a computer-controlled turbidostat for the culture of planktonic algae

A Computer-Control ed Turbidostat for the Cu Planktonic A

institute of Ocean Sciences, P.O. Box 6000, Sidney, B.C. V$L 4/32!

Scripps institution of Oceanography, La lolls, CA, 92093, USA., and iet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 9 1 109, USA

and K. B. Denman institute of Ocean Sciences, P.O. Box 6000, Sidney, B.C. V8L 482

Hilt, S. H., M. IS. Abbott, and K. L. Denman. 1985. A computer-controlled turbidostat for the culture of planktonic algae. Can. I. Fish. Aquat. Sci. 42: 744-753.

We designed, built, and tested a microprocessor-controlled turbidostat that maintains phytoplankton cells at nearly constant biomass concentrations without nutrient limitation of growth rates. There are fundamental limits placed on the achievable degree of constancy by physiological changes in the organisms being cultured. The sensor responds nearly linearly over a wide range of cell densities, extending well into high pigment concentrations (approx. 20 rng ChB aim3). The microprocessor-based controller eiiminates the need for time-consuming and delicate set-up and modification of operational variables and provides for simple implementation of virtually any logical feedback algorithm to control cell concentration. AB- though the result was not unexpected, we have shown that growth rates measured in the turbidostat are equivalent to corresponding measurements in a batch culture growing under identical conditions, until such time as the populations develop differing physiological and physical properties due to their different environments.

Nous avons consu, construit et teste un turbidostat commalade par microprscesseesr, qui maintient les cellules phytoplanctoniques a des concentrations de biomasse presque constantes sans quTi y ait limitation des taux de crsissance par les substances nutritives. be degre de constance qui peut &re atteint est limit6 fondamentalemerit par les changements physiologiques qui se produisent dims les organismes que Ikon cultive. Le detecteur repond presque de facon lin6aire pour une vaste garnrne de densites cellulaires, s'appliquarat bien de fortes concentrations en pigment (environ 20rng/rn3 Chl a). Le dispositif de commande par microprocesserrr elimine la necessite d'une rniseau point longue et delicate et dkne modi%ication des variables op6rationnelles et il permet dktiliser pratiquement tout algorithme logique de retroaction poker contrbler la concentration des cellules. be resultat ktait prkvisible, mais nous avons dernontre que les taux de croissance mesurks dans le turbidostat sont equivalents aux mesures correspondantes enregistrees daws une culture par lots se developpant dans des conditions identiques, jusqu-au moment oO les populations acquierent des propriet4s physiques et physiologiques distinctes en raisgsw de Beur habitat diff6rent.

Received June 29, 1984 Accepted January 1985 (J7848)

he continental shelf region off southwest Vancouver Islmd supports Iage areas sf persistently high phytoplankton biomass and primary productivity (Mackas et al. 1980; Dewman et al. I98 I), which result mainly from

topographically controlled upwelling (Freeland and Denman 1982). Because these regions had such high nutrient levels (typically 5-10 mmol N/m3 in the euphotic zone throughout the summer) and phytoplankton biomass (values greater than 15 wag Chl alm3 were common) (Hi11 et al. 19825s, 1982b, 1983), the growth rate of phytoplankton was not nutrient limited, and the

resent address: Department of Oceanography, University of British Columbia, Vancouver, B.C. V6T lW5.

major factor limiting potential productivity was probably Bight availability. Thus, we initiated laboratory studies to determine the effects s f differing Jight eonditisns on the maximum growth rate s f local phytoplankton species. In p a r t i c u 1 ~ ~ we were interested in the effects of fluctuating light simi%x to those experienced by phytoplankton being moved up and down through the euphotic zone by various water motions (Denman and Gargett 1983).

We needed a system that would allow us to culture algae continuousHy at or near the maximum growth rate of the population, and that would provide us with values for the growth rate with a minimum of operator intervention. Since we intended to study the response of algal populations to changing

744 Can. /. Fish. Ayuat. Sci. , Vol. 42, 6985

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

conditions, the system also had to allow us to measure growth rates under non-steady-state conditions (i.e. under programmable light fluctuations).

The chemostat is a relatively simple and widely used continuous culture apparatus, but it cannot be used reliably to grow algae at or near their maximum specific growth rate and, more importantly in this case, it is not useful in the study of transient effects, because a chemostat, by definition, requires the achievement of a steady-state relationship between growth and dilution for meaningful information to be obtained (Munson 19364; Watson 1932%. Batch culture techniques are simply too labor-intensive to consider. A turbidostat, on the other hand, maintains microorganisms at a nearly constant concentration by the controlled replacement of culture with fresh medium, and the rate of replacement is directly related to the specific growth rate under all conditions. Thus, the response of growth or production rate to changing external conditions is, in principle, easily determined. Because commercially available turbidostats were expensive and the choice was severely limited, we designed and built our own system. This paper describes the results of that process.

