GALIFORlnA STAIJ.'E lli'HVERSITY, NOR'l'ERI:CGE
EFFECT OF
A
PREDATOR ON SPECIES DI"\JERSri'Y
\l
A study under conditions of
continuous cultivation
A research proj~ct submitted in partial
satiEfation of the req~irements for Lhe
degree of Master of Science in
Biclogy
by
--
Daniel SteptEn SvJee:Jey
January, 1979
f.he project of :Janiel Sweeney is
approved~
California 3taTe University, Northridge
.;ar~i..i&.ry, 1979
:,.."'"._~~·-. . . . .~.......... -.. ..~----~ .. - ........._ · __________..__._,._,___,_, ______ ,..._.._._....,_,___--.., __________________.______,.....__ ~~ .. -·-~~1
;;,.-•.•
-·..-.-~·-L<-•·~•-•r
....__,., •. ___
...- _ _,___,_._,.. .....
~-....,...._- --~
-.,.,~--·-
__ ,.,..
._.....__~.~-·•
,..,_.,_. __
~·-•
.._...,,
-~
,.,.._.., .__._, _
__..,
-·-••-_,,__,.,_...,._.~,-·_.~~-,
••-~o·~~.•,-..--•-··•·•'><·_,....,.......,~---- ~·
!
AC Kl'W\'JLEDGEMENTS
To Dr. Charles R. Weston I am indebted for his
patient assistance,- critical
results~
~valuations
and exuertise in continuous
of my·
cul~ivation.
also thank Dr. Charles Spotts and Dr. James Dole
for their assistance in my research, as well as,
serving on my com:nittee.
Lastly, I exnress my graditude and love to
my wife and children for their patient forebearence
of my long absences from them while conducting my
research.
I
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'I'ABLE OF
. .. . . , . . . ... . ........ .........
CONTE{JTS . . .. .. . . . . .... . . .. . . .... .. . .
LIST OF FIGURES
ABSTRACT
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INTP.O:L)iJC.TION
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l\1=-:DIA ..... ..............
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CONTINUOUS
0::
.. . ....... .. 66
fi-1ATHSrv;ATICAL ':!"E:COFY •••••
A PP.ARJ\'l'US ••••••• " ....
..
IVlAT~RIALS
lt:iErr·I:ODS AND
ORGAJ~IS!v1S
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CulCIVr,'~':
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........... ..... .. ......
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ANALYTICAL !ViETEQijS ••••••
~
.!\?~ALYSIS
POPULATION
I:'
J..·v
I\I:~LT~Jr~
••••••••• ~ • a • • • • • • • • • • • • • • • • • • • • • • • • • •
INITIAL BATCH CUL'.L'UN.:S •••••••••••••••
STEADY STATE A?TEI'-'iP'I·S ':liTH YEAST
S'I'EA~Y S'TATES OF
S~~'EADY STATES OF
EACTE.RIA • • • •
••••••
20
••••
'"'1
c.~
• ·•
.........
PF.O:OZOA
13
18
COMPETITION BETWEEI,; ::31\CTERIA
IN Tllli CtrE:~~lOSTP/I •••••••••••••••••••
sr_L'EADY STATES - BOTH 3ACTERIA
AND PROTOZOA • ' • • • a • •
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4
BIBIIOGni-\PH!.
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-----------~--------~--·------~-j
LIST. O.f.' .!.'IGURES
FIG'TR.ES
-,
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2
DIAGf\Ai':.
OF
'='
SPECLt.\.L
.'-}
STEADY STATE OF
BA'l'C~'
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CtTL::''JR.E APPARATIJS ••••••• ':...'{ .
PSEUODr~~O:-~AS
FLUORESCEHS- AT DI.t<'l<'ERiiTT
SUBSTRATE CONCENTRATIONS ••••••••• 24
ESC"f'...ERICHIA COLI •••••• 26
5
STEADY STP.TE OF
6
1•JASHOUT OF TETRAHYME:·-JA PY1UPORr;IIS
7
T. PYRIFORHIS AND P. FLUORESCENS IN
STEADY STH.1'E .••••••••••••••• "·· •••• 31
8
T. PYRIFORMIS,
\'liTH OUT P_REY ...................... ~ 29
P. F'LUORESCENS, &
1<
OLIINT-s·T.t.A>_...,_P.lE
._, I y ~'i., m
.!::!.•C
•••••••••• 34•
·--------~-----------·----------
v
i:FF:SCT OF A PR2DATOR
0~~
SPECIES DlV ~~RS 1 TY
A study under conditions of
continuous cultivation
by
Daniel Stephen Sweeney
N:aster of Science in Biology
N.ay, 1978
A study was made of the food web formed from a
Teterh;ymen~
: nrotozcan,
uvrifcrmis, two bac~seria., Pseudc:-
'
. , .
1 •
;' mo:-~as 1..t..uorescens .an d _,.::,scnerlct!:!.a
£.2.:::..!,
ar.c.- a g.tucose
~,
~
;
l
i
•
•
m1n1.mal medium in chemos tat culture.
An attempt ;-as made to
.further substantiate the idea that species diversity withi
~in a cmr.munity can be dependent upon predation.
The fJys-
'
'
tern was divided into simpler
narts, first by demonstrating
-
; that each bacterial species in pure culture would achieve
steady-state growth on the substra.te, whereas the protozoa
1
could not. In batch .and
steady~state
growth studies of
, _pure cultures o:f the two ··bacterial species,·. P. · fluorescens
· was~the·more successful.
l P ..
fltJ.O:CP.sc.:c~ns
;. escens•
and E. coli in the chemostat,
~onulation
~
~
In competiton studies between
E·
flucr-
level was 100-to 1000-fold greater.
Interestingly, E. coli's steady-state population increaf->ed
·vi
-
;annroximate.ly
t10-:fold
when -P, fluorescf:ns was a.dd,ed.
