COL TtV A'fKl!f OP OF AUl.Alt WITR OTIIER
FR1!3B-WATE.R MICROORGANISMI 1M '1&11 fROPICI
JOra• Jorgen.Mn ud laclnto Coant
~mio de Cabo Jlanco,
llaiqMtia, D. F.,
Tbe iDdu.etrlallntereet la tbe l&rc•·~teale eulthatloe~ of mJerMiaae
fticb bu been mutiNt • tM Ulllted ... anllelaewtaere atnce tlle publlllltbt
or tM wortt olS,.C.br :uMS Ida e»-worker• wn.' tM nnaUle CJ)lQ:.
may ~ the patllkatkla of work earrled oa for maay
ne1~ tn an the eulttfttice or heteroseneau• com-
'"of freab-water mk:roora&atlma. Tbe followlac .-zoraliOD ot tldl
- Wlll refleet more the lulatent •tnaat• to tleYelop a be1teally .aund
\d11: thaD ta.e preaentatkla of preelee bAochmical data. Jt ta hoped, bow•
...-, th.at the 1iptflcance ol tbeM otMenaUOnl on the •• of microalpl
nu.r .. ta t.~u autrltien wlll oUHt tbe eompvatlve gr01ane .. of our
aaetiaods and data.
Our WOt"k at Cabo Blanco wltb trellb-wat.~ mJeroorpaama la a aequel
m tbe P.....arvatbo Late Project~ wtúeb datM back to ltl2, and Whlch coctemplat.
ct the larce-~teale u .. of the plzJtoplankton of Lah Maracatbo for
· ttM extnction ol Yitamln.t, notably cvoteDe, and for proce .. tnc to ob*•hl
prote!De. Tbere wu tn the Scancttnntan eountrtee 1n tbe early tbirtiea
coaelderable lnter..-t 1n aettt.nc evotene at Jow coet for ue u aa dditlYe
prodact ID 'DU'¡'&IiDe and for the winter fetcitnc ot da1rJ cowa. lince 1t
wu lumw11 U\at ra.,.,. leaYee or acelled cvrote u a .ource ol
-tto-•itamla A, lt ., .. thousht tbat .ome YUJ abur.c'zet tropical folia~•
1'A¡ht be und u a 1n matertal. Jor¡IUIG undMtoot t:o a •am- * of aucll matertal• ln V-ne••la 1n 1832 to cet a J"CCICh •••• al tMtr
~1~e avotene cODteat. ,._ method conalstld al~ 1ft co•aputQ¡ tbe
lle!t8lty of the oranse-,.now ttneturea obtalnabll from tbe vl«t material
• fttrac ... with gaeoUne and wbllequently ab1kln« out chlorophyU wilh
• aqo~eo,• eohUon ol cauttc .,aa.
CWLftYA1'1)Jf OP ALGAE IN 'l'HJI! TIIOPICS iet
fttl te 01'\MM teeta were. made •loolc tiM •horea of Lue Mar.caibo,
wtdclt olteD., eepeeiiiiJ ta DI'Gt'Diftl cahu, sbowe lar¡e areu COftl' •• witt.
YetJ ••u• pb}'toplanldon coloQiea held in wspea.lon elo.e to tM Rl'fact.
Tbe idea occW"red to .Jorc•n•eo that tbt• practtcany unUmtted .upply of
cblor~'Uic meterw could bt obta1ned u a concentrate bJ lar¡e-eealt
flltrat1oo of tlle ,...J', and c:OQJ.d then be r~ced to a thtck fiuid. U a conUn~
·• proceu of catrlfuptioa could be nolYed. 1t could nat be re~
to a poa 111' oe a ne.am drum tlrter ud ntraeted witA a •uttablt
IIOl•aaA w .Uale C&I'CÜnt-rtcla Upidu.
On 111•ral tri¡)e. ~ tM •~ore• of the late, moet otten ln dugout
c:uo.a, mlcJ'Ot'lgal aurfue coloate• oo tbe becalmet wateu we.re .eeoped
up with • calaba~ di¡)per .c1 filtertcl through a couple ot old ftlt hat..
