficult to judge
from the appearance of a culture when the right mean between a deve-
lopment too small for accurate analysis and too large on account of
autolysis, has been reached.
In Table VII I have collected the results obtained with these
three substrates; only those cultures which have been analysed after
a short time and in which not too much of the sub.strattgt; has been used
up, show a percentage of recovery of more than H0%.
Certainly the carbon balances of the acetat(\ succinate and butyrate
cultures are not as satisfactory as those of the lactate and malate
cultures. Nevertheless the percentages of recovery actually obtaiîied
with the three former substrates, together with the fact that in the
1-cultures no metabolic products other than COj could be found,
point towards a total conversion also of thes(gt; substrata's into cell
material and COg.
Chapter IV.
rrcliniinarr survey «Î the oxpprlmentiil resiilts ohtainiMl.
The ex|K'riments described in the two foregoing chapU-rs enable
us to give here an outline of the metabolism of the p. s. b. in orgatiic
media.
In the first ])lace the exjieriments re])()rtelt;l in (Chapter 11 show-
convincingly that a jM-rfectly nonnal developnuMit of the p. ». h. is
possible in media eontainiiig, Iwsides the necessary inorganic salts,
only one simple, nitrogen-free organic ccmipound. The most important
point in this connection however is, that these media are free from
oxidizable 8tdj)hur compoiunls. The significance of this fact can not
1m' easily overratlt;'d, since an authority on the subject like \i'{nlt;Hjrad.Hk ;f,
even as n^centlv as 1 !gt;31, still expre8slt;«d the opinio»» that the develoi)n»ei»t
of the p. 8. b. was absolutlt;«ly co»»fi»»ed to mineral media eo»»tai»ii»»g
hydrogei» sulphich' (44).
We must rememlH'r i»» this co»i»iectioi» that »vim A'»*/(2K) already
made some jm-liminary observations, which contradict«'«! ]\ tnlt;}{fralt;lMk )f'H
stat«'mei»t. Thes«' ohHlt;'rvatioHK however wen' n'stricted to a small
numlM-r of orga»»ic media, mostly «gt;f a c(»m|)lex »»atun-. My ow»» exigt;eri-
raents fully corroboratlt;'d those of van Xiel and mon'ov«'r they showlt;'«l
the suitability of a larger number of simple orgai»ic eon»poundH as
substrates for the p.s.b. Men'with the probien» of tlu- n»etalM»HKn» of
the p./f.b. i»» orgai»ic media had iMfonie accessible to a (piantitative
chemical treatn»e»Jt.
On the metabolism of the inuple sulphur bacteria in organic media. 149
In the meantime it has to be kept in mind that the exjjeriments
referred to above yielded another fact of primary importance, viz. that
also in this heterotrophic metabolism the cooperation of radiant energy
is an essential factor. We must conclude from this fact that photo-
chemical processes play a part in the conversion of the organic sub-
strates, which is a most remarkable phenomenon. Besides the p. s. b.
and the Athiorhodaceae, no heterotrophic organisms arc known in whose
metabolism radiant energy plays such an important part. Moreover,
with the Athiorhodaceac radiant energy is an essential factor for develop-
ment only under anaerobic conditions; they are able to develop in the
dark when oxygen is available [cf. van Niel and Muller (2i))]. The
p. 8. b. however, l)eing strictly anaerobic, do not develo]) in the jjresence
of oxygen. .Among the autotrophic organisms the green plants and the
coloured sulphur bactcria are known to need radiant energy for their
develoimient; this external source of energy renders possible the reduc-
tion of CO2, the only source of carbon for organi.sms living autotrophi-
eally. Now the p. s. b. are able to develop autotrophically (in the
presence of oxidizable sulphur com])ou!uls) as well as heterotrophically,
in both cases only when radiant energy is available. This suggests the
possibility that there is some connection l)etween the metabolism under
autotrophic and under hetlt;.rotroi)hic conditions. In the lU'xt chapt^-r I
shall rettirn to this ]gt;oint.
The fact that the p. s. b. convert their sul)strate8 ])ractically
completely into cell matlt;gt;rial and relatively small quantities of CO,,
is certainly not less remarkable than the necessity of radiant energy.
Whilst aerobic organisms ngt;ay convert u]) to .50% of their substrate
into cell moterial [cf. the fignn^s given for moulds by Waksniav (4(),
]». 417)], anaerobic organisms a» a rule convert their suhstrat^'s only
for 10% or less into cell mat^'rial and for 5M)% or nu)n. into mi'tiiholic
products. In order to grosp the full meaning of these foets. we must
realiz«' what the function of metaboliKm is. We can consider this function
to Im* twofold; firstly to provide the building stones for the cell mat^M-ial
(assimilation)'anlt;l secondly to provide the organism with the neci'ssary
energy (dissimilation). Since aerobic dissiniilation processes (i.e.
dehycirogenatior.H with oxygm as a hydrogen accepti.r) yield more
energy than ana«'robic ones (i. dehyjlrogcnations with organic
HuhsUinc-s or sulphat-e a« hydrogen actvptors) jht unit of suhstrat*-
conHumed, it is easy Ugt; understand that a«gt;robic organisms diHsimilatlt;'
a smaller fraction of tlu'ir stilmtraU. than anaerobic ones.
The p. s. b. appan-ntly lt;lo not lt;lerive the energy they need from
a chemical eotiversion of their substrata's. This question will 1«. dealt
with latlt;gt;r on.
Finally the experiments have revealed another phenomenon to
which we must pay attention, viz. that from the various substrates
different amounts of CO2 are formed. This is brought forward clearly
when we consider the amounts of COg produced in relation to the
amounts of substrate consumed. The figures collected in Table VIII
denote the number of millimols of COg produced pt-r millimol of sub-
strate consumed for the various substrates, both of ^ and of 125ccm-
cultures. The figures for the butyrate cultures are negative because in
this medium COg is taken up instead of being formed. At the bottom
of the table the mean values for each substrate are given. Since the
amounts of substrate consumed in the pjTuvate cultures are not
accurately known, no figures for these cultures have been included.
Apart from the very low second value, the figures for lactate do
not differ too much. The acetate, malate and succinate figures are also
fairly uniform, but between the highest and the lowest figure for butyrate
there is a considerable difference. When we compare the mean figures
among each other, we see that those for lactate and acetate do not
differ very much, but that the mean figure for succinate is distinctly
higher than that for lactate or acetate and that the malate mean is
again distinctly higher than the succinate mean, whereas the mean
figure for butyrate stands out by its negative value.
Table VJIJ.
Xum})er of milllmols of (quot;O» produced jht millimol of snljstrate consunuHl
for the cultures treatlt;Hl in Chapter III.