:i.n
~
.
~
.the .food
we~ study.
predation ·of the protozoan on tr1e
:two bacterial resulted in s teady.:.sta te growth of t;oth
i
;t.he nredator,. less numerous prey ,and more ::1umerous prey.
;This study seems to indicate that the competitive exclusion
l~rinciple
may be altered if a predator is feeding upon
oo competir,g
;
!-
..'
;
l
urey who otherwise may not coexist.
;
..
~-~..--- ~~~--·
,________ ,.,_,.. _.,_.._.._,.,~.~-·--..--··~~-~·~-·-··~~-··--~~~··~··-~--··¥~·--'-..~-__,, -~---·- .-- -~~------- -·--~-------. . . . ·~-·---···rl-··""'"' •·r;
J
I~~]'RODUCTION
Th.e struggle for survival hetween spec'ies has been
a
topic for scientific investigation even prior to the
work of' Darwin (1859). Later, the idea developed that
no ·two species, as far as was then known, could coexist
if both possessed identical ecological requirements.
Thereforellf slight differences in the requirements of one
suecies:,, w·hich might escape notice at first, would tip
the scales of survival in its favor, thus ensurir.g its
survival and the other species' extinction.
Later,
mathematical equai.ions were cieveloped by Lotka (1925)
and Vol terra ( 1926) which descr.i bed relationships bf:tween
two spec:ies growing on the same food source.
'l'hese
equations: predicted that eventually only one species
would exist in the community if two or more species
.possessed identical ecological requirements.
To s·ee if the mathematical models of Lotka and
Volterra: actually predicted a real
ecolog-~.cal
system
). Gause (l9Jli·) set up an experiment wherein Paramecium
aurelia an(l Paramecium caudatum fed upon bacteria in
:. a closed' system.,
~audatum\
then
th~
P. aurelia survived, whereas
E·
died out since its rate of increase was lower
other protozoa.
Since this first experiment
others ha\re supported Gause's findings, namely, Park
2
r-Ha:~ s:.ar:·-·( 197 ;-;,-Gil p~~·-::~-:;-:~-;~-c ~-~~9-;;·;-:-·-;~·~~:···:::~·-·--·i
Sutherland (1976), Culver (1976), and Yesner'(l977), etc.~
in what has come to be known as Gause's principle or the
competitive exclusion principle.
In modern terminology.
this prir.ciple states. emphatically that two noninterbreeding populations cannot occupy the same ecological
niche in the same community, hence there will be the
eventual disappearence of one of the species,
Following this line of thinking it was then proposed
.that maybe different species which were competing for
the same substrate could coexist if a predator were
p~esent
to prey upon the more successful competitor,
according to Paine, (1966, 1971).
In this study-a microbial steady-state system was
maintained by a chemostat.
The microbe's nutritional
requirements are more defined then higher organisms and
therefore easier to manipulate.
The chemostat maintains
steady-state growth conditions which are easier to define.
and analyze according to, Jannasch (1965), Herbert, et
al, (1956), and Pirt (1975).
Starting with the foundation laid by Monod (1950)
and Novick and Szilard (1950), on the prediction of
th~
steady-state parameters of the chemostat others have
refined them, such·as, Herbert, et_ al, (1956) and built
upon this foundation new models on the role of a predate:·
in maintining snecies diversity rather then competitive
3
;-_,._..____
/
.. _.,_,.___
.c--~-·~~~~~-----·~--------~--~ --·-----·~--~~.~~-~·•~.·.~-~ -~--~,
I exclusion
••~-~·"-·~~-;--
of a species. as stated by Yang
l
! Villarreal,
·~1 arreal,
V1..!.
~
al, (1975): Comins
Lt
l
!!_,
&
.. _. ..
~~€~-·:--••··--~-·-·•~•
.. ,.,,
~-~--~.--......--~
..
I
&
\'l'eston (1969);!
Hassell (1976):
' 1 9 77;.
\
\-'-
Testing these models to demostrate that a predator
can partially account for species diversity were carried
out in the chemos tat under
steady-sts.~~;;e
Tsuchiya, et al, (1972); Jest, et
al~
conditions by
(19?3); Drake
&
Tsuchiya (1976): Bader, et al, (1976); Dent (1976);
Boncmi, et al, ( 1976) ; etc.
All of these ·researchers
either attempted to correlate their results to the model
proposed by Lotka (1925) and Volterra (1926) or Monod
(1950) or else derived new equations or models in attempts
to fit their results.
f
i
.I
t
The object of this research was to find out if two
competing microbes that would normally result in the
extinction of one and the incr~ase of the othe-r could be
maintained together by a predator within the same
chemcstat. None of the previous papers have satisfactorily
demonstrated this role of a predator in maintaining
'species diversity, and thus offering an alternative to
competitive exclusion.
'
--·-·--------'-·"··-··.-.-~---.. --'-;;---------.,.------ ..._J
)
t£ETHODS
AND
IViATERIALS
ORGANISI•~S:
The tiliated protozoan, Tetrahymena pyriformis, was
:purchased from Turtox, Inc., in a pure culture of 0.1 percent protoeose peptone (Difco).
The bacterium,
Pseudomona~
ltluorescens, was isolated from soil and identified as
I
icram-negative rod which produced a soluble fluorescent
:pigment, and would not grow at 42° C.
The bacterium,
]Escherichia coli, was isola ted on Ef.~B a.gar by its metallic
l
:green colony and further identified as a Gram-negative
\facultative anaerobe which produces gas from lactose
!broth, indicative of lactose fermentation.
'
)
The yeast,
!Saccharomyces cerevisiae, was isolated from Fleischmannfs
l"Active Dry Yeast", a commercially available baker's
1
i
....
;yeas~...
Serratia marcesens was obtained from the SUN
j
~iology
Department culture collection.
i
i
~1EDIA:
!