System Design for Turbidostat All turbidostats consist of three basic elements: a device to

measure cell concentration (sensor); an arrangement of vessels, piping, pumps, valves, etc., to hold, mix, and transfer the culture and the diluting medium (plumbing); and a mechanism to control the concentration of the culture, usually through the controlled replacement of culture with new growth medium (controller). In this section we describe these elements as implemented in our turbidostat.

Sensor The sensor is the central and most critical element in the

turbidostat. Although there are other ways to sense cell concentration, we chose to design an optical sensor. The measurement of cell concentration can be made within the culture vessel itself, or culture can be circulated from the culture vessel through an external sensor. The first choice has the advantage that a circulating pump system with its attendant piping and wiring is not required, and that the algae are not disturbed by pumping. This type of sensor arrangement does, however, have several serious disadvantages: (1) slight move- ment of the culture vessel relative to the measurement system may completely change the optical parameters of the system; (2) algae growth on the wdls of the culture vessel can unduly affect the optical signal received by the sensor; and (3) some means of compensating for changing ambient light is necessary.

To avoid these problems, we decided to design an external sensor. There are two basic optical methods for measuring cell density: measure the light scattered from the cells or measure the light transmitted through a fixed thickness of culture. The latter method is implemented in the beam transmissometer, a well- known optical instrument where the light intensity measured at the sensor (I,) is related to the source intensity (Io) through the following relation:

I, - I. exp (- cL)

where L is the path length between source and sensor and c is the beam attenuation coefficient. This coefficient includes attenuation due to absorption of light by water and by particles in the beam path, as well as attenuation due to scattering of light out of the beam.

As a turbidostat sensor, the beam transmissometer has two deficiencies. First, in order to minimize errors at high cell concentrations, the optical system must be designed so that the sensor detects mainly transmitted light and only a small amount of forward scattered fight (it is not possible to eliminate forward scattered light from the beam). Second, the sensitivity of such a device increases with the optical path length (until the fraction of light transmitted = Ile) so that, in order to achieve satisfactory sensitivity, a physically large sensor may be required. However, this conflicts with other design criteria: that the sensor should be as small as possible to minimize the time that cells are sppt of the culture vessel and that the surface area of the sensor should also be kept small to minimize growth on the sensor walls.

Using s ca t t ed light to measure the cell concentration results in a simpler optical arrangement because scattered light is now detected rather than excluded. Consider the intensity of light scattered from a small volume of suspension a d measured at some fixed angle to the optical axis. The intensity of the scattered beam is itself attenuated with distance L from the scattering volume, as shown by the following expression for measured light intensity I, of the scattered beam related to the source intensity Io:

I,,, = iob(N) exp (- c(N)L) , where c(N) - c, + CPN is the attenuation coefficient for the scattered beam and b(N) = b, + BPN is a linear approximation for the initial scattering at some given angle; N is the cell concentration, P is the geometric cross-section of the cells, C is the fraction of light lost through scattering and absorption by a unit geometric cross-sectional m a of the cells, B is the fraction of incident light scattered at the appropriate angle by a unit geometric cross-sectional area of the cells, md L is the distance between the detector and the scattering volume. The constants b, and c , are the coefficients for the initial scattering and subsequent attenuation of the scattered beam for pure water.

We obtain an expression for the sensitivity of the intensity at the detector to cell concentration by differentiating and substi- tuting for b(N) and c (N) in the above relatatim:

dI,,,lcEN = IoP(B - CLb(N)) exp (- c(N)L).

Keeping L small maximizes dI,l&. Note that

lim L-0

and thus, I,,, increases linearly with N, provided L is kept small (i.e. L < llc(N) and L 9< BI{@~(N))). In a transmitted light sensor, I, decreases nonlinearly with N. Note also that in the absence of scattering particles (i.e when N = 0), there will be a small amount of scattered light caused by scattering from pure water, and then as N increases, I, will increase nearly linearly. I, will always be much smaller than Io, and therefore, what has to be measured is a large fluctuation in a low-level signal, which is easier to do accurately than to detect a small fluctuation in a high-level signal as in a transmitted light sensor.