UctAte |
Aceute |
RucclnalP |
Malate |
Kntyrata |
0.38 0.10 |
0.89 |
1.28 |
-1.24 | |
0.05 |
0.23 |
0.84 |
1.36 |
-0.69 |
0.37 |
0.16 |
0.71 |
1.04 |
-0.51 |
0.33 |
0.15 |
1 0.62 |
1.12 |
— 0.81 |
0.23 |
0.09 |
0.63 |
1.31 |
— 0.62 |
0.38 |
0.20 |
0.87 |
1.18 |
— 0.54 |
0.35 |
0.13 |
0.59 |
— 0.49 | |
0.20 |
0.21 |
0.55 |
-1.03 | |
0.61 | ||||
innan 0.29 |
mean 0.17 |
mean 0.70 |
mean 1.22 |
moan — 0.74 |
The significance of these charaet«'ri8tic differences among th«- mean
figures for the various substraU-s iM'comes clear when we consider the
various oxidation levels (»f the Hubstratlt;gt;s. In this conneeticm it WH'nis
desirable to introduce a measure for the oxidation level of a compound,
i. e. for the degree in which the average carbon atom in the compound
is oxidized. It is obvious that this stat^ of oxidation must be compared
with the state of oxidation of the average carbon atom in a standard
compound. As such I should like to take the average carbon atom in
a carbohydrate. Since every organic compound can be supposed to
be derived from a carbohydrate with the same numl)er of carbon atoms,
either by addition or by substraction of hydrogen atoms (eventually
under uptake or loss of water), it seems indicated to accept as the unit
of oxidation the removal of 2 H-atoms and to define the quot;oxidation
valuequot; of a given compound as half the number of hydrogen atoms
involved in the said transformation, divided by the number of carbon
atoms present in the compound. When hydrogen has to he withdrawn
from the compound, the oxidation value will Ik» taken as negative,
in the oi)po8ite case as positive.
We can now calculate the oxidation values of the various substrates.
Lactic and acetic acids have the empirical formulae of carbohydrates:
CaHfiO.,, resp. CaH^O,; therefore their oxidation value will he 0.
Succinic acidnbsp;possesses 2 H-atonis less than a C^-carhohydratv
(C^HgO,), so its oxidation value will be = 0.25. The empirical
formula of malic acid is C^H^O^: l)y loss of 1 HjO this becomes
or 4 H-atoms less than a C^.carl)ohydrate, so that the oxidation value
of malic acid will Ix- i « , nbsp;Finally we get for butyric acid:
c,h«()j-|-2hj() c^h.jo, - C4H,()4 -tH. Oxidation value:
2/ __0 rgt;0. If we arrange the various sulwtrates according to
their*oxidation values, we oi)tain the following series:
hutvric lt; lactic aiul acetic - succinic lt; malic acid.
We slt;'e innnediatlt;'ly that the anumnts of CO, prodm-ed ju'r unit
of Huhstrat*. consiuned vary in the sanu' way. Now we nnist In-ar in
mind that, when (M), is split off from a substance, the oxidation value
of the pr(Kluct of this n'action will Im- lower than that of the
Htibstance itself:
(-M,cocoon CiriCMO fCMV
In this n.action the oxidation vuhie is lowc-red from -f 0.;i:t (pyruvic
acid) to 0.5(gt; (acetJildehyde).
Realizing that the sttbstrat«' is practically coniplet^'ly convertlt;Ml
intlt;. cell mat^Tial, we arrive at the conclusion that, when CO, is produwd.
the oxidation value of the ivll material taken as a whole will Ik- lower
than that of the Huhstrat«.. W hen however the synthesis of cell mat^'rial
from the organic «ulwtraU' inchules an uptake of CO,, the .)xidation
value of the tgt;..|| material will Ik« higher than that of the organic substrate.
Now we can calculate the mean oxidation value of the cell material
in cultures with the various substrates from the figures in Table VIII.
I shall give the calculations for lactate and acetate as an example.
The mean value for the number of millimols of COg produced per
miUimol of lactate consumed is 0.29. We therefore substract 0.29
(COg) from CgHgOg:
r 3.0(1 H fi.OO O 3.00
C 0.29nbsp;O 0..58 -
r 2.71 H 6.00 O 2.42
Now we add so much HgO that we obtain equal numbers for oxygen
and carbon:
C 2.71 H 6.00 O 2.42
H 0.58 O 0.29 -
C 2.71 H (5.58 O 2.71
and calculate the excess of hychogen as compared with an imaginary
carbohydrate of the same carbon content: 6.58 - 2 X 2.71 = 1.16.
This figure divided by 2 gives the number of oxidation units that the
cell material is more reduced than this imaginary carbohydrate: 0.58.
This figure, divided by the number of carbon atoms in a quot;moleculequot;
of the imaginary carbohydrate and providetl with a minus sign, gives
tho mean oxidation value of tho coll material in lactate cultures:
— 0.58/2.71 = —0.21.
For tho moan oxidation value of tho coll matlt;'rial in acetiito cultures
we got tho ff»llowing calciilation:
C 2.00 II 4.00 () 2.(K)
(• (1.17nbsp;() 0.34
('1.83 H 4.00 () l.(t(S
H 0.34 () 0.17
(• 1.83 M 4.34 () 1.H3
— 2X 1.83 TT-0.68. Oxidation value of tho coll material:
0.68
— ■ . =0.19.
2 X 1.83
In the samo way wo obtain tho following moan oxidation vahios
for tho coll matlt;'rial in tho othor substratlt;'8:
Though the oxidation values of the cell mat4'rial in th«- various
substrates are not identical (moroovor Table \'III shows that. oR|gt;ooially
for butyrate, there are differences among the individual cultures with
the same substrate), they indicate clearly that the cell material taken
as a whole is always a little more reduced than carbohydrate. Now the
cell material of bacteria in general will consist chiefly of proteins^
[cf. the figures given by Hopkins, Peterson and Fred (13) for Clostridium
acetobutylicum and Lactobacillus Ijeichmanni]. For the composition
of the average protein we may take the following figures: C 52.(1%,
H 6.9%, N 16.3%, O 22.5%, S 1.4% [cf. Oppenheimer {30, p. 121)],
from which we obtain after division by the atomic weights: C 4.38,
H 6.85, N 1.16, O 1.41, S 0.04. We can ehminate N and S as NHg
and HjS, substituting for one molecule of NH^ or HgS one molecule
of HjO, which gives: C 4.38, H 5.79, O 2.61. The oxidation value
calculated from these figures is —0.07.
Besides proteins bacteria will contain small amounts of sub-
stances which have a much lower oxidation value than i)rotein8,
e. g. fats, lipoids and, in the case of the p. s. b., the red (carotinoid)
pigments.
The»«quot; facts make us understand why the oxidation vahies fouiul
for the cell material of the p. s. h. are .slightly negative.
In conclusion we may say that the differences in the amounts of
COj produced or taken up in cultures with the various substrates are
in agreement with the observation that these sub»tratlt;^s are practically
completlt;'ly converted into cell material, the oxidation value of which
is approximately the same with all substrates.
in the next chapter 1 shall try to give a more detailed analysis
of the metabolism of the p. s. lgt;. in organic media.