The medium, T2, used for the maintenance of T.
'
!
j
pyriformis was 1. 0 percent prot-oeose peptone {Difco).--
The media:, P, for early batch cultures was composed of
i
.0 gm yeast extract (Difco) arid 2.0 grn of glucose in
9oo
ml distilled water.
~nd
later batch cultures consisted of (per liter): glucose,
The medium, MM, for the chemostat
i
!
(NH 4 ) 2so 4 : 1.0 gm; MgS0 4 "7H 2 0, 0.05 gm;
trace element solution, 1 ml. Autoclaved separately from
~.10
)
or 0.05 gm:
4
5
)
and subsequently added to the cooled chemostat medium were
the phosphates
consis~ing
13.4 ml; o._s
KH 2 Po 4 ,6.6 ml.
N;
of (per liter):
0.5
~ ~a HF0
2
4,
As a precaution against
:contamination when thev.were added to the chemos"tat
v
!
.
imedium the phosphates were added through a 0.22 micron
'
imillipore filter.
!
The trace element solution mentioned above consisted
Znso 4 .?H 2 0, 0.44mg; CuC1?.2H 2 o,
;o.0135 mg; coso 4 .7H 2 o, 0.024 mg;_Mnso 4 .H 2 o, 0.0165 mg;
:of (-oer iiter):
O.P8 mg;
The medium used for viable counts in batch
'
}
aTJd
steady-state culture was Tryptic Soy Broth with Yeast
1
\extract and glucose in the concentrations (per liter):
•
I
:Tryptic Soy Broth without dextrose (Difco), 5·5 gm; Yeast
:extract {Difco), 1.0 gm; agar agar (Bacto), 15 gm;
:glucose, 2.0 gm.
The agar was added after the Tryptic
Broth and Yeast extract were dissolved.
,Soy
The glucose
'
'was
autoclaved separately in 100 ml. distilled water and
1
)then added aseptically to the sterile medium.
i
f The medium used for differential counts between E. col:
i
)
~nd
P. fluorescens the medium was eosin methylene blue
i
-
l
. L'"·~~~----~---~--·-------
·;
!
'
..
c
I.
Theory
The theory of continuous culture in a chemostat as
develoced by Nonod (1950), Novick and Szilard (1950),
:and Herbert, Elsworth, and Telling (1956) has resulted
:in several formulas to describe the kinetics of s~eady-
From these formulas which elucidate
state growth.
;the parameteres of' steady-state growth there are three
;critical constants.
First,? max , the maximum growth
rate ctt saturation levels of substrate.
Second, / .... is
~.,
a saturation constant numerically equal to the
;c:::ncE·:~tration
:~qua}
suc~trate
at which the specific growth rate,/.U., is
::a one-half ocf the maximum growth rate.
Tii.i.:cd, '!.,
~the yield ccr;stant which is the dry v;eieht of orr.'.i:-~isms
jform~d per gram of' substrate used.
These constar:ts can
;be determined in either batch or steady-state cu1t.ures.
·The dilution rate, D, in chemostat is described in
\
.'•h
"~ e formula.6 D= f/v, where f
is the flow rate of fresh
1rnedium i:!'":.to the culture volume, v.
~
•
oJ
•
~3pecl.fl.c
growth
rate,~,
:.log
.n :n {· s/Ks +
·
e 2/t.a. ·== ,.,..,~
-.
and s
l.S
s) •
In steb.dy-sta te the
is equal to D.
Also, D=
where td is the doublinc; +'
. . 1me,
the ste<:tdy-sta te substrate concentratior:.
Also,
·the StP.ady-state uonulation level, x, is ::: Y(sR - s)
;,. {s- -
g. ,
... D
where sR = substrate cone en,u
s (D/r-m
'
tration entering the culture vessel.
~-
rt
.1<:
=
)-
,,
r'
~
f
'
'
~
r
)
W'i.th these equations derived by Monod (1950) the
'
steady-state concentrations of microb2s and subtrate
in the culture vessel can ta predicted for any value
of the d'ilution rate and concentration of ir. coming
substrate. provided the values of the growth constants
m' K5
.,
and Y are known.
These equations describe
completely the behavior of a continuous culture running
under s:teady-s tate conditions.
II.
The Apparatus:
In. the type of chemostat used, (refer to Figure 1),
gravity .flow was counter-balanced by a capillary resistance, L,. tc assure constancy of medium flow.
of
)
This method
metering tiedium into the growth chamber allowed rates
of (J.O ml/min} f·cr long periods of time.
Medium, oxygen,.
and organism population was completely mixed by amagnetic
stirrer, rfi., and telfon co a ted iron stirring bar, SB.
! Hu1nidified
sterile air was provided by a pump, AP, whose
output af air (300.ml/hr) bubbled through water, AH, and
then through a sterile cotton plug, AF(CF), which in turn
~passed
through f'ritted glass air stone, A, into the
! medium.__ Constancy of' air flow was maintained by a
'
l
'hydrast:atic back pressure system (illustrated in Figure 1 ).~
/Temperature constancy was maintained at
jo 0 c
by a combined·
:heating and pumping unit, H, with a thermostat.
Also, the-
· temnerature of incoming medium. as well as, the temperature
)
'.....of ·the gro\rlh chamber was kept __cons·tant
by
-·-- ..
~
~-..-~·-........_..,...,......_~~~-~·-·-~~-·..:....---~----~--·~·------~-----·~-----~-·--· ·--~-
;,..._.~~-~----····
--···-~···-~
ciz:c~J_?:.:t!.Dg __ .... ~
·-~ ---···~ ~
7
.
.
--------------,-------
8
___________~--··""-~----~~--··~-··~---·~-'-'o-0 ~:....~.--.
,----..,..-·-···-----~-~---~·_.."