Tbe qtaalltitaUve Jttld at tbe best wu 1320 me (d.rJ wei¡ht) per Uter of
Thla ter m, whtch means llttnllJ HtJdek úter," le nted br
the nattvt l.lalbtrmtn to deacrtbe thoae parta of tht la.ke water contaisUAg
myriads of p1Jlhead-ttzed coloniee of mJcro•copic al¡ae wlthln M tacb or
two of tAe n.rface. Tbe 44tbiek water" le d»peraecl tomewbat by tht tnd~
wind that aprlnlt up after tea o'cloek ln.the morning, and then the )ield of
al¡aa 11 leu. One collecU.oe from the·agttated "thlck water., eoetained
about .00 mc/1, but 150 to 200 mg/1 waa tbe usual yield.
WbeD tJwt drled material waa extracted w1th topplnjJ-plant gaaoUM,
aolull0111 of llpidea were obtaifted wtth an orange color up to 6 Umtl u
lnt•tM u the beat from leavu. Tbe Jllicroalgal cbloropbylla clid DOt 10
lnto Ws eol vent, a faet ftieh gaft tM mate!' !al an advantage onr foUq•,
trom wb1ch chloropbyU WJ 1 ahrays obtained wlth tbe carotenoid eomplu.
A metbod of lsolattng carotene aQd carotenotd8 waa developed, throu¡h tbe
dt.conry that the wbole eoapla could be adaorbed .on lead tultide, leav·
1na the colorleas llpidea la tM solvent. Typic~l rhombtc cryatalt of c;a,oteftt
were later obtatned abmdanUy fl'om a1m11ar natural mleroal¡al -terlal.
Tbe common yellow unthophylla u well as red analoguea from tbe ~
MJXOt*yceae were also reeognlaed and obtatned 1n separate aolatioes. ~·
To utlllae tht defatted material, it wat plaontd t0 de.elop a conU.OU.
proctle of HpaNting protelna from ca.rbohydratea by pualng .a gu tbnilSb
a mlxed eoluUoc o! both and then .,_••me tht froth lnto petrole-om ettwr 01'
heuu O'Nr water. The protelns woWd rematn colloldally attacbtd ka tllill ·
petrQIMun .tta ar 1llJrer, whlle the c&J'btalllrdratn woWd go lnto tbe 1IPiiat.
Accompanitd u tts.r would be bJ a sJMU amouat of protamSn, tbe carbob)'
Clratea couk' be IAWiHd iJl a termea•Mioo lndw!try or aa clllture medl\Uil
for J•eta 01' fuaai.
lll tbe year 1U3 U. Vane•elan eo.ernRUtat pant.d JorgettMn p1tenta
ol '"* ecope fOI' UM p.repanüon ot pb~ fOl' l.Aduatrlal purpc¡11a
1~2 GROWTB OF ALOAE IN MA88 CULTU.R.I
and for tbe extractlon or carotene from a~ae. A twenty-year exclualve
cooceutGa for the lDduatrlal t~ae of tbe pbytoplankton on the southern hall
of Late Maracaibo wae stgned betweeu Jorgeuen and ...oeiatea and tbe
Government in 1~38, and lt was raUtted by the national Coasr••• 1n 1938.
UDder tht. conceaaion a tu wu to ha ve been paid only on .the weilbt of
material hanested from the late and oot on sub8equent inerease by cultintlon.
Therefore 1t was plannM to multiply natural harvesta of phytoplaokton
from Lau Maracatbo by pullll)lng them lnto baslns o! ferWized
water of proper pH and to use carboll dtoxide from flue gasee u a source
of carbon. Tbe cost of obtaintng a tu-free yield in thls way was to be
compared with the coet of obtatntrc the .ame quantity of material by_filterlng
a larger quantlty of lake water, and paying tax on the whole amou
At firat the projec::t wu recetved wtth skeptictsm and enn wtth rtdicule
in Europe and the United States. It wu compared to gettt~ gold out
of sea water. Almoat aU btochemists constdered 11$-llver olla cheaper
and better 80Urces of vttamin A tban tbe pro-vitamin carotene. Recent
research at the time had tndtcated that vltamin A from animal sourcea
was superior to the pro-vitamin in both human and animal nutrltion, as it
went more readlly 1nto hepatlc storage.