Because of these considerations, we chose to design a sensor employing scattered light detection, and because scattering by particles is strongest in the forward direction, we designed the sensor to detect forward scattered light, but not transmitted light. The physical design of the sensor is shown in Fig. 1. A conical sensor interior minimizes both volume (36.3 rnL versus 100.5 mL for a cylinder of the same maximum diameter) and surface area (9 1.1 cm2 versus 125.7 cm2 for the cylinder). We

Can. J . Fish. Aquat. Sci., Vok. 42, I985

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

OUTLET

WINDOW

Fig. 1. Vertical cross-section of the optical sensor.

chose a very bright light-emitting diode (LED) which emits red volume A& divided by the area dA and multiplied by the light (635-nm peak wavelength with a halfwidth of 50 wm) as forward scattering coefficient B defined earlier. Thus: our light source. Light levels within the sensor are very low compared with light levels in the culture flask during lighted periods, and the wavelength of the LED is near the edge of the action spectrum for most phytoplankters. In addition, at typical pumping rates used in our experiments, cells average less than 2% of their time inside the sensor. Thus, we expect that the effect sf sensor light on photosynthesis will be negligible, even during dark periods.

A precision current source provides power to drive the LED, so that the f o m a d voltage of the LED reflects changes in the power output of the device and provides a reference signal for the controller. This circuitry is not temperature compensated, since temperature is a controlled variable in the turbidostat. The LED is designed to concentrate most of the light flux into a cone with half-angle 12" around the optical axis. The detector, a PIN-type photodiode operated in the photovoltaic mode, provides a signal that is preamplified by a high-input impedance amplifier and then further amplified and low-pass filtered prior to measurement by the controller. The geometry of the sowceldetector arrangement and the optical stop ensure that the

B,, = (NPBdxd4)ldA = NPBdx

Of course, the scattering coefficient is not going to depend solely on the geometric crass-section sf the particles. Other factors such as refractive index and geometrical shape are also important (e.g. Van de Hulst 1957; Deimendjian 1969). However, for the present purposes we will ignore these complications and let later experimental tests reveal whether this approximation is justified.

We can now obtain the intensity of light scattered h m the small volume, dl,,:

where I ( % ) is the intensity at x. However, I ( x ) can be related to Ho through the attenuation coefficient as outlined above, and we can write:

dl,, = NPBBo exp ( - c(N)x)dx.

The scattered light itself wi13 be reduced by attenuance before reaching the sensor, so that

side walls of the sensor and the source itself are not "seen" by the dl,(x, dr) = NPBIo exp (- c(N)x) exp (- r(N)(L - . x ) ) d r , active area sf the photodiode, other than through diffraction. Thus, virtually the only light detected by the photodiode is the where d m ( x , &) is the light detected at the sensor due to halo of light surrounding the source caused by scattering in the scattering in the small volume &A at a distance x from the forward direction by pure water and by cellis in the culture sousee. Finally, integrating from x = to x = L gives medium. The contribution due to scattering by pure water is minor, except in cultures where the cell concentration is very 8, - HoNBBL exp (- c(N)&).

low (Jerlov i 976). Later we will show results of calibrations of the sensor with If we consider only single scattering and neglect scattering by different types and sizes of particles, and analyse the validity of

pure water, we can develop a simple theory to describe the this model. operation of this sensor. The probability P,, of light being scattered in the "fonuard" direction (because of the geometry of Plumbing the sensor, this includes angles between 2 and 15" from the optical axis) by particles in the small increment &at a distance x The turbidostat plumbing is shown schematically in Fig. 2. from the source is given approximately by the ratio of the sum of Culture vessels are 3-L boiling flasks, which sit in a tempera- the geometrical cross-sections of particles within the small turc-controlled water bath. Cultures are stirred using a Teflon-

746 Can. J. Fish. Aquar. Sci., Vol. 42, 1985

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

WAKER BATH QVERFLQb.8

FIG. 2. Schematic arrangement of the turbidostat.

coated 7.5-cm magnetic stir b a rotating at 68 rpm. All piping and valves are of Teflon. The pump circulating culture through the external sensor is a piston-type fluid metering pump operating at a stroke rate of 1725lmin with flow rate adjustable from 0 to 1008 mumin. We typically use a flow rate of 300d lmin , so that the sensor volume is replaced approximately $ times per minute, and Wow speed through the piping is about 25 c d s . The pumps removing culture and replacing medium me also fluid metering pumps, with the culture removal rate set to be slightly greater than the medium replacement rate. Thus, the amount of culture in the flask, usually set to 2 L, is determined by the level of the outlet pipe in the flask.