<••<
••
~.--~-- . .~·"• ,_.,A<-·>~--------'~--'~"·•-••
"'
·-···~
-- ~-
'
heated water through ihe jackets which surround the
chamber, C, and capillary flow resister, 1.
Volume of the growth chamber,.was approximately
2000 ml.
in which the medium volume was 1250 ml.
The
chemostat medium was pumped to an overflow reservoir, OR,
from a 1R liter pyrex carboy thus insuring a constant
hydrostatic head, HH, as well as, recycling the excess
medium back to the carboy.
Once in the overflow
reservoir, medium passed through a J.O meter coil of
capillary
~ubing,
L, which had an internal diameter of
0.5 millimeter thus creating the necessary capillary
resistance to the gravity feed system.
)
The capillary
' coil was connected to the medium inlet through a pyrex
side-arm dripper, i'JlD.
This dripper was designed so that
there was no direct connection between the incoming
medium and the actively growing culture in the chemostat's growth chamber.
Without this set-up it would be
possible for back contamination of the reservoir.
The
growth chamber had openings in its stainless steel lid,
L, for the dripper, air, A, thermometer, T, overflow, 0,
inoculation and sampling, IP.
The overflow was designed
so that the escaping air could only escape through it
while carrying excess medium.
The chemostat medium was autoclaved at 15 psi for
60 minutes without the phosphates to prevent formation
)
of. toxic hexosephosphates.
The phosphates were added to
- - ____l __
-
-·---
- -- ---
--
_.
- - ---
·---
-----:-;-
--- -
- -- -- - -- -
'
I
lthe cooled carboy through a Hydrosol Stainless
~illipore
I
~ilter with a 0.22 micron filter.
With all lines from
I
(the carboy shut off a vacuum system was applied to the
i'
:cotton filter.
This caused the phosphates to be drawn
I
!through the millipore filter into the carboy.
~acuum
This
system was also used to initiate siphoning between
'
ithe number one carboy and a new carboy attached in series
j
:to sustain the duration of runs.
i
Initially, it was
i
!believed that the medium nesded an antibioticfu order to
:maintain sterility, , especially when adding on a new cariboy.
:l
I
It was found not to be necessary and discontinued.
.1.ne flov1 rate
111'
.,
jcylinder under the overflow and measuring the volume of
escaping medium in a given time interval.
i
l.
-·
I
-~---------·
.•~~
---
-·--
10
f;
if
;·
. .--~-------~-- -- , _.,_,___ t···---·-··-----------·----..-· ··~.---..--------·----~·---·-------------- -~·-···------------···--·--···
t
'
11 L. --·~Tf-lQDc::
,.J.J_YTIC·11.
1
l'lir.... ·•• . v;
ANA
Population Analysis
Sterile population samples were obtained by wiping
the overflow line clean with a clean paper towel then
flaming it with
95~
ethanol.
Without this flaming pro-
cedure there were usually contaminants introduced into the
i
samples, esnecially when.low dilution rates were used in
I
plating.
I
I
The samples were diluted by serial dilution
technique and plated on various nutrient media.
'
The
population of the chemostat or batch cultures was determined by differential counts on plates of Tryptic Soy
B.coth Agar, Eii:B, and 150 micrograms per ml. of te·tra-
cycline in Tryptic Soy Broth Agar.
'rhe total population of Tetrahymena pyriformis, the
ciliated protozoa, were determined by visual counts of
samples ranging in volume from 0.01-1.0 ml. depending
upon the population density.
Samples were placed on
watch glasses then counted with a binocular dissecting
microscope at 20-80 power .
.)A Max
The maximum growth rate, ,M..Max
batch culture conditions.
,
was determined 'by
Pseudomonas fluorescens,
Escherichia coli, and Saccharomyces cerevisiae, were
grown in 250 ml. culture flasks containing 99 ml. (used
to facilitate serial dilution technique) of medium.
bacteria and yeast were grown in chemostat medium with
\
/
The
!
-t~~-..-..----·~--A·
--------·-------------------------~
--"- - - - - - - -
-
-
-\'
~'
11
r1·
• • -·-··
)
.
~·••~• ,_._..,._J~· -~~~-~~- ·f:-~~~-_,...,,_...,._- ..,.-••v•·~..._~ •. ,_ ··---.-~-------~-~- .... ._. ..._.._,. • - · - - - - - - .,..~------~--~·-'"'"~•• ~ -----~"'-•---~-....~--~.-.-- ~-~·---
o. 2% glvcose.
Al-l cultures were grown for a sui table
,
length of time in a water bath shaker set at 250 RH.r, witt
a temperature of JO 0 C to ensure that exponential grov?th
had begun.
These exponentially growing cultures were
then innoculated into new medium with identical environmental conditions.
All batch cultures were done in
triplicate simultaneously for each organism.
An average
was then obtained from three cultures for the r~
n Max·
I
I
.!I
)
~
'i
1
J
!~-~--·__.._ ________ ~--:-··-------·-----·-·---- -----------~
-1
12
)
'
;------------FIGURE' ·t-:·-·--·-DIAGRAl'f:. OF.CH:2fiiOSTAT
(
'
Chemostat as used in this study. Symbols
(sequence following the medium flow): R,
res·erv·ol.r \13 liters): IV:, magnetic stirrer;
S, stopcock valves; V, one-way valve; AT,
air trap with one-way valve~ P, pump~ OR, overflow reservoir. SP, sampling port; L, capillary resistance with jacketed water bath; H,
submersible heater;water pump; MD, medium
drippers; 0, overflow port; PH, pressure head;
T, thermometer; AF, air filter.; A, fritted
glass •ir stone: IP, innoculation port; MP,
millipore filter; CF, cotton filter; CM, hooded
ground glass couplings (male); CFe, female
hooded ground glass coupling; }LI{, hydrostatic
head;: C, growth chamber; SB, stirring har; L,
stain.less steel lid wi.th rubber 0-r.ing.