AhHlvHls «f the molalH.llHni oI Iho purple sulplmr ».nrleria in (»nranie media.
Some 70 years ago Pasteur discovered that microorganisms exist,
which are able t(. develoj) in a medium c«)ntaining only one sinii.le,
nitrog..n-free organic elt;mii)ound. Still the important consequence of
this discovery, that thes«' heterotrophic organisms must l.uild uj) the
wide varietyquot; of .gt;rganic substances which are jiresi'iit in their cell
matlt;'rial, exclusively from this one comi)ound, has seldom Ikvii realized.
When we bear in niind that the comiM)siti(m of microorganisnis in
general is aln.tit as complex as that of tho higher plants [cf. tho data
' llucteria pnHluring larp« ..mount.« of roll wall material of a .«pIh..
hydnito nHtiinlt; will form an oxo«gt;ption.
collected by Buchanan and Fulmtr (%)], we realize how remarkable
the synthesizing power of such a small single-celled organism is. We
are still more impressed in the case of the p. s. b., which, in synthesizing
their cell material, use their organic substrate in such an economical
way, that only small quantities of COg appear as waste products. But
the synthesizing power of the p. a. b. appears in its most impre.ssive
form in a mineral medium containing oxidizable sulphur compounds,
where the cell material is built up exclusively from carlwn dioxide.
The same holds for media in which these sulphur compounds are
replaced by leuco-dyes hke leucomethylene blue, because these leuco-
dyes are only dehydrogenated to the corresponding dyes, which further
remain intact. In these cases the synthesizing power of the p. s. b.
equals that of the green plants.
These introductory remarks may suffice to eluciflate the remarkable
character of the phenomenon which is usually designated with the
simple term of assimilation. It .seems well worth while to enter into
a more detailed consideration of the chemical processes underlying the
synthesis of cell material.
Nowadays there can be scarcely any doubt that acetaldehydlt;-
(fat formation) and a-ketoacids, especially pyruvic acid (synthesis of
aminoacidsi) are the building stones of the complex organic suhstance»
which constitute the cell material [cf. A7wyivT (17) and QuaJiWl
To thes«. we may add simple sugars, especially glucose, from which a. o.
the })oly«accharide8 of the cell wall must Im- fornu'd.
When we consider this view|H)int to Im- generally ap])licah|c- for
an understanding of the synthesis of cell matlt;Tial, itquot; is clear that in
every 8plt;'cial case we have to account for the formation of thes.- quot;huil.
ding stonesquot; from the substrate. It will U- useful to illustrate this with
a few examples.
As such I choose the metal)olism of different organisms with
lactic acid as the organic sul)stratlt;'. This substance will Im- dihsimilatvil
in some way under lilHTation of energy. An aerobic organism will
dehydrogenate lactic acid with oxygen as a hydrogen acceptor. iM.r
' The rarlKJn chain of pyruvic acj.l itself i« f„uu.l in aia.une and serin.,
and Hly ,n tyro«n,c an.i tryptophane. However, als.. the amin..Hci,iK whi. h
are milmtitutiou pr.).h.ctH of valeric acid (arginine an.l histidine).
acul (leucuie) and ghitaric acid (glutami.uc „ci.l) „„. m.gt;Ht likely .leriv.Ml
from p.vriivie acKl. iKX-auHo the reactivity of this Hulmtai,..,. niak.viit .'.xtren»elv
Huitrtblo f.ir cot.phng rwictions with .)thor carlnuiyl co,„,KnindH I.Mi.linu to
the formation of a-ketcMci.U with more c.mplev earlMin cluiiiiH. such as
occur in the alK)ve mentioned aniinoacids.
the removal of lactic acid in muscle tissue during the aerobic phase
Kluyver (ll) has suggested the following scheme:
CHsCHOHCOOH O—► CHjCOCOOH HjO,
CHjCOCOOH—CHjCHO -l-COj,
CHjCHO Hj(»^CH3CH(()H)j,
CH.COH ()—»► CHjCHOH H,(),
^H \)n y
CH,CHOH.±;:CH,OHCHO.
0
Tho same chain of reactions may occur with any aerobic organism
thriving on lactic acid. Of courslt;» it is quit«' ixissible that part of tho
acetaldehyde is dehydrogonat^'d to acetic acid (which can bo further
dohydrogenated or Imlt; oxcrot^nl). Also the glycolaldohydo may bo dehy-
drogenat^'d. However, tho maiii feature of tho scheme is that the
organism in question can obtain the quot;building stonesquot; pyruvic acid,
acetaldehyde and carlxihydrato, from tho substrate by a series of
dohydrogonations with oxygon as an accoi)tK)r, in combination with
H docarlwxylation and a hydratntion. It ai)j)oars from the sclu^nu«
that acetaldohydo and carlMihyihato are obtained froni tho substrata«
via pyruvic acitl, so that wo may call this cimipound the quot;mother
Hubstanco of tho coll matlt;gt;riar'.
It is evident that othor suitable hydrogen acco])tlt;)rs can take the
place of oxygen, e.g. nitraU- in tho case of tho denilrifififig (Hicteria
«quot;lt;1 HulpliaU« in tho caso of tho «ul}}hate reduciiuj Ixirteria. The last
tnontion«Ml inicnM)rganisms aro highly aiuiorobic and as far as w(gt; kiiow
«ulpliat«' and other nHiucible sulphur com|K)unds aro tho only suitable
Hccoptors for thom [cf. linarn (2)].
Wo 800 then that in tho almvo niontioned caslt;gt;s th«« quot;building
Htonosquot; occur as intonn«Hliat«gt; pro«lucts in the dissimilati«)n pnKvss.
We may ox|H'ot that in tho ca«o of the /). h. h, the quot;building 8tlt;m«gt;squot;
aro likewim. obtaimMl fnmi laotic acid by dlt;'hydroglt;'?uition, the diffen«noe
^■ith tho alK)Vo montionod organisms In-ing only, that thes«' quot;building
Htonosquot; are converted oxclusivoly into cell maU'rial, instead of lK«ing
convortrd t« a largo oxt4'nt into wasU- pnxluct« in a dissimilation
process. The natun« of tho aoci'ptor uslt;«lt;l by tho /». h. will Ik« disouswd
Inter on.
Tho Hch«-mo for tho aHsimilHti.)n of lactic acid aids to tho under-
«tanding of the assimilation of pyruvic acid as wvll. Tw«. h-aturos of
the metabolism of the p. s. b. in pjTuvate cultures may be explained here,
viz. the formation of traces of acetylmethylcarbinol and the amount of
volatile acid, which is not as negligible as in the lactate, succinate and ma-
late cultures (compare Table II with Tables I and IV). It appears from the
scheme that in a pyruvate medium the first step ^—apart from an assimila-
tion of the substrate as such — is a decarboxylation of pyruvic acid to
acetaldehyde, a reaction which is easily brought about by a great
variety of living cells. Therefore the suggestion lies at hand that this
reaction will proceed with a higher speed than the assimilation of the
acetaldehyde or its dehydrogenation (in the hydrated form) to glycol-
aldehyde. This will lead to an accumulation of acetaldehyde which will
favor other conversions of this highly reactive substance. It is a well-
known fact that for instance yeast will convert an excess of acetaldehyde
into acetylmethylcarbinol [cf. Kluyver, Danker and Visser't Hooft (20)].