Controller

Most turbidostats described in the literature (e-g. Maddux md Jones 1944; Sorgeloos et al. 1976) have used electrsme- chanical control systems. A typical system would use the elec- trical imbalance between sensor and reference voltages, re- flecting an increase in cell concentration, to turn on a relay that activates pumps or opens valves to remove culture and add medium. These systems often have problems with "chattering" of the relay when cell concentration is near the set point or, if a time-delay relay is used, sverdilution of the culture. In addition, they are difficult to set up, and changing variables such as set point or pumping time is not easy. An attractive alternative is control by microprocessor and associated hardware and software. Although the initial investment of time and money is probably higher, once the system is set up its characteristics are determined completely by the initial values of system variables (e.g . pump time, set point) entered by the operator at the start of a run. In addition, the logical algorithm describing feedback between measured cell concentration and pumping rate is

infinitely variable, since it is determined by software. Recently, Sinnett and Davis (1983) have described a programmable turbidostat based on a microprocessor, which is used in pollu- tion studies to control the concentration of suspended sediment in a seawater tank in which mussels were grown. They have used it to reproduce the variation in suspended sediment concentration found over a tidal cycle, demonstrating the value of programmability.

We used an Intel SDK-$5, a single board microcomputer development system based on the widely used $885 $-bit microprocessor. This board contains memory, central processing unit (CPU), a simple keyboard and display, and most of the support devices needed for the application. The addition of a few chips for analog to digital conversion, multiplexing, decoding, intempt generation, and permanent program storage is all that is required to built a sophisticated turbidostat controller. A block diagram of our system is shown in Fig. 3. The components sf the controller are as follows:

ABCIMUX - the reference and sensor voltages pass selectively through the multiplexer (MUX) to the analog to digital converter (ADC), which on command from the CPU measures the voltage and provides an $-$it number corresponding to the voltage to the CPU.

INTERRUPT CIRCUITRY - this circuitry intempts the CPU when a measurement cycle is due, and is reset when the CPU responds to the intempt.

ADDWSS DECODE - provides enabling signals to the ADC and the MUX, and a reset signal to the intempt circuitry.

I10 - this is a single line, controlled by the CBU, which is used to turn a relay on or off.

CHART RECORDER -this dual trace chart recorder is used to keep a running record of the analog sensor and reference

Can. 9. Fish. Ayuat. Sci., Vol. 42, 1985 747

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

CHART RECORDER I ,, -,,.,,-,, 4

SIG REF'

FIG. 3. Block diagram of controller electronics.

voltages. The event marker provides a record of the time and number of pump cycles throughout a mn.

At the beginning of a mn, the operator is pmmpted to enter values far several variables, as follows: (a) "BIN" for boxcar averaging; (b) the length s f time between intempts (typically 1 s); (c) the time the pump will be on for each pump cycle; (d) the time to wait for complete mixing after a pump cycle; and (e) the level of cell concentration to maintain.

The conmllling program is intempt-driven, which means that the CPU spends most of its time waiting for an intempt signal. On receipt of an intempt signal, the CPU executes a sequence of operations and then waits for the next intempt. This sequence is shown in Fig. 4 in the f o m of a flow chart. Because of the high operating speed of microprocessors, the CPU spends almost all of its time doing nothing, even at a sampling rate of l hz. Thus, many turbidostats could be controlled by one CPU with little additional electronic hardware, and each turbidostat could have its own control algorithm and set points. To avoid pump cycles triggered by spurious high levels of

raw sensor signal, we "'b~xcar'~ averaged both sensor and reference signals. This entails keeping a list in memory of the W/B most recent measurements of sensor and reference voltage, and computing the averages of these M readings, which are then used in the subsequent determination of cell concentration. The mixing time after a pump cycle is required to avoid overdilution of the culture, since it takes some time for the complete mixing of the diluted culture and for the fully mixed culture to appear at the sensor. Having presettable values of pumping time and mixing time allows the operator to tune the response sf the turbidsstat to the characteristics of the cell culture under examination.