(
'
'
~-------~-. ~. .--,..-···--..---·. ~-- . -----·-~---~--...,.-·,. .·-··--·,_.._·-·-~-· ---·------..-.---------·---<-'"<!
'---
13
)
rc
f
I
I
1
.I
I
I
I
I
I
l
~
i
[
F.
f
r
[;
f
II
I
r
I:
I
i:
l
It
I'
I'
'.
' '
~ ~-. ~--·------"' --.
___
__...
_,__
_......_._
-------------··---
-----
1:
'------------+
-····---·-·-·
FIGURE 2:
Diagrams of special comuonents designed and used
in the chemostat lettered A through E. A- oneway
valve with ground glass seat and !'loat (ao:), .
direction of medium flow (b•); B- oneway valve with
air trap capped with a rubber cap (c') as used on
vials through which an hypodermic needle is inserted;
C- medium dripper in~erted through stainles~ steel
lid (i•); D- male/female ground glass couplings for
joining carboy bottles in series; E- overflow port
activated by medium height (h') and incoming air
pressure (e•) thus forcing the medium outside (g')
through capillary tubing (f').
(
·---------------------------------------------,~.-..-..~J
---·-----~
15
~._--·-~---
i
l
A
8
-
----r t..'''
c
~­
j
I
...... ··-· ..................................t . -----· --.. . . . ------------------·-----·----..----- .............---:~------------------------- ------·------------·-··:
FIGURE J: .~
Diagram of batch culture equipment used in
own laboratoryN This equipment was
designed and usad in place of a constant
temnerature shaker incubator. Temnerature
constancy was maintained at J0°C. ~ 0. 5°C.
by a 100 watt tungsten lamp within an insulated box. Aeration through air stone was
300 ml/minute by a aquarium pump.
author~
l
•------
i
·-·~---~..-~J
l
f
I
I
17
i
f.
F
!
}
!
'......,. __"'
-~-
·~~-·-~--..,-~-----­ -----~~~··---~
- - - - ----
~ ~------·-~----~-----~---.J
I~ITIAL
l~
CCLTURES:
b~TCH
labora~ory 1 equipment
tte author's rrivate
·~·~is
design€d .::.nd then built at CSUN.
-~•
l
'
worf..,~:!
-,
that the
sa +... ~s f
0
0
actor~
~red a tor
was
equipment, l''igure
J.y enougr.
' t o tes't' +h
'
'
,,. e nypo-r;,nes1s
0
L
would i.:'rey upon tho:; successful
!~rey
to an extent it could :.ot utilize c.ll of the available
nutrient therefore allowir.g a seco·:·:d orga:r:ism to co-
Initial batch
cultur~s
to determine
h•.r - 'l
glucose shewed it
tP
for
cerevisiae was n3 hr. -.L
3aceU!~~omyces
~e
1•
3
~..
J
-,.-;..,r-;reac.
.•..- ..... ...
.......
.
Jis expected_,
P. fluorescens outgrew S. cerevisiae J!l batch'cult:ure
when beth were intrcducec - . . . approxir.:a.tely equal numbers
into" the above rr.cdium at 30° C. After. inoculation dilutioi·
nlatings were made every thirty minutes thoughout expo-
nential growth of' the mixed culture.
1~
Next, batch cultures were run with the predator,
f
l Tetrah;y-:rr.ena oyriforrr.is, present with its prey, P. fluor'escens and the
l S.
i
u~successful
nonprey,
s.
cerevisiae.
cerevis iae can;:ot be prey for :.r,e 'I'. p-yriformis s ir.ce
it is too large to pass
Dilution platings were
throu~h t~e
do~·le
predator's gullet.
as described earlier with
•:._, the results
.. . .,_______ indicating
.
--- that
,___ ::i. cerovisiae•s no·pulatior.
·-•••'
·~~~'<oA---·-~--..,._~_,.,._
~-~--~
·~--··-~-.-- -----~--~~-
-·-~----
oo·.•~--<0 - - -~~~-·.:._ --~-~--~-!!---~--------~
·
-·-····--~--~·--··"·---------~ --~--~-·-··
i
'~-- -----------------;----------------------------~-------
'
I
---------------------- ------~--- ------------------- -------------·--------------------------,]
I~-."'as incre;ising ±"aster with the predator then without it.
l
l 'fhe
~
P. fluorescens population showed a slight drop when ·
' grown with the predator as cmr;pared to batch culture run£
;without it.
!n the short period(2-3 hours) of these
; batch cuiture runs there was no significant change in
~
)the population of the predator.
r'
''------------------------------·-----------------------------------------------~-----
lj
20
)
The yeast, S. csrevisiae could not
reac~
steady-stat~
in the chemostat under any conditions tested.
Neither
0.1 3m nor 0.05 gm glucose/liter concentrations of mini-
mal medium with dilution rates from 0.10 to 0.15 and a
temperature of 30° C enabled the yeast to avoiS) washout.
Attempts were made where yeast was inoculated
simultaneously into the chemostat with P. fluorescens
and T. Qyriformis or after
formis had achieved
f·
fluorescens and
s~eady-state
were tried where the inoculum of
conditions.
s.
!· pyriAttempts
cerevisiae was equal
to or much larger than the inoculum of P. fluorescens and
even second inoculations were made within the same run
)
after the first ingculum had washed out.
.tng.
. . h er concen +t-ra +.,J.on
•
~
c ....r g.1.ucose
cou ld not be used to
see if S. cerevisiae would survive since it would result
· a nopu~atJ.on
, · o r tne
· bacter1a,
· aoove
·
1 o8 celLS
. . 1m~· wnere
·
J.n
by-product inhibitior. could occur voiding the basic chemo1
stat assumption.
Increased temperatures could not be
used since 30° C. was optimum for the predator.