Another possible consequence of an accumulation of acetaldehyde is
its dehydrogenation to acetic acid instead of to glycolaldehyde. Though
acetic acid is also assimilated by the p. s. b., it is quite acceptable that,
as long as pjTuvic acid is present, it will be left untouched, since it is
much less reactive than the latter compound. Assuming that the volatile
acid formed in the pyruvate cultures was indeed acetic acid, its apjx'ar-
ance in these cultures becomes quite comprehensible.
Before giving schemes for the assimilation of the other organic
substances which I have used as substrates in my experiments, I must
say a few words about the assimilation of the p. s. b. in mineral mlt;'dia
containing oxidizable sulphur compounds. Here, as with autotrophic
organisms in general, the organic cell constituents are formed exclusively
from COj. This involves a reduction of CO^, which we may assume
to yield CH2(gt; as the primary product. Apart from the experimental indi-
cations of the occurrence of formaldehyde as an intlt;gt;rmediate prmluct
in the COj-assimilation of the green plants and of certain autotrophic
bacteria, a theorc^tical chemical consideration of COj-assimilation in
general naturally leads to the assumption that CHjO is the primary
product «)f this process. Whether we are dealing with the convorsion
of COj intlt;j hlt;'xoseH (in the case of the green plants), or into pyruvic
acid (in those cases where no hexoses or their derivatives ap]Ngt;ar as
storage products, hut apparently a direct conversion of CO, into cell
matlt;'rial takes place), we are facing the problem of thi- formation of
compounds with more than one C-atom jxt molecule fnmi a 0,-com-
pound of a much higher oxidation value. Th«- obvious coum* for such
a prlt;Kgt;ess to take is a reduction of the CO, to a C,-comjMMind having
approximately the same oxidation value ax the Cj- or Cg-com{)ouiids
that are ultimaU'ly formed, i.e. a reduction to CHjO. This com]K)und
needs not be identical with the stable formaldehyde as it is kept in the
laboratory, but may well be a reactive form, which, on account of its
reactivity, is immediately polvmerised to sugars or involved in other
reactions. The formation of pyruvic acid from CH.^() may proceed a.s
follows:
3C'H,0 -►CHjOHCHOHCHO (glyceric aldehyde)
CHjOHCHOHCHO CH,COCH (OH), (methylglyoxal hydrate)
(•HsCOCH(OH), -r ace. -^OHsCOCOOH H,-accgt;.
This conversion may take place with the p. s. b. in mineral media;
acetaldehydc can be formed from pyruvic acid by decarboxylation,
whereas sugars may be obtained directly from CHjO by pol_%-merisation.
Now that I have (h-awn up a scheme for the formation of the
quot;building atonesquot; from lactic acid, it is an easy task to do the same for
the other substrates in a similar way. Also acetic, succinic, malic and
butyric acids will have to be dehydrogenated in order to obtain pyruvic
acid from them; with this compound we have reached the scheme given
above for lactic acid.
For the conversion of acetic acid into pyruvic acid the following
scheme has often been suggested [cf. for instance Widand (42, p. 2(W)]:
2IICCOOH Hi;c.—gt; H ()()CC1I,(MI, COO II llj-aco.,
/
W
II IInbsp;II I.I
IIOOCC-CCOOll BCC.—♦ IIOOCC ('('(toll II,-HOC..
II 11
11 11
||()()C(';=(;C()()II iijOji^HoocciijCiioiicooii.
n(gt;(»C('ll,( ll()ll(quot;0()II : HOC.-* llOOCCHjCOCOOll II,-ai!«.,
llOOCCIIjCOCOOll—► CIljCOC'K)!! 00,.
The dehydrogenrttion of acetic acid to succinic acid has Ikmii
|k)stula(ed for a wide variety of aerobic atid facultjitiv(gt;ly analt;«rohic
orgatusms: for muscle tissue by 77gt;«7i6«Tf/(US) and Ahlffrvn (1) and for
bacteria i)ynbsp;('.ill); for mouhis igt;y various investigaUirs [cf. Jirrn-
Alt;it/er(4)]. Also U w-Z/ifMi (41) has expressetl himself in favor of this
mlt;Kle of l)i(K«hemical oxidation of ac«gt;tic acid. A purely chemical con-
version of aet'tic acid into sticcinie acid wa« effected i)y Moritz and
^VolllfnsU-in (25), tjsing iKTsulphate as an oxiiii/.ing agent, which n'sult
w»w confirmed by Jiernfuiwr and Slriti (5). \ ery few ohligatlt;gt;ly analt;gt;robic
organism« can thrive on acetic acilt;l as the only source of (^arhon: ajMirt
fnmi the p. a. h. (and proi)ably the AthiorhtxUtceae) only the methnnr
producing bacteria [Sohngen (36)] and the suljjhate reducing Vibrio
Eubentschickii [Baars (2)]. Whereas the former ferments acetic acid
to methane and carbon dioxide, the latter dehydrogenates acetic acid
to COg with sulphate as an acceptor; also here the first step may well
be a dehydrogenation to succinic acid.
The dehydrogenation of succinic acid to fumaric acid, followed
by a hydratation of the latter compound to malic acid, by quot;resting
bacteriaquot;, was proved by (pastel and Dampier Whetham (34). The
same processes probably occur with moulds [cf. Bernhauer (4)].
The dehydrogenation of malic acid to oxaloacetic acid and the
subsequent decarboxylation of the latter to pyruvic acid was first
postulated by Quastel (31) for Bacterium coli and Pseudomonas pyocyanea
and seems indeed the most probable way of conversion.
The assimilation of succinic and malic acids is also explained by
the acetic acid scheme.
With butyrate matters are more complex. It is {xjssible to conceive
of an application of Knoop's well-known theory of ^-oxidation of fatty
acids [(21): cf. also Coppock, Subramaniam and Walker (9): Kay and
Raper (15)], in terms of dehydrogenation and hydratation, as follows:
H Mnbsp;H H
CUsÇ-ÇCOOH -f acc. CHjC CCOOH Uj-acc.,
H H
H 1!
riisc=cc()0n 11,0:!^;: ciijcnoncuacoon,
CIIsCHOIICIIjCOOH -f-acc.-* riljCOCHjCOOll Hi-i»««:.,
ciiscornjcooii -f HjO—»► cnjcoon -f ciijcoon.
We would thus have obtaitH'd acetic acid fr(mi butyric acid by
withdrawal of 4 H-atoms and by addition of 2 molecules of H,().