Tests and Results with Turbid~stat

We calibrated the sensor by measuring its output as a function of the concentration of several different particles: Lycopode'eem pollen, the living diatom Tha&assiosira profwmda, silicon carbide grit (106B8 x ) , and Micrslite 10 1 microspheres (Pierce and Stevens Chemical Corp., New York). Particles were dispersed in particle-free aqueous media: filtered artificial seawater for the diatom and HSOTON II diluent (Coulter Electronics) for the nonliving particles. The Lycopodium pollen and the T. profun& were of a nearly uniform size, but the silicon carbide grit and the microspheres were mot; in these cases we reduced the size range considerably by a combination of filtering, settling-out , and centrifugation. Particle size spectra were measured using a model ZB Coulter Counter in conjuwc- tion with a Norland Inotech model 5300 pulse height analyzer; particle concentration was measured using the Coulter Counter. We show size spectra of the particles used in the calibration tests in Fig. 5 and the results of the calibration mns in Fig. 6. The maximum csncentration of T. prsfunda in these calibrations corresponds to a pigment concentration of about 16 mg Chl a h 3 . The curves in Fig. 6 result from a least-squares fit to the data of the model derived above for the optical response of the sensor:

6, -- K + IoNPBL exp ( - c (N)L)

where K is an offset value added to the model to account for differences in the optical properties of the aqueous media used. The results of the fitting process are given in Table 1, where mean cross-sectional m a s were derived from the measured particle size spectra. The statistical validity of the fit was tested using the method outlined in Bevington (1969), by which the significance of adding an additional tern to an existing fitting

748 Can. /. Fish. A q u a . Scb., Vol. 42, I985

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

COMPUTE CELL

FIG. 4 . Flow chart showing software algorithm for control sf cell concentration.

FIG. 5 . Komdized size spectra sf particles used in tests of the optical sensor.

equation is measured using the chi-square statistic. In this case we compare the 3-tern model with a simple Z t e m linear re- gression to the data. In all cases except Lycspsdiurn pollen the %ern fit is better than the 2-tern fit at the 95% confidence level.

Using the mean cross-sectional area and the estimated values for the fitting parameters, we can derive the variables C and BOB. To assess the significance of these values, we used the method described in Silvert (19'79) to map out the 95% confidence regions for the parameters. Figure 7 shows these regions in ( C , PoB) space for the four different particle types. A large elongated ellipse indicates that the variables have large and correlated uncertainties.

To test the ability of the turbidostat to maintain a constant cell density, we grew T. profunda at several different concentrations, controlled by the turbidostat, in artificial seawater medium (Harrison et al. 1980) with a11 nutrients in excess. These runs were typically 5-10 d in length. Particle density and size spectra were measured 2 or 3 times daily except on weekends. Data from a typical run are shown in Fig. 8. and a summary of data from three different runs is shown in Table 2. Since particle size was observed to vary markedly with time, we perfomed another experiment taking measurements of particle density and size spectra at roughly 3-h intervals over a period of 27 h. These data are shown in Fig. 9. During all of these tests, the algae were growing under light-dark cycles ranging from 6 k light : 18 h dark to 12 h light : 12 h dark.

Early in the development of this turbldostat, we wanted to ascertain whether the time series of pump activity could be used

Can. J . Fish. AqnaaP. Sci., Vok. 42, 6985 749

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

TABLE 1. Results of nonlinear fit to the model equation for the optical response of the sensor. - - - - - - - - -

Pariameter estimates Reduced Mean cross-sectional

Substance N chi-square m a (cm2) Blank IoB C

Silicon carbide 9 0.340 13.5 X lo-" 51.8 382 2.48 Lycogsdie'urn pollen 9 0.383 585 X 1W8 54.9 367 0.5'9 Mierolite H 0 B 2 2 6). 363 44.2 X lo-" 51.8 450 0.86 T* profunda % 7 0.184 8.49 X lo-' 54.3 880 1.62 T. profunda 2 12 0.959 16.5 x 10-' 51 .% 985 1.56

CROSS - SECTIONAL AREA (crn2/rn~)

FIG. 6. Sensor output versus total geometrical cross-section for several particle types. Solid lines result from a Ieast-squares fit of the 3-parmeter m d e l sf sensor response to the data.

to give an 6binstantaneous9' measurement of specific growth rate which would be the s m e as that measured in a batch culture growing under identical conditions. We preconditioned a large batch of culture in the turbidostat for 48 h, and then divided that culture in two. Half remained in the turbidostat and the other half was allowed to grow as a batch culture; the subsequent growth in the two cultures was then monitored for the next 12- 30 h. Time-integrated growth rate as directly measured in the batch culture and as computed from the time series of pump activity in the turbidostat were then compared. The results are shown graphically in Fig. 10 and 1 1 , where time series of accumulated cell biomass (as indicated by the total cross- sectional area) and mean cell cross-sectional area are plotted for two experiments. The increase in cell biomass in the turbidostat is of course inferred from the amounts actually pumped out.