A certain intereet in the project had been shown by an American pbarmaceutical
manufacturlng company ae early as 1934, but by 1940 netther
tbat concern nor any other was eufflcienUy interested in tM nutrltional
or therapeutlc poss1billtles of phytoplankton to wa.nt to engage in their development.
ID that year the chemical department of a large food industry
expres.ed the view that the project was sound sctentifically but wlthout
commercial interest. 'nle concesslon was canceled by the Venezuelan
Government in 1941, because the propoeed industry h.ad not been started
wtthln tbe two years stipulated, wtth an addlttonal year of grace.
The Culture. at Clbo Blanc;o
The Maracaibo Lake Project had received considerable publiclty in
Venezuela and there was perhape a eertaln perplexlty 1n government cirdea
u to why foretgn a.nd dome8t1c capital had not come !on-ard lo deftlop
tt. ID July 1941 Jorgen.se.n recelved permlaeion to maintain at the
CabO Blanco Leproaarium, at the expense of the Ministry of Health and
Social Welfare, a fatr-stsed experimental culturr of tre.U-water mieroorpntams,
and to test thetr nutrlUonal value, in co-operation wtt:h Dr.
Jeetnto Connt, in cues o! low general health among the pattents. Tite
nutr,donal use of tbe cultivated alga! "soupe" would at the same time
permJt observat1on1 as to the course of the disease in cases that were
aggranted or statlonary in spite of treatment wtth chauhnoogra oU.
lt wu declded to carry on the cultures ln such a way as to simulate
u nearlJ u possible the natural ~currence of freah-water phytoplankton,
which dlrectly or indirectly •usU. ina an almost endlees varlety of animal
CULTIVATION Oi' ALGAE IN. 'nlE TROPICS 113
We. h was thou¡bt aleo tbat tbe nrlety of nutrlUonal factora would ineTHII
wtth tbe oumber of IJ1)6Ciea.
As culture ve.sela we used bowls made locaHy of Ullllaztd baked red
clay. Tbey measu.red 40 cm bl d1ameter at tbe top and 20 cm in depth, and
tbeir capadty was about 15 utera. Eaeb bowl was placed on a cement pillar
1 m 1n hel¡ht. Evaporatton from·tbe poroua aldea kept the water at a
temperature of Je• C, even tbough the bowls were exposed to direct tropieal
The water u.ed wae the municipal supply of the ~n of Maiquetia. lt
wu alicbtlJ allalline and calcareoua and at the time waa untreated for the
destructlon bf microorcanlsms. It eontalned a nrlety of phyto- aod sooplaotton
ll!liCU.. Mieroalgal bottom sedimenta trom an open cement water
tank were used as aeed material, but algae from local mud.boles were later
obtatned and culttvated to provide additicee' apeciee for wh1eb our cwtw'e
medtam wu favorable. Cbroococcaceae Mtd ~ystaceae were frequently
obtaJ.ned from licbena by placlng these in ftltered eartb decoctiona to Whkb
a clar1fted broth obta1n8d by boUJ.ng m.tcroalgal "aoupa" bad been added.
Tbe aerobtc fungue would prolUerate at the su.rface, and tbe alga! a-belot,
baYlng been released from its captivlty 1n the fungal hyphae, would muh '•
at the bottom or in suspens1on. The algae thue obtalned, if capable of ra¡. J
multipUcatlon lA our medium, would be incorporated in the complex of t <e
Durq the ttret two years we uaed aolutlons from a commerctal 6-10-8
fertilizer to matntabl in the bo•l• a nutrtent eoncentration about 1 per cent
above the natural eontent. We have slnce, bowever, used various other
t,pee. Any natural water to whlch any commerctal fertilizer has been
ldded will nstain aome sort of microalgal complex. (We are at present
aucce..tully uai.ng a commerctal preparatlon known u ".Plantabbs" torether
with ammonlum acetate to produce very dense cultures of a cornpla
rlcb 1n lipi<lea.)