In connection with the |k)ssihility of /^-oxidation of butyric acid
I tried to grow the thre«' ty])e8 of p. s. b. with Na-a- and /9-hydroxy-
hutyrate (two 30 ccm-cultures of each typlt;' with each com|M)und) as
substrates, with the result that only the Chromatium anlt;l Pnendomonas
types developlt;'d in /î-hydroxybutyrate and that n«ine of the ty|K'H
develojM'd in a-hydroxybutyratlt;'. Now theHlt;' two tyjM'H grow w«'ll
in butyrate, whereas the Thiocyntia tyj)«' d(K'H not, so that this result
is in agrelt;gt;ment with the aKHum|)tion of /5-oxidatio!i and slt;M'ms to exclude
the igt;osHibility of «-oxidation. However I only dispowd of small
quantities of a- and /î-hydroxybntvric acids, which wen* by no means
pure and fnmi which I prepared the Na-salts via the lia- and ('a-salta.
For this reason I do ?iot want to attach much value to th«»se exix'riments.
Now there is an other waj' possible for the assimilation of butjTic
acid. viz. a conversion of the methyl group into a carboxyl group,
yielding succinic acid. Yerkade and his collaborators (39), who found
that undecyhc acid is oxidized to undecane-diacid in the human
Ixxiy, introduced for this mode of oxidation of fatty acids the term
quot;(a-oxidationquot;. In terms of dehydrogenation and hydratation this
conversion of butyric acid might proceed as follows:
H H
CH —CCHjCOOH acc. —♦ CHj^CHCHjCOOH Hj-aoc.,
^H H
(•H,r-CHCH,COOH HJO^CHSOHCHjCHJCOOH,
H
HOCCHjCHjCOOH acc.—► (gt;HCCIIjCH,COOH Hj-acc.,
()HCCn,CHjCOOH nsO.±;rnOCCHjCH,Clt;)()H,
HO
H
HOCCHjClIsCOOH acc. —► HOOCCHjClljCOOH Hj-acc.
Hf)
In this way succinic acid is obtaiiu-d from butyric acid by with-
drawal of « H-atoms and by addition of 2 molecules of H^O.
A chemical conversion of butyric acid into succinic acid was
fffectlt;Hl l)y Cahrn niul Hurtki/{H), who oxidized butyric acid with
hydrogen }x»roxide. Slani, Suhrawani^m and Waikrr ('M) isolat^'d
Nuccinic acid from a culture of Aspfrgtllvji niger on Ca-butyratlt;' and
mentionelt;l the iMissibility that it had (.riginatod din-ctiy from butyric
acid by oxidation of the methyl grmip. I have sought very carefully
for traces of succinic acid in l-cultures with butyratc« as well os with
ttcet«t4', which wen- start^'d esix-cially for this purjKists but no indication
of the presence of this acid couhl 1kgt; fotiiul. This may nu'an however
that succinic acid and the other i)ossible int«'rm(«diatlt;gt; productlt;lt; are
more .lt;asily attackelt;l than butyric and ac(«tic aci(b themselves, so tiiat
they do n('gt;t ap|H.ar in the medium, but artgt; further convert^'d insi.le tho
cells jvH 8(M)n as thoy an- forme«!.
Tho two ways «)f assimilation of butyric aciil outlined alMivo soem
tho most ao(M.ptablo »)nos. As yot it is imiH)ssiblo to say which of thon»
is to b«« pn'fiTH'd.
In tho schonu's givon in this chapter 1 i»»vo di8cuss(gt;d how tho
various substratos can »h- convortod inU) tho quot;building stlt;mesquot; ..f tho
coll material by dehydrogenation, «locarlM.xylation aiul hydratation.
Besides the quot;building stonesquot; and COg. no other comi)ounds are formed
from the substrates by the postulated reactions.
Now dehydrogenation of a substance will lead to the formation
of compounds with a higher oxidation value than that of the substrate
itself. In Chapter IV I have shown that the cell material taken as a
whole has a lower oxidation value than all the substrates used in my
experiments, with the exception of butyric acid. Therefore the rise of
oxidation value caused by the dehydrogenation of the substrate must
be compensated in some way or other in the course of the assimilation
process. Such a compensation is obtained by the decarboxylations:
oxaloacetic acid (ox. val. 0.75) -»■ pyruvic acid (ox. val. 0.33) -»■
acetaldehyde (ox. val.—().50).
However we can not account for the formation of highly reduced
substances containing long hydrocarbon chains (e. g. the higher fatty
acids and aminoacids like leucine) by means of a decarboxylation of
less reduced compounds. The most acce])table scheme for the fornuition
of the higher fatty acids is the following one [cf. Hachn and Kinttof (11)]:
a coupling of two molecules of acetaldehyde, yielding acetaldol, which,
by splitting off water, passes into crotonaldehyde. This unsaturated
aldehyde is hydrogenated to butyric aldehyde, which in its turn
combines with acetaldehyde, etc. Thus we see that in this process un-
saturated compounds act as hydrogen acceptors.
A hydrogénation is moreover an essential link in the ])roce88 of
the formation of aminoacids from ketoacids [cf. Knoop and (hsterlin (22)] :
K. cocoon 4-nbsp;H. c xii. cooii n.o.
K .C - \H .COOH 2 I! —► K . C11 X M, . COO 11.
Now that we have c(mie to the conclusion that the assimilation o
the organic substrates involvlt;'s a series of ({«'hydrogénations a« well
as a series of hyilrogenations, it is logical to c(»nibin«' th«'He ])roe«'sslt;'H
to an oxidoreduction ])r«)cess in which the hy«lrog«'n of the substrat«'
is transfi'rn'd tf) organic acceptors, which an' the pn'cursors of the
nu»re reduced cell constituj'nts. Th«'se a«'c«'ptors are «h'riv»'«! from
dehydrog«'natilt;m products of th«' siibstrat««, viz. from th«' quot;buihling
stonesquot;; for instance the unsaturat«'«! ald«'hy«l«'s from acetaldehy«!«'
ami the in)inoalt;-ids via k«'toaci«ls from pyruvic acid. Howlt;'ver, the alMtvo
mentioned acceptors can not Iw the only on«-« which e«im«' into jilay
in the (lehy«lr«)genati«)n «)f the substrat«'. We have selt;»n in ('hapt«'r III
that in butyrat«- cultun's is taklt;'n up fnmi the nic«lium. 't his mi'auH
that C( gt;2 has Ix'en hydrog«'natlt;-d, i. «). w. that CO^ hnn artrd as a ht/drwjrn
arrej)tor. Th«' only jMmsihr«' hyjlrogen donators for the hydrogénation
«»f the COj an' butyric acid its««lf or com|M)un«ls d«'rive«i frtmi it; k«i
we arrive at the conclusion that here CO^ is reduced by an organic
substance. Since the oxidation value of butyric acid is lower than that
of the cell material, this substrate has to be dehydrogenated to a certain
extent by a quot;foreignquot; acceptor, i. e. by an acceptor which is not a
derivative of the substrate'» own dehydrogenation products. We must
therefore conclude that the jiresence of COg in butyrate cultungt;s will
be indispensable for the development of the p. s. b. The following
exjx'rimental results confirm the correctness of this conclusion.