Discussion

The goodness of fit of calibration data to the nonlinear model for sensor response indicates that the model gives a good description of sensor behavior. Thus, we have confidence that our assumption that light scattering can be related simply to cell cross-sectional area is reasonable. The variables C and loB can be estimated directly from the fitting parameters and may be useful for the optical classificatisn of particles. C , when multiplied by the h o w n values of N and P, gives the beam attenuation coefficient, describing an inherent optical property of the particles. loB has no formal optical analogue, but can be

RG. 7. Boundaries of 95% confidence regions in (@, BOB) parameter space for the particle types used in sensor tests.

thought of as an integration of the volume scattering function P(8) over the forward angles between about 2 and 15", times the source intensity Ho (unfortunately, there is no simple way to measure I. with our system). The region of integration for any particular particle depends on its position within the sampling volume of the sensor, so that the quantity B is an average. However, since the averaging will be the same for all well- mixed particle suspensions, one can intercompare the forward scattering properties of different particles. C and B are interrelated, since beam attenuation depends on both the abso~tion of light and the scattering of light away from the beam axis. Figure '7 shows that the 93% confidence regions in parameter space for the different particles do not overlap. At first glance, it appears as if one should be able to distinguish between particle types by their "'C - loBV signature, but Mie scattering theory shows (e.g. Kattawar and Plass 1964) that scattering and absorbance of light by particles are highly dependent on both the particle size and their complex index of refraction. Thus, a given signature arises through a nonunique combination of particle size and physical composition.

In any case, calibration results show that the sensor is sensitive to small changes in particle concentration and that its output is approximately linearly related to particle concentration, making it suitable for use in a turbidostat system. The value of sensing scattered rather than transmitted light is immediately apparent when computing the attenuation of light by these particles that would be measured by a transmitted light sensor of the same physical size. For a value of 2 f a C , at the highest concentration of T. grs f~nda used in the caHibrations, transmitted light would be reduced by only 9% compared with an increase in scattered light of 238%. The value of 9% is also a

Can. J . Fish. Aquat. Sci., Val. 42, I 9 5

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

3 - 1 1 MARCH 1983

DAY OF MONTH RG. $. Data from an 8-d run of the turbidostat with Te probtda with a 12 h light : 12 h dark cycle. Curves show temporal changes in the total cell volume and the mean cell cross-section.

I LOCAL TIME ( h )

FIG. 9. Changes in toVal cell cross-section and mean cell cross-section of T. profunda over 27 h, with a 12 h light : B 2 h dark cycle. Vertical axes are expanded to emphasize the observed changes. Dividing total cross-section by the mean cross-section per cell gives a mean cell density in cells per milailitre (about lo5 cells/nm%, during this experiment).

theoretical maximum, attainable only in a "perfect" beam calibration curves, and nonoverlapping 95% confidence regions transrnisssmeter. for the parameter estimates. This difference occurs because the

A problem with using any optical sensor to measure cell scattering properties s f particles are size-dependent, and these concentration is demonstrated in Fig. 6 and 7. Two separate two cultures had different mean sizes. Mie theory predicts that calibrations using T . profun& gave two slightly different as particle size increases, the forwad asymmetry s f the volume

Can. 9. Fish. Aquat. Sci., Qol. 42, 1985 75 I

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

TABLE 2. Summary of results from experiments with T. profnnda to test the ability of the turbidostat to maintain cultures with variable cell cross-section at constant cell concentration. The mean concentration was obtained by integration over a volume frequency spectrum obtained with a Coulter Counter.

Mean cell Mean cell Time density cross-section

Run (h) N (mrnyp~) SD % (pm2) SD %

T B 165.7 16 3.38 0.72 2 B 8.00 0.79 10 T2 192.5 19 1.94 0.47 24 8.11 8.88 11 T3 244.1 12 1.99 0.24 13 8.91 1.0 1 1

scattering function increases. Therefore9 larger particles will scatter more light into a particular forward solid angle thaw will smaller particles. For these two cultures of Ts profuncia, the equivalent rnean spherical radii were I ,@ and 2.29 Cam. Using data from Kattawx and Plass (1967) we computed the halfwidth of the angular intensity function (the angle at which the scattered intensity is half that at zero degrees) for these two cases to be 6 and 4", respectively. The sensor will therefore "see" more light from the 1ager particles than would be accounted for by the increase in geometrical cross-section done. This will be interpreted by the controller as an increase in cell concentration, and the suspension of larger cells will be overdiluted. This effect is demonstrated in Fig. 9, where the daily increase in mean cell size is coincident with a daily decrease in cell concentration, despite the turbidostatically determined "cell concentration" being held constant.