Dilution
~rates could not be adjusted any higher or lower since it
!
l
! was
found that the protozoa achieved steady-state with the
bacterium only within a narrow range.
The yeast was dropped as a competitor in favor of
Escherichia coli since it was impossible with this
i
.I
il
l
I
l
particular strain of yeast to achieve steady-state
)
_E~!~~-i.~-~~1:].~. __w tt..h ~.!! __~h~-n~~~-s .??:ry_ peirC:I.met e rs.L... -.-----..-------------- __j
21
~----,·-----~-----------·--------·--····-·--·-··-·~- .....- - · · - · · - - - - - - · - - - - · · - - · · - - · - - - · - -
I
•""-><'-'
.. - · . . . . . . . . . . . . . - · · · ·
s•rEADY-s'rATES cF BACTERIA:
Initially it was felt that the only way ·to maintain
sterility of medium in the chemostat was by using a
medium containing an antibiotic.
Also, since
E·
fl~--
escens is a cream col.ored colony on Tryptic Soy Agar and
rather hard to differentiate from other common contaminant~ i~
young (~4 hour) platings, Serratia marcescens
was chosen because of its production of the red pigment
prodigiosin which makes its colonies easily differentiated.
Both
dropped.
Df
these original parts of the experiment were
First, it was found that when contamination
took nlace ·in the chemostat the magnitude of contamir:ation
was either so large as to have within it sufficient
nurr.bers of microbe-s to insure a mutant present that was
resistent to the antibiotic, or the contaminating organisms were already resistent, i.e •• fungi or mutant
bacteria from the adjoining Microbiology class laborator- .
ies.
Second, S. marcescens after only 24 hours growth in
the chemostat at a temperature
= 30°
C., dilution rate
= 0.32, and "P" medium showed heavy wall growth which
does not result in steady-state growth.
Steady-state growth of P. fluorescens was
achieve~
at two different substrate concentrations - 0.1 gm and
0.05 gm glucose in minimal media as shown in Figure
The
/kn
)
~m
l.
4.
for 0.1 gm glucose/liter= 0.1) hr- 1 , whereas the
for 0.05 gm glucose/liter = 0.10 hr- 1 • The last
___ __j
22
)
runs in the chemostat were at this lower substrate level
,
:!
so that population levels would not be self-inhibitory.
:j
II
Steady-state growth of E. coli at the substrate
concentration of 0. 05 gm glucose resulted in a mu maximum'
= 0.14
.
hr.-~.
See Figure
)
)
'
'-------·----~-----~--·-------
5·
FIGURE
4:
Comparison oi' growth for _E. fluorescens at
glucose concentrations of 0.05 gm/biter and
0.1 gm/li t.er with temperarur:= = 30 C., 1 and
dilution rates = 0.10 hr-
and O.lJ hr- ,
respec·tively ..
.I
f
·-i -..
~ ...
..,__.
...
______________
-
i
---·-··-------.-1
10
9
,.-..
--
.. - - - £ - - . A - - J - A -
-~·
. \'
:~- ..
-·-·-·
• -•-•
0.05 gm/liter glucos~
•-•-•
0.,10 gm/liter glucosE:
.· ..
_.,.
6.
10
.. ~-:~
"J-
,,.
0
24
48
72
96
120
144
168
192
216
240
.(
TIME (hours)
;
~--
...
---~-i---··~--------~--·--------------_...--.-----·--··-----~---~-·-~--·~-~·~-----·---
...
:l
-.1
25
FIGURE
5:
Steady state growth of~· £2.ll a~ 1 o.os gm/liter
gluco::>e, dilgtion rate = 0.14 hr , and temperature = JO C.
·
l'--·---------·-
__________________ jl
26
:::;
10-'
:..
,::)
:X:
~
:g
a.
·:::>
.~
1J.
10'
..
-·~- ·
I
0
24
I
I.,l.ftj
i
120
I
I
J
144 168
192
216
240
Tltr!E (hours)
___________,_j
27
)
STEADY -.sTA'l'ZS GF .PROTOZOA:
An attempt to grow T. pyriformis in the ' chemostat
on 0.05 gm glucose at J0° C., and a dilution rate of
j
0.10
I1
.of
hr-~
Figure 6.
resulted in T. pvriformis washout as shown in
However, itwas possible to grow the protozoa
in the chemostat when prey was present, such as, P.
£luorescens (e.g. Figure.?), when the dilution rate was
1
0.15 hr -1 or less. When the dilution rate was 0.16 hror greater T. pyriformis could not maintain steady-state
even though the bacteria could.
)
)
·~---...,------··-·------1
(
FIGURE 6 a
T.
~yriformis
washout in
O.Of gmjl1ter glucose, JO
hr-
~he
chemostat at
C., and 0.10
dilution rate.
-.:"
!
L~.--------
-~
_j
)
a-:--o-o
T. pyriformis
a'\
a
0
0
8
12
16
20
24
~8
TIME (hours)
'
i..----------------
J2
30
Steady state growth of !· pyriformis and P.
fluorescens in the chemostat at 0.1 gm/ 1
liter glucose, dilution ra.te of O.lJ hr- ,
and JO
C..
·
-
. '),
"'--.-.---·-·-·-.----··---------------------------------'!
r
"
•
31
)
· A-A---4
-
= P.
.
·':
,__
------
--A
/~
fh~ore;:cens
...
.
A-A-- ---• -.&
..
I
I
I
•
~a-----ri----a
,..;
~~
/.
~
N
0
\,
~
;P:::
a/~/
~
ii-4
0
~
P:l
~
:s
~
1
10
')
.
'
.
,.~~~·--~·~--~·-----·--~·----~--~·----~·----·----~--2
0
-
4
··~·~-"'-------·-·---------~·
6
8
10
12
TIME (days)
14
16
18
·~....:...-~----~-- ~--···--------·-··---· --------~--~....... ..1
.
r-----~------~----------------~----------- --··--------------~------------------------------------------~--------·-·-----------·.