ButATate cultures without special addition of XaHCOg, notwith-
standing the fact that these cultures were adjusted to the usual initial
Ph, scarcely .showed any develoj)ment. When NaHCOj was added
afterwards, the cultures immediately develojied in a normal way.
I performed this experiment with nine cultures, always with the same
result. The very slight development observed in cultures withotit the
addition of NaHCOj must be attributed to small amounts of COg
introduced with the inoctdum and, in the form of NajCOg, when
adjusting the pu. But this development did not yield more than 10 milli-
grams of dry bacteria per 100 ccm of medium. When 0.02 % Na H COg
was added, the yield of dry hactlt;'ria iM-came 4 5 times as large. This
«'ffect of NaHCO;, was not due to its ])oising action, lHgt;cause the p„
in the cultures withotit Na H CO., had not changed froni its initial value.
'the hydrogénation of COj will yield jirimarily ClIjO, which als«)
^ill he used for the synthesis of cell material; ther(gt;fore in the hntyratlt;gt;
tquot;ulturcs there are two sources of bacterial stihstance, viz. butyric acid
'ind COj.
\\ hen COj can act as an accei)tor for the dehydrogenation of butyric
acid, Mi(. Huggestion lies at hand that COj can do the sani«' for th«gt;
dehydrogenation of the other stibstrates'. However, in cultures with
the other substrata's is formed instlt;gt;ad of l)eing taken tip, so that
we have no «lirect proof that also in these cns«gt;s COj acts as a hydrogen
acceptor. Nevertheless there is an indication that, at least in acetate
and succinate eidttires, COj nnist ])lay a ]mrt as an acceptor in thlt;'
quot;Hsiinilation process.
On p. ir)4, 1 hav«' pointed out that the synthesis of tiie greater
»Majority of organic clt;«ll constitut-nts probably passes the ]»yruvic acid
stage. \\hlt;«n we look at the schem«'given on p. 157 for the conversion
of aeetlc and suceinie acids into pyruvic acid, we slt;«e that in the ease
' I want to iMiiiit out timl i*i»i .V(V/(2«. p. 102). when lt;lealinn with the
Kri.wtJi of llip /I. „. h. ill iirjjanic miMlia in the )il»sence of sulphur eoni|gt;oiin(lH.
«Ireaily HuuKivlt;tlt;Hl tlwit liirtic acid inijrht function as ii hylt;lromMi «lomitor
for CO,.
of acetic acid 0.5 mois of C Og are produced per mol of substrate converted
into pyruvic acid and that in the case of succinic acid 1 mol of CO, is
produced per mol of substrate converted. When we l)ear in mind that
part of the pyruvic acid has to be decarboxylated in order to obtain
the other quot;building stonesquot;, we see that the numf)er of mois of CO,
produced per mol of substrate assimilated to cell tnaierial, must be even
larger than 0.5 resp. 1. Actually I foiuid that the average number is
0.17 for acetic acid and 0.70 for succinic acid (see Table VHI). The
only way to explain this discrepancy is to assume that a hydrogénation
of CO2 proceeds simultaneously with the assimilation of these sub-
strates, i. o. w. that also here CO, acts as an acceptor for the dehydro-
genation of the substrate.
The formation of p\TUvic acid from lactic acid does not involve
a decarboxylation; in the conversion of malic acid into pyruvic acid
1 mol of CO, is split off per mol of substrate converted into pyruvic
acid, but in the assimilation of malic acid to cell material 1.22 mois
of CO, are split off per mol of substrate assimilated. Therefore there
is no indication that CO, will act as an acceptor in the dehydrogenation
of lactic and malic acids, such as there is in the case of acetic and
succinic acids. But it is quite possible that the amount of precursors
of highly reduced substances is insufficient to effect the dehydrogenation
of the whole of the consumed substrate, so that also in the assimilation
of lactic and malic acids the acceptor CO, must come into play.
The conceptions given above may well apply t(j the metabolism
of the Athiorhodaceac also. Thes«« organisms neelt;l organic substivncea
for their development; they can thrive aerobically both in the dark
and in the light, but anaerobically only in the light. Inn Niel and / (29),
when discussing this remarkable situation, arrived at the following
conclusions (I.e. p. 262): quot;In case the metalmlism of [rurplc bacteria
in organic media under analt;'robic conditions and with the supply of
radiant energy is chemically similar to the metalK)li8m under aerobic
conditions in the lt;lark, we are dealing with processes in which the de-
hydrogenations involve«! in the synthetic processes are carried out
with oxygen as the final acceptor in the dark, whereas in the light the
oxygen is replaced by some othf-r com|M)und which n-quin-s ttctivati«»n
by ralt;liant etiergyquot;.
With a view to the results obtained in this pajM'r, the suggestion
lilt;'s at hand thot this quot;other comiKuindquot; will Im' carbon dioxide. The
Athtorhfxi/iceae ik»«slt;'ss the sanu' system of pigments as the p. s. b.,
so that we may exjK'ct them to Ih' also capable of using (ÎO,, under
uptake of radiant energy, as an acceptor for the hytlrogi-tj of organic
com|K)unds.
Now that we have duly considered the chemical side of the assi-
milation, we must devote our attention to the energy side of this process.
Kluyver (17) has made it acceptable that the chemical processes involved
in the synthesis of cell material are spontaneous reactions, i. e. reactions
accompanied by a decrease of free energy. Now the assimilation of the
p. s. b., in mineral media containing oxidizable suljihur compounds as
well as in organic media, involves the use of COg as a hydrogen acceptor.
We may expect that a dehydrogenation of these sulphur compounds
and of organic substances with COg as an acceptor will Ix* accompanied
by an increase of free energy ; if so, the aiklition of radiant energy will
be a thermodynamical necessity in order to make these reactions
proceed. As yet it is impossible to say whether radiant energy will have
the same function in one or more dehydrogcnations of the organic
substrates with organic acceptors like those mentioned above. However
this does not seem very probable, since most of those oxidoreductions
will also occur in tho synthesis of cell material of ordinary heterotrophic
anaerobic organisms, which do not require the coo|X'ration of light
energy.
In connection with tho eiu'rgy roquiremonts of the assimilation
process, a few remarks alwiut tho iM)S8iblo other energy requirements
of the p. s. b. may bo made. Hesidos for tho jM'rformanco of inU'rnal
and oxtormd mochanical work, energy will Ik» nocossary for the quot;maintlt;»-
•lancequot; of tho organism. Tho various physico-chomical structures inside
the coll, which aro inlt;lis}Hgt;nsal)lo for its oxist»'noo as a living unit, nml
a constant stijiply of onorgy to proU'ct tlu-m against a breakdown.