A sensor measuring transmitted light would have a similar problem, although the correlation between mean cell size and con@lled cell concentration would be positive rather than negative. This would come about because in any beam transmissometer the entrance aperture angle is nonzero, with the result that both transmitted light and a portion sf forward scattered light are detected by the instrument. When particle size increases, transmitted light will decrease because of the geometrical increase in cell cross-section. However, at the same time the forward asymmetry of the volume scattering function increases and the sensor '6seesq' an increasing amount of forward scattered light. The net effect is a smaller change in transmitted light than would be expected from the increase in geometrical cross-section, and the controller would underdilute suspensi~ns of larger cells.

Cell size changes impose a fundamental limit on the ability of any turbidostat to maintain a constant cell density. The effect is not small: for example, during the 27-h experiment monitoring cell size changes, at one point rnean cross-sectional area increased by 27% over 9 h; over the same interval, controlled cell concentration decreased by 17%. To put these values in perspective, during this same period the culture was growing at a rate of roughly 15%/h, so that in theory, uncontruHled cell density would have increased by almost 400% over this 9-h period. Errors due to changes in cell size then appear to have a minor effect on inferred growth rates.

We note that rnean cell size is not the only factor that will affect the scattering coefficient of a suspension of cells. Other physiological effects, such as shape change (e.g. diatom chains breaking into smaller subunits, or increasing in length through division) or changes in cellular pigment concentration, will change the optical properties of the suspension, and thus the apparent cell concentration. Therefore, a simple optical sensor, no matter how well designed, will allow cell concentrations to be held ody approximately constant.

En Table 2, the standard deviation in cell concentration in the

first two cases (TI and T2) is roughly twice that of the mean cross-sectional area. There ape at least two factors that can explain the increased variance in addition ts that accounted for by changes in cell size. First, several of the readings of cell counts were taken either shortly after shaking the flask, so that the controller was in the process of reducing cell concentration at the time of the reading, or at times (such as the end of a prolonged dark period) when the eel1 concentration had dropped below the set value. This effect can be quite large. Hisr example, if in run T2 we removed only two values taken when the sensor reading was significantly higher than the set value (those values

8.020

31 AUGUST 8982

Q - TURBIDOSTAT (predicted)

I 1

2 4 6 8 T I ME ( hours)

38 AUGUST 1982 1': A

E - FLASK

A - TURBIDOSTAT

T l M E ( hours)

RG. 10. Changes in mean cell size (closed syn~bols) and total cell cross-section (open symbols) of DureaBiella tertiolect~ in parallel runs of batch culture and tuhidostat over 8 h under constant light. Time origin was 09:34) local time.

Can. J . Fish. Aquaf. Sci., Vo'ol. 42, 6985

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

8 - 2 SEPTEMBER 8982

TIME (hours)

1 - 2 SEPTEMBER 1982 55

e- FLASK ' .\

0 4 8 12 16 2 0 '24 28 32

T I M E (hours1

FIG. E 1. Changes in mean cell size (closed symbols) and total cell cross-section (open syrmbols) of B. FerFiolec~a in parallel runs of batch culture and turbidosta? over 3% h under constant light. Time origin was 08:30 local time.

can be seen in Fig. 8, the third and fourth points), the standard deviation of cell concentration is reduced from 24% of the mean to only 14%. A second source of variance in cell concentration is the formation of bubbles on the LED inside the sensor. These bubbles, which are often impossible to remove except by disassembly and cleaning of the sensor, appear to change the beam pattern created by the LED and cause unpredictable small changes in sensor output. This problem adds perhaps 3 or 4% to the total variance.

The results of the pipfallel flask tests (Fig. I0 and I l ) are encouraging: as expected, growth rates measured in the turbidostat closely parallel those in the batch culture, at least for the first day of growths In the experiment of Fig. 10, which ran for 6 h, the cell concentration inferred from the turbidostat pumpout record is almost identical to that in the batch culture throughout. Mean hourly growth rates were 3.$2%/h for the turbidostat and 3.14%/h for the batch; growth rates computed using only initial and Anal values (final predicted value for the turbidostat) and assuming exponential growth are 3.36%/h and 3.05%/h for turbidostat and batch, respectively. In the experiment of Fig, I I , which ran for approximately 31 h, cell concentration predicted from turbidostat growth closely parallels that in the

batch culture except for the first few hours in which there was no growth in the turbidostat. Mean hourly growth rates were 3.7%/h and 2.8%/h for turbidostat and batch, respectively; the corresponding computed exponential growth rates were 2.9%/h and 2.5%/h. Growth rates in the turbidostat are consistently higher than those in the batch culture. This result is to be expected, since cells in the turbidostat are not as constrained by competition for light and nutrients as those in the batch cultures. Evidence for the increased competition is seen in Fig. 1 I , where mean cell size in the batch culture continues to decrease at an accelerating rate after hour 23, while that in the turbidostat begins to level off. Growth in the turbidostat clearly begins to outstrip that in the flask after this point as well.