I CONPETITON BETwEEN
~1.1
At a dilution
BACTERIA IN THE cHEt-mSTAT:
~
rate of 0.14, temperature of 30
0
c.,
and glucose concentration of 0.05 g/liter E. coli achieve(
h
steady state at approximately lO:J cell/ml, as shovm in
Figure 5.
P. fluorescens 'tras then added to the culture
in approximately equal
rrJ.rilbErf~
"{2
hours later.
Vlithin
24 hours P. fluorescens 1 population concentration increased from 4.5 X 10
4
to
6.6 X 107 cell/ml and achieved
steady state whereas E • .£.Ql.ils population increased from
1.1 X 105 to 2.5 X 105 cell/ml.
\t.fithin the next 24 hours
the puJnping apparatus i>'hich adds fresh medium from the
reservoir stopped.
I cannot bo certain for how long the
pur:-:p i.'ras stopped, but estimate the length of time the
. !
gro-v;th chamber of the cbemostat \-las not at steady state
to be 1-2 hours before the pump was repaired and steady
state conditions again initiated.
days the
pu~p
On three subsequent
again failed and steady state conditons had
tobe reinitiated.
By determining the amount of overflow
collected bet\':een sampling it was found the do·vm time of
the pump averaged·45 minutes.
The last time the pump was repaired it did not breakdown again but vJ"ith the shortness of tL"Tle the chemostat
could not be run until E. coli either r:Jas vmshed out, .
as predicted by batch culture/·max
;U
, or achieved steady
state vlith P. fluorescens.
;
- - - - - - - ______________________________,J'
33
FIGURE 8:
Eventual· steady state growth of T. pyriformis,
P. fluorescens, and E. coli after intermittent
non-steady state growth-;-T:'"e., the pumping
of medium stopped four times for short periods
between days 7 to 11.
i
.
...
-~..__....__...,.. ....... _.i
·'·
~
'j.
~
c---c---a ~ T. pyriformis
o-·-o-tJ =E. coli
A--A- --.i
\
r-i
~
.
= P.
fluorescens
0
/\
Q
.:_
0
2
c3to
N
\
'~
0
E-l
0
0::
0..
li.
0
Ul
g51o1
P=l
:E
:;::::,
z
~·:·~~
.... ~''t:
2
J
~
5 6 7
TIME (days)
1.0
r
..
_;
-·"·---..
~.·-~~-----.-~-·--·----~··-~__,.,.- -----~-6----~----~-~~~--~·----·---·~------·--~-~--.
STEADY-STATES - BOTH BACTERIA AND PROTOZOA:
.,
Steady-state was easily achieved in mixed cultures
' of the pr.otozoa and the faster growing bacteria,
f·
Fluorescens, hereafter referred to as the more abundant
more successful utilizer of the substrate.
See Figure 7•
Steady-state was achieved in the chemostat where
the more successful and less successful bacteria were
competing for the limiting substrate of glucose when
the predator was present feeding upon both prey.
This
is shown in Figure 8, where the conditions for steadystate growth were:
= 0.05
gm/liter, temperature
= 30°C., an~ dilution rate= 0.14 hr- 1 • Population
levels were:
E. coli=
glucose
P. fluorescens
= 1.5
X 107 cell/ml.,
5.4 X 105 cell/ml., and!· uvriformis =
5 X 10° cell/ml.
i
~
.
----~-~--·--~---~-.)
-·<•~-----.c·~-----~~--
... __ ._. ·- "'"' •-~·-•• • -·-· -'~··--·-• ••
-<w••
'--~-·•-· -·"-•~.............-~--~---~~·---~- ,_,...._,..,..__.--.-·~1
DISCUSSION
Species diversity within a
co~~unity
as a result of
a. predator has been postulated,· theorized, rna thematically
modelled, and seemingly observed and recorded by several
researchers.
Seemingly observed and recorded, since it is
imnossible to concisely identify in the natural environment any particular organism's ecological niche.
With
this in mind it is no wonder that this area of Ecology
is attracting more research with the concomitant flood
l
:of new pubLished papers.
Unfortunately, either the papers
are from what the author deems the "Aristotelian" scientists who 1ike to think up new models but never seem to
have any interest in attempting to see if they fit the
real world, or from the "Field" scientists who try to
fit thesH models to the real world.
Occasionally, someone·
attempts an experiment, such as, Tsuchiya et al, (1972),
Jest~
al, (1973), Drake & Tsuchiya (1976), Dent et al,
(1976),. Levin et al, (1977), and Chao et al, (1977) which
may begin to unravel some of this food web.
My goal was to utilize the simplest possible organ- .
isms, i.e-. microbes, and in a well definable environme.nt,
t
i~e.,
the chemostat at steady-state conditions, in an at-
,.
;tempt to discover whether or not a. predator, T. pyriformis,
1
is
respo~sible
for the coexistenc3 of two bacteria,
f·
.fluo~escens and E. coli, which otherwise could not coexist.:
Previous work has been done which demonstrated that
37
an ameboid predator and its bacterial prey could coexist
in a chem.ostat at steady-state according to Tsuchiya et
al, (1972} and Dent et al, (1976).
This coexistence was
also demonstrated for a ciliated protozoa, Tetrahymena,
according to Jost et al, (197J).
in this research paper that
It has been demonstrated
.T.· pyriformis will achieve
steady-state conditions with its prey, P. fluorescens,
at JO 0 C., a dilution rate of 0.15 hr -1 or less, and an
inflowing glucose concentrat.ion of 0.05 gm/liter (Figure
?).
Going a step further than some of the above papers
it was demanstra:ted that T. 2yriformis could not maintain i taelf in ·the chemos tat with glucose and mineral
salts as shown in Figure 6.