A7uytyr(lK) has |Mnntlt;'d out that this quot;maintonancH'quot; nuiy Im« effect»»«!
by an oxidonnluction process which ostablishos imtontial diff(»n»noes
at tho intlt;gt;rfacoB which are tho essential olemonts in th(»se physio«)-
ohomieal structuros. Tho various dissimilation jirocessos which wo
oncountiT in living colls are finally iu)thing but oxidon'lt;hictions, but
the samo also hollt;is for the assimilation jirwossos. Then»fongt; it lt;!olt;»s
not seem oxohided that tho photosynthotic process taking plaolt;» in
the colls of tho coloured Hul/thur iHicUria not only ])rovilt;los tho building
quot;t^mes f«ir tho cell nu»tlt;'rial, hut also maintains the stnjctunlt;s inside
these colls which angt; oHwntial for thoir oxist4gt;noo. This nutans that as
long as assimilation proclt;M«ds. thoso bacU«ria will not quot;nee«!quot; a dissinula-
tion i)roolt;«sH.
Htit what will hapiMMi in tho ihirk ? In their natural onvironnient
the p. H, h. aro Bubmittrd to diurnal |M«ri(Kls of darkness and Ihoy
cortainly lt;lo mit dio or pass into a n'sting stag«' ovortught. Neitln-r can
We expj-ct that thoy will I*' ahlo to survive a in'ricn! of st'veral hours
without sonu' pnKM'ss taking plain? inside their colls, which maintains
Arehlv fOr MIkrobioloicia. iM. 4.nbsp;12
the potential differences in their physico-chemical structure. Similar
views are expressed by van Nid (28, p. 104^—107). He considered the
possibility that some fermentation process might occur in the dark,
but he refuted this idea because the p. s. b. are not able to develop in
the dark, not even in media containing yeast extract, pyruvate or
glucose. However I think that it is quite possible that an organism
can effect some special fermentation process, without being able to
synthesize its cell material from the fei-mentation substrate without
the aid of an external source of energy. Such a special fermentation
process might be the conversion of sugars into lactic acid. It is quite
conceivable that the p. s. b. are able to quot;maintainquot; themselves on a
lactic acid fermentation in the dark, but are able to develop only in
the light, because they need radiant energy for some of the dehydro-
genations involved in the synthesis of cell material.
In connection with this possibility an observation made by Derx
[cf. van Niel (I.e. p. 106)] is of importance: p. s. b. kept in the daylight
stained violet with iodine at the end of the afternoon, whereas early
in the morning no such staining could be observed. These facts point
towards the production of a reserve earbohydrattgt; in the light, which is
metabolised in the dark. Supposing that the bacteria indeed quot;maintainquot;
themselves in the dark by a lactic acid fermentation of a reserve
carbohydrate, one might expect that the lactic acid formed will be
assimilated during daytime. Such a situation can In* com])ared with
the discharge and charge of an accumulator; when the hactlt;'ria are
kept in the dark for a long time, the accumulator will Im« totally
discharged and the bacteria must either die or paas into a resting stage.
However, further investigation will lgt;e required blt;'fore anything
more defitiite regarding the dissimilation of the p. a. b. can Ih' said.
In summarizing the considerations given in this chapter on the
metalMjlism of the p. s. b., we can say that all evidence is in favor of
the assumption that with theslt;' organisms also organic substaricos
can act Jis doiuitors for the hydrogénation of COj, and that also in
organic media, one or more photosynthetic pr(M'esslt;'H are ijivolved in
the m«-talMgt;lism of the p. s. b.
This metalgt;olism is thus linked up with that of the p. s. b. in mineral
media, where oxidizable sulphur clt;miiM)undH act a» donators for the
hydrogetuition of (JO^ and with photosynthesis in genlt;Tal. In the
equation :
CO, 2H,A CH,() 2A 11,()
we may now replat-e H^.V by organic sulwUinces nu well a« igt;y H,S
or HjO.
Summary.
1.nbsp;The purjde sulphur bacteria are able to develop in media con-
taining only one simple, nitrogen-free organic compound, in the absence
of oxidizable sulphur compoinids.
2.nbsp;Radiant energy is indi.spensable for development in these media.
3.nbsp;A quantitative chemical investigation has been carried out of
the metabolism in cultures containing lactate, pyruvate, acetate,
succinate, malate or butyrate as the organic substrate.
4.nbsp;In these cultures practically no metabolic products other than
relatively small amounts of CO^ have been detected; in the butyrate
cultures COj is taken up instead of being formed.
.'i. By determining the carbon content of the bacterial substance
synthesized in the cultures, it has been shown that in all probability
the substrate is completely converted into cell material and COj, i. o. w.
that the assimilation predominates in the metabolism.
6.nbsp;The differences in the amoimt of COj formed (or taken up)
IH'r unit of substrate consumed in cultures with different substrates
are caused by the (Ufferent oxidation values of the various substrates,
the average oxidation value of the cell material of the bacteria being
approximate !y the same with all substrates.
7.nbsp;Since a consideration of assimilation in general leads to the
insight that the greater majority of organic cell constituents is formed
^fom the substrate via pyruvic acid, the ways in which this ivcid can
formed irom the various sjibstrat«'H used in the exiK«rim«'nts have
IxM'n discussed.
K. The conversion of (he substraU's into pyruvic acid involves
one or mon» dehy«lrogenation« ; a considlt;gt;ration of the hydrogen acci'ptors
which may effect this dehydroglt;'nation shows that CO, must play a
prominent part as an acclt;'jgt;tor in this process.
Iîgt; coimwtion with |xgt;int 2 this leads to the conclusion that
photosynthj'tic processes are involved in the njetwbolisni of the purple
f^^dphur badrria in organic nx'dia.
10. In the equation h»r photosynthesis in general:
CO, I 2n,A-gt;lt;'n,(gt;4-2A ll,0.
HjA may now n«plac«Ml hy «»rganic sulwUnces an well aw i)y M,S
or H,0.
I.itoriilure citfrf.
1)nbsp;U.Ahlgrnt, Skiuid. An-h. f. rhj-siol. 47, Suppl. I, IH26.
2)nbsp;J.K. Hwrn. Diiw. IWft H»»quot;-nbsp;HaimtUirmn, Die farbloson
»nd roten Krhwef..ngt;«kterien. Jenn H»24. 4) K. Hrmhaurr, Die oxy-
dativon (Hminnen. Berlin M».'»2. 5) K. HmJiaurr u. U . .SVm». Hiorhom.
Zoit«|.hr. 21«. 21», IH.12. — «) A'. Huchavnn antl E. I. Fiihner, l'))yMiology
12*
-ocr page 44-166 F. M. Muller: On the metabohsm of the purple sulphur btuiteria etc.
and Biochemistry of Bacteria, Vol. I. Baltimore 1928. — 7) J. Buder,
Jahrb. f. wiss. Bot. 58, 525, 1919.