Acknowledgments We thank P. J. Hmiscsn, J. R. Forbes, and K. A. BeMacedo

for advice and assistance throughout this work. Two anony- mous reviewers also provided helpful constructive criticism.

References BEVINGTQN, P.W. 1969. Data reduction and error analysis for the physical

sciences. McGraw-Hill, New York, NY. 336 g. DEIWEND~AN. D. 1969. EBectrornagnetic scattering on spherical polydisper-

sions. Elsevier, Amsterdam. 290 p. BENMAN, K. L., AND A. GARGETT. 1983. Time and space scales of vertical

mixing and advection of phytop8ankton in the upjxr ocean. Limnol. Oceanogr. 28: 801 -8 1%.

DENMAN, #. L., D. L. MACKAS, Pi. J . FREELAND, M. J . AUSTIN, AND S. H. HILL. 1981. Persistent upwelling and mesoscale zones of high productivity off the west coast of Vancouver Island, Canada, p. 514-521. In F. A. Richards Led.] Coastal upwellim. American Geophysical Union, Washington, BC. 529 p.

FREELAND, H. H., AND K. L. DENMAN. 1982. A topographically controlled upwelling center off southwest Vancouver Island. J. Mae. Rcs. 40: 1069-1093.

HAMSQN, P. J., R. E. WATERS, AND F. J. R. TAYLOR. 1980. A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J. Bhycd. 16: 28-35.

HILL, S., K. DENMAN, D. MACKAS, AND H. SEFTQN. 1982a. Ocean Ecology Data Report: Coastal waters off southwest Vancouver Island. Spring and summer 1979. Can. Data Rep. Hydrog. Ocean Sci. 3: 118 p.

1982b. Ocean Ecology Data Report: Coastal waters off southwest Vancouver Island. Spring and summer 1980. Can. Data Rep. Hydrogr. Ocean Sci. 4: 103 p.

HILL, S., K. DENMAN, D. MACKAS, H. SEFTON, AND R. FORBES. 1983. ocean Ecology Data Report: Coastal waters off southwest Vancouver Island. Spring and summer 1981. Can. Data Rep. Hydrogr. Ocean Sci. 8: 93 p.

JERLQV, N. G. 1976. Marine optics. Elsevier, Amsterdam. 231 p. KATTAWAR, G . W . , AND G. N. PLASS. 1967. Electromagnetic scattering from

absorbing spheres. AppB. Opt. 6: 1377-1382. MACKAS, B. L., G . C. LOUTFIT, AND M. J . AUSTIN. 1988. Spatial distribution

of zooplankton and phytoplankton in British Columbia coastal waters. Can. J. Fish. Aquat. Sci. 37: 1476-1487.

MADBUX, W. S., AND R. F. JONES. 1964. Some interactions of temperature, Iighe intensity, and nutrient concentration during the continuous culture of Nitaschia closterium and Ternaselmis sp. Limnol. Oceanogr. 9: 79-86.

MUNSQN, R. J. 1970. Turbidostats. p. 349-376. Pn Vol. 2. Methods in microbiology. J. R. Norris, and D. W. Ribbons [ed.] Academic Press, New York, NY. 432 p.

SILVERT, W. 1979. Practical curve fitting. Limnol. Ocemogr. 24: 76'9-773. SINNETT, J . C., AND W. R. DAVIS. 1983. A programmable turbidostat for

suspended particles in laboratory aquaria. J. Exp. Mar. Biol. Ecol. 73: 167-174.

SQRGELOOS, P., E. VAN OUTRYVE, G. PERSOONE, AND A. CATTQIR- REYNAERTS. 1976. New type of turbidostat with intermittent determination of cell density outside the culture vessel. Appl. Environ. Micmbiol. 31: 327-33 1.

VAN DE HULST, H. C. 1957. Light scattering by small particles. Wiley, New York, NY. 470 p.

WATSQN, T. G. 1972. The present status and future prospects of the turbidostat. J. Appl. Chem. Biotechno1. 22: 229-243.

Can. J . Fish. Aquat. Sci., Val. 4 2 , I985

Can

. J. F

ish.

Aqu

at. S

ci. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y Y

OR

K U

NIV

on

11/0

9/14

For

pers

onal

use

onl

y.

a computer-controlled turbidostat for the culture of planktonic algae

Documents