Feed-Forward Control of Dynamic High-Density Microalgae Cultures Using
Model Based Predictive pH Control and a Novel Biomass Sensor
Jun Wang, Benjamin Geveke, Ryan Johnson, Wayne R. Curtis
[email protected], [email protected], [email protected], [email protected]
Keywords: Algae Sensor, Biofuels, High-Density Algae, Process Control and pH
control
ABSTRACT
Control of pH in algae cultures is complicated by the presence of CO2 and the
typical use of buffers. As a result, the stoichiometry of proton secretion and uptake has
not previously been explicitly measured. In this work we measure the stoichiometric
coefficient for proton utilization for photosynthetic growth on different nitrogen sources.
Based on our observation, we purposed our explanation of underlying biochemical
process associated with nitrogen incorporation into proteins. Further experiments are
then performed to demonstrate the surprising prediction that growth on strong acid
(HNO3) or strong base (NH4OH) can be nearly proton exchange neutral, though can only
be implemented with careful bioreactor fed batch control. Additional improvements in
algae bioreactor operation including a variable-intensity LED biomass sensor are also
noted.
INTRODUCTION
The pH instability is an inherent obstacle in the growth of continuous high-density
microalgal cultures. Because of the cost and toxicity, traditional brute-force ‘feedback’
pH control method is not suitable for large scale algal cultivation. Besides, due to the
preferential assimilation behavior of algal nitrogen metabolism, the balanced media
method, which typically works for high plants, does not applied to algae. So far,
extensive studies have shown that this imbalanced pH is mainly the result of the
metabolism of different nitrogen sources. Starting from the stoichiometry, a phototrophic
growth of algae could be expressed in the following general equation:
1
Here,
represents the biomass composition and is normalized by setting
carbon as unit, and x, y, z are the the biomass composition of hydrogen, nitrogen, and
oxygen, respectively. According to the above equation, the stoichiometric relation
between imbalanced proton (pH change) and biomass accumulation could be clearly
defined. With the knowledge of this relation, one would be able to control the culture pH
through correct dynamical feeding of different nitrogen source. The concept is to
implement a feed-forward control strategy in which nutrient addition is predicted based
on anticipated growth based on measurements of light, temperature, etc (Figure 1A). We
therefore set out to accurately measure the stoichiometric coefficients of this relationship
by careful metering of nutrients, acid and base as well as including correction for biomass
composition measurements.
METHODS
Cultivation: The main flask is cultivated under a controlled environments that has a
temperature between 25~30 Celsius degree, light intensity between 250~ 400 μE/m2/s
with a 16/8 light/dark diurnal cycle. For the measurement part, pH is controlled by
computer program through dosing acid/base, with the amount of acid/base being recorded
(Figure 1B). For verification part, pH is not controlled by external interference. For the
cultivation with HNO3, NH4OH, and NH4NO3 as nitrogen source, these chemicals are
batch fed into cultivation flask based on current growth rate as indicated by pH changes
and optical density (OD) measurements. OD measurements could be measured online in
a flow-through cell which provided for separation of interfering gas bubbles (Figure 1C).
Detailed media is provided as the (WFAMC) media in reference 1. Two gas
concentrations, 5% CO2 and 0.6% CO2 are used to grow culture.
Inoculation: Chlorella vulgaris with culture number 2714 from UTEX culture collection
is inoculated at a 0.1 optical density, all cultivation are then stopped after reaching 2.5 optical
density.
Determination of stoichiometric coefficient:
The optical density as well as the dry
weight is measured during and at the end of experiment. Biomass composition analysis is
performed to obtain the mass percentage of C, N, and H. One the other side, for the measurement
part, the pH is maintained constant and the total amount of acid/base doses are recorded through
2
balance and pH controller. For verification part, if pH is not balanced during the experiment,
titration will be performed at the end.
Figure 1. Various undergoing research & development at Curtis’ lab towards large scale
algal cultivation system. A) Design of the feedforward control system; B) Experimental setup
for stiochiometric relation measurement; C) Optical cell density sensor developed at Curtis’
lab; D) Continous algal cultivation system using photobioreactor.
RESULT & DISCUSSION
To measure the stoichiometric coefficient accurate, a highly-instrumented algae
culture flask was set up so that minimal amounts of acid and base could be introduced via
a actuated solenoid (Figure 2A). This was combined with simple mass balance on the
mass of a titrated acid or base solution for directly measuring the addition of H+ or OH- to
the culture as it would grow on KNO3 or NH4Cl. Combining this time-dependent
measurements with growth as well as endpoint biomass composition (Figure 2B), the
stoichiometry of the growth reaction was directly measured.
3
Figure 2. Setup of and the growth vs relation. A) Experimental setup of stoichiometric
coefficient measurement, consisted of cultivation flask, controller and acid/based delivery
system; B) Comparison between algae optical density (diamond) and imbalanced proton
(square) of acid/base vs light hours for KNO3 (Red) and NH4Cl (Yellow) media, under 0.6%
CO2 gas.
This work therefore quantifies the relation between the change of pH (imbalanced
proton) and nitrogen assimilation with measured stoichiometric coefficients. In the
absence of heterotrophic metabolism, the proton imbalance is not only dominated by, but
also