8) E. Cohen and W. H. Hurtley, Biochem. J. 11. 164, 1917. - 9) Ph. D.
Coppock, V. Subramaniam and T. K. Walker, J. ehem. Hoc. 1928, p. 1422.
10)nbsp;Th. W. Engelmann, Arch. Xéerland. 23, 151, 1888.
11)nbsp;H. Haehn u. W. Kinttof, Cham. d. Zelle u. Gewebe 12, 115, 1920. —
12) J. HesUnga, Ree. Trav. chim. Pays-bas 43. 551, 1924. - 13) E. IF.
Hopkins, W. H. Peterson and E. B.Fred, J. of biol. Chem. H.i, 21, 1929. —
14)nbsp;A.W.K, de Jong, Ree. Trav. chim. Pays-bas 1«, 2.'gt;9, 1900; 20,
81, 1901; 20, 382, 1901; 21, 191, 1902; 21. 299, 1902; 23. 131, 1904.
15)nbsp;H.D.Kay and H.S. Raper, Biochem. J. 16, 465, 1922; 1», l.^S,
1925. — 16) 0. Klein u. O. Wemer, Zeitschr. f. physiol. Chem. 143. 141,
1925. - 17) A.J. Kluyver, Arch. f. Mikrobiol. 1, 181, 1930. - 18) Derselbe.
The chemical Activities of Microorganisms. London 1931. — 19) A.J.
Kluyver u. H. J. L. Donker, Chem. d. Zelle u. Gewelgt;e 1.3. 134. 192Ö. -
20) A. J. Kluyver, H. J. L. Donker u. F. Visser't Hooft, Biochem. Zeitschr.
161, 360, 1925. - 21) F.Knoop, Samml. chem. u. chem.-techn. Vorträge,
neue Folge, Heft 9, 1931. — 22) F. Knoop u. H. Oesterlin. Zeitschr. f. physiol.
Chem. 148. 294, 1925; 170, 186. 1927.
23)nbsp;J. B. tender Lek, Diss. Delft 1930.
24)nbsp;H. Molisch, Die Purpiu-bakterien nach neuen Untersuchungen.
Jena 1907. — 25) C. Moritz u. R. Wolffenstein, I^r. d. chem. Ges. 32.
2531, 1899.
26) C. B. van Niel, Biochem. Zeitschr. 187, 472. 1927, - 27) Derselbe,
Diss. Delft 1928. — 28) Derselbe, Arch. f. Mikrobiol. 8, 1. 1931, — 29) C. B.
van Niel and F. M. Muller, Ree. Trav. liot. Nóerland. ÄH, 245, 1931.
30)nbsp;C. Oppenheimer, Grundriß der Physiologie, Teil I: Hiochetnie.
Leipzig 1925.
31)nbsp;J.H. Quastel, Biochem, J, IH. 365, 1924. - 32) Derselbe, elwnda
1», 641. 1925. - 33) Derselbe, elwnda 20. 166. 192«. - 34) J.H.Quastel
and M. Dampier Whethain, ebenda 18, 519. 1924,
35) J.Smit, Diss. Amsterdam 1913, - 36) N. L. Söhngen. Diss, Delft
1906. — 37) H. B. Stani, V. Subramaniam and T. K. Walker, .7. cliem, Soc.
1929, p. 1987.
38)nbsp;T. Thunberg, Kkand. Arch. f. Physiol. 40. 1, 1920.
39)nbsp;P. E. Verkade, M. Elztu, J. van der Lee, H. H. de Wolff, A. Verhüte.
Sandbergen and D. van de Sande, I*roc. Kon. Akad. v. Wetensch. Sä. 2.51,
1932.
40)nbsp;S.A. Wakstfutn. Principles of Soil Microbiology. I^iltiinore 1927. —
41)«. Wieland. Krgebn.d. Physiol. 20. 477. 1922. - 42) Derselbe in C.Oppen-
heimer, Handb. d. Biochem. d. Monm-hon u. d. Tiere 2. .Tena 192ft. —
43) S. Winogradsky, Zur Morphologie luid Plijwologie der Schwefellmklerien.
1^'ipzig 18HK. — 44) Derselbe. Bull. lt;le I'lnst, Pasteur 2». 679. 1931. •
/ii
/I
-ocr page 45-De roode Zwavel bacteriën zijn mixotrooph.
De roode Zwavel bacteriën zetten hun organische substraten quantitatief
om in bacteriemateriaal en koolzuur, welks hoeveelheid afhangt van het oxy-
datieniveau van het substraat.
3.
Ook bij de — onder invloed van het licht verloopende — ontwikkeling der
roode Zwavelbacteriën in organische media wordt koolzuur gereduceerd.
4.
De Californische Coniferae — voorzoover zij een beperkt areaal hehlien —
zijn te beschouwen als relictendemen.
Dp door Heme gevonden reactie van methylclyoxaal met acetylazijnzuur
geeft een ongedwongen verklaring van het verhand, dat bestaat tusschen vetaf-
bratik en koolhvdrnatstofwisseliiig in het dierlijk lichnam.
M.Heme. Zeitschr. f. physioC Chem. 18». 121. 1930; 248. 1931.
H.Stöhr u. M.Heme, ehmda 20lt;i. i. 1932; 212, 111, 1932.
Het homologiebegrip van .Jacobshagen verschilt in wezen niet van het
historische h(gt;moloffielH.grij), zoodat de definitie van Gegenbaur boven die van
Jacnbshagen te verkiezen is.nbsp;, , ■ m- t • •
E. jZobshagen, .Vllgemeine vergleichend« Formenlehre der Tiere. Leipzig
1926, 8. 73—132.
7.
De infectie van Hazelnoten met NemaUuiiH»-a Coryli IVglion gescliiedt iloor
Blökende insecten.
De voorstelling van limütau^ over hei. ontstaan der polygnen in de plant
verdient de voorkeur boven die van Emde.
/v. Hernhauer. Hiocheni. Zeitschr. 24», 199, 1932.
H. Emde, Helv. chim. .\cta 14. 888. 1921.
9.
Voor het eleotrcHjapillaire von»chijnsel van limiuerel heblnM» FreumUich en
Saliner. en liikerman ven bevredig-Mule verklariiig rgquot;'ven.
H. Freundlich u. /i. SöUner, Zeitschr. f. physik. Chem.. Abt. A, 1J8. 349, 1928;
152. 313. 1931.
.ƒ. J. liikerman, ebt^nda 153. 451. 1931.
D.» door Clark «.«.n-en verklaring van het v.M.rkomen van Diatomoeön in
«edimenten. die bulten de ,gt;o..Ih.reken onlMjuu. zijn. iR te verweriM-n.
liruce is. Clark. J. ot (iwl. 1«, 583, 1921.
Het verdient «nni ftanlM.veling. • in het inteniationale wetenHchapiK'lijke
verkeer, n^t KnJ.l-ch. I)«it..ch en tVansch. een vierde taal te gebruiken.
■jm-
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