Tbe ferUUsed water gave a sUghUy acid reacUon.. Llberal add1tlona of
clarified deeoetioll8 boom well manured garden eoU or barnyard topso11
were made to each culture veasel twtce a wettk. Thc cultures were eUrred
nnee a day with a dlpp&r to keep the most pbototropt.c epecies from shutUn¡
otf the Ught from Ule specles at the lower levela. The water lost by evaporation
wu replaced cSaJly.
Tbe typical complex consi.sted of bottorn ¡rowt.ha of Myxophyceae, moatly
Chroococcaceae and matted aggreptea of alender fllaments oí HomocystiDeae
with infUtered cella of spherlcti ¡rHn algae. ~ystaceae eucb as
Cblorella, Qecyetta, and .Ankietrodeamna, and 1ome Scenedesmaceae occurnd
denaely ln sufll)enaion. On the aurface we generally had very beavy
194 GROWTH OF AI,GAE IN MA8S CULTURE
proliferations oí a atrongly phototropíc green euglenotd witbout a Oagellum,
but with a red eyespot close to the anterior tip. lt moved by gyrating the
anterior part ot tts exceedtngly elasttc body. tt would encyst readlly,
forming a dark green motionleas sphere. Its "chloroplaf:lls" seemed to
be Chlorella cells harbored In lts cytoplasm.
CUiates such as Colpidium and Vortlcella and at times Paramecium
were usually abundant among the algae. Rotlfers were rare, but microcrustaceans,
such as the copepod Cyclops and several species of Cladocera,
would be present from time to time, th'e former at the bottom and lhe latter
closc to the surlace. On the sides of the bowls the larvae oí a species oí
Chironomus would build tltcir sheltcr tubes Crom algae caught in their silkspin.
The intense r~d color of these .. blood. wigglers," be !.n~· due. to hemoglobin,
might indicate the presence of hematopoietlc ractors In the micro
on which they tived.
We called the entlre _material "plankton" to facilitate reference ~d by
reason o! its similarity to the natural comple"' otJresh-watcr microorgantc
lifc commonly known by that name.
A fair allowance .for error must be made as re(':ards our identification
of algae. Thcre is as yet no biol~lst ln Venezuela with special trainiug in
microalgal taxonomy whom we could consult. tt is quite possible that some
of the microorganisms called by us Myxophyceae bclong to groups difiicult
to class1Cy because of charactertstics that place them as transition forros
between bacteria and algae.
For the purpose of harvesting, wc took advan~age of the natural descent
of thE' cells to the bottom during the night. In the f:'arly morning, before the
upward phototropic moverr.ent had started, we siphonerl úff Crom 80 to 90
per cent of the water and hrushed thc precipitated material írom the sides
of the bowls into the bottom. In this way, we obta!ned from E>ach bowl every
9 to 15 days an average of 1.5 liters oC thick "soup" containin~ about 2.5
per cent dry weight o! microorgantsms. Thc water slphoned off wal'> refertil.
ized and returned to the bowls after each harvest.
Thc net amount of cellular material ln the daily harvest was estimated
by dryin~~ 20 ml of thc «soup" ar.d wetghing the resldue. By the end of 1943,
we had 200 culture bowls in use with a da ily average of about 3000 liters of
water. We harvested during that yE:ar a total oí 8060 liter..; of "soup" contalnin~
an estimated minimum of 204 kg of microorgantsms. One bowl,
each harvest from which was dried on a framed glass plate 0.~ m2 in area,
gave a total of 1368 g in one year.