It was also demonstrated
·-
1
that if the dilution rate exceeded 0.16 hr-- that even
in the presence of abundant prey could not prevent T.
oyrifo!'mis from being washed-out of the
chemostat~
I was
unable, because of the lack of time, to demonstrate that
the T. _pyri.formis could achieve steady-state conditions
with the other prey used in this study,
l it
~·
coli.
However,
had been demonstrated previously that E. coli will
?
l 'lchieve
s·teady-state growth in the chemos tat with the
' amebae._ Dictyos.t:elium discoideum by Tsuchiya et al, ( 1972)
and Dent et al ,, (1976), or with T. pyriformis by Jost
:' -"""t al '
--
( 1- 9 ....( -:lj }
•
i
-
It has been demonstrated by this research that either
of the bacteria alone in pure culture can achieve steady-
l
.·s--ta·te growth-within' 'the--necessary parameters necessary
for the predator's survival in the chemostat (Figure 4
and 5) as has been demonstrated numerous times by earlier
:workP.rs.
An attempt was made to demonstrate that P. fluor'
\escens would displace E. coli from a mixed culture in
l
ithe chemostat which I do not believe was conclusive.
!·Ihe displac-ement of one bacterium by another was supposed!ly demonstrated by Jost et al, (1973) when E. coli was
!introduced into a steady-state culture of Azotobacter
withi~
:vinelandii.· They found that
!
:coli had been
i~troduced
that the A. -----vinelandii ,_nonulation
...
8
,·wer:.t from slightly under 1 o
•C
J days after the E.
cell/ml down t.o 1 o5 cell/ml.
They never actually reached
washout~and
graph could be interpreted
as an approach to steady-
istate.
None the less, they assumed
in fact their
that~·
vin~landii
\
;would washout of the chemostat.
~y
experience with eon-
itaminants in the chemostat as early as 1971-1972 has
:demonstrated that mixed cultures can last much longer
'
l
ithan this.
Thus, I feel the question remains, was A.
~inelandii
displaced or could it have survived at some
j
!lower population level?
According to Figure P when P.
1
'fluorescens, was introduced into a steady-state populatior
. f'
'0-
~
t, •
, .
£.2.:!:1:.
+'
p
vn,e -=-
0
fluorescens' population increased
:slightly over one thousand fold.
1
'
'"'XDP'•+...,~lv
;_,. . ) ...,.c v '-".\...~ .....
_'.; _______
~_-,,_...,-~,.._..,
R
.::. •
coli
---
showed a
Concurrently, and un-
~0-fold
__.
increase over its
_____ _...,. _______.____________
'
'
,~,~--~·~-----~------··--·u;
39
E. coli population the pumping mechanism became erratic
resulting in steady-state growth
interrupt~d
by short
periods of batch culture conditions (approximately 1-2
hours per 24 ·!;lours). ilii th little time and the prospect
'
of once ag~~ aborting a run (which with this chemostat
;,
is more often than not) the predator was introduced into
\
r/
the culture.
Whether or not P. fluorescens, stimulated the growth
of E. coli, is not conclusively proven or disproven.
This would be the next logical experiment to attempt.
Also, would the two bacterial prey species have reached
-··
steady-state growth as a mixed culture without the
presence of the predator?
This would also be answered
in follmv-up experiments •. Recent papers by Pirt (1975),
and Aris and Humphrey {1977) state that when-two organisms compete for the same substrate without preying on
each other, that it is possible for
to occur.
st~ady-state
growth
If the growth curves (plotted as suecific
growth rate vs. substrate concentration) «crossover" at
the coordinates of nutrient feed concentration and
dilution rate, (Z,O), then the two organisms could be
maintained in the chemostat.
Aris and Humphrey (1.977)
'present qualitative phase portraits drawn for Jl distinct
tY,pes of situations.
i
---··---~-tiA{
40
Steady-state· growth has been demonstrated uride.r
conditions of continuous cultivation in a mixed culture
of the ppedator, T. pyriformis; the more numerous prey,
P. fluorescens; a.nd the less numerour prey, E.· coli.
Whether of not oscillations or dampened oscillations
would have occurred cannot 'be determined by results as
shown in
Fi~Jre
Jost et al, (1973) managed to main-
8.
tain steady-state growth of a mixed culture of a predator and two prey· for eight days until an explosive
onset of wall growth terminated the experiment.
It was
shown in theory by Yang and Weston (1969) that the level·
of resource concentration rose as the consumer
populatio~
was reduced by the predatcir and that an additional consumer could coexist with the original consumer
by~using
the resources released by predation.
Attempts to use
s. cr::revisiae as a non-prey compe.t- ·
itor of the bacteria, P. fluorescens, were completely
unsuccessful.
s.
cerevisiae could not be maintained
in pure culture in the chemostat at dilution rates and
glucose concentra};ions of one-half that reported in the
literature by Jenkins (1963).
Possibly, the yeast
needed more then glucose and mineral salts to achieve
steady-state within the parameters of this
experiment~
It would be interesting to discover what it does need.
so
th~t
th~
relationship.betwe~n
a successful prey,
unsuccessful non-prey ancl a predator could be
.
studied~
j
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I had hoped by studying a.food web, consisting of
a substrate fed upon by two bacterial species which were
in turn fed upon by a predator, to further substantiate
the idea that two competing species will not necessarily
demonstrate the principle of competitive exclusion, but
may coexist.
Interestingly, most recent models of the
coexistence of two
speci~s
in a chemostat, as cited in
this discussion, seem to imply that species diversity,
at least in the chemostat, does not
dation.
d~pend
upon pre-
Perhaps, whether or not this is the case could
be demonstrated by using
~·
coli and P. fluorescens
since they may cdexist without a predator in the chemostat.
Once again, as I have always taught my students in
junior high school, in science when you have answered
one question you have only opened one door to find
several others closed behind it.
-i·;ire of teaching science.
This is why I never
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