The cultures were from time to time invaded by Cladocera. and in
such cases the water was clear alter harve~tin ~ anct had to h · re-inoculated,
but ordinarily no reinoculation was neccssary. We malntaine<t ('mergency
se~ cultures, howcver, ln severa! extra bowls. The water lost by evaporatlon
in these was a!ways replaced with fertilized t~arth decoctions, and
they wue r.iven carbon dtoxtde in the formol ordinary ftoda water. It waa
CULTIVATX>N OF ALGAE IN THE TROPlCS
·obeerved that slale soda water e.xcelled the gaM<>Us product as a etimulant
for repr~uc:UQn, perlla¡» because of the greater ab111ty of the water to retaln
c~bon1c ac1d. The 14soups" harvested from these bowle at monthly
lntervale were of extraordinary volume and thicknees. Harvesta of 4 llters
of "soup" w1th 45 g dry weight of rrJcroorgantsms to the Uter were commonly
obtained. This gave usa rough idea of what might be produced in
large culture baalns of appropriate constructton with the use of earth decocUone,
a euilable fertlllzer, and carbon dloxide.
AdmlgislraU.On to Leorous Patient¡
During the ye~rs from the beginntng of 1942 to the end of 1946 these
plankton "eoups" were used aa accessory food for leprous patients. The
harvested "soup, waa boUed for about 20 minutes and a líttle salt was
added to tmprove the flavor. The patlents drank it willingly. The taste,
whtch was not unpleasant, varted with the speetes complu:.
The patients before recetving the "soup" were In poor general health
and ln an advanced stage of leproma!ous leprosy. They were of all ages:
37 were from 8 to 20 years, 26 from 20 to 40 yPars, and 17 from 40 to 70
years. The daUy dosage of plankton "soup" was 400 ml for the cruldren
and 600 ml for the adulta. The length of tre~tn1ent for an individual varied
from one to three years.
Observation of the eflect of the administration of the planl<ton "soup"
naturally was interfered wlth by the well known spontaneous variation in
the condlUon of leprous patlents. Certalnly no m effe-cts resulted lrom it.
ln a majorlty of the cases there was a marked improvement in energy, in
wetght, and in general health, all of which seem~d to reeult f:-om the plankton
"soup" rather than from any other factor in the treatment of tite patients.
lt was not possible to arrange a control group of patients that would
rnatch those fed the plan.kton "soup." The control had to be the record of
the patients prior to treatment and the general experience here that lepromatous
leprosy does not improve spontaneously in children.
In some cases of severe anemia and emactaUon we employed a ccncentrate,
obtained by bolllng the ordtnary "soup" down to about one-third of
lts volume to contain about 7 per cent dry wetght of mlcroorganlsms. An
outatandtng case waa that of a 33-year-old woman in thie leprosarium, whose
wetght increased from 41 to 57 kg during one year when she recelved 500 m1
of the concentrate datly.
Here at Cabo Blanco we are now endeavoring to produce large quantittea
of concentrates of a ·cértain fractlon of mtcrcalgal Uptdee which seem~
greaUy to accelerate the leprous reacUon and the passege of active lepromatous
leslons to ctcatrlcial forms. Recent observatlon.s duri.ng the experimental
treatment of a prevtously untreated case of very eevere lepromatous
leprosy 1n a 12-year-old glrl point toward the poselbillty that the active
196 GROWTH OF ALGAE lN MASS CULTIJRi
microfact.or involved 18 a strongly hypoph.asic red carotenotd from tbe
Myxophyceae. The production of thls pigment by these :ntcroalgae is
greatly 1ucreased v.·hen they utUise arr.moatum acetate as a source of
The ptgment wm be tried pr~.ferentially ln a group of norJeper~ who
ha 1e been pro ved 'by a negaUve Mltauda te8t to be naturally suaceptihle to
the diseue. Shculd this tt>st cballie to pos tUve after adm1nJatratlon of the
carotenoid dur!ng a ccrtaln period, tt n:11ght be possible to prevent U\e
spread of leprosy 1n countrles where 1t la endemtc. by giving to susceptible
pel"IOns a natt1ral res!.stance factor which they are, perhapa hercditArlly,
unable to produce in their skin tissuee from common carotenoid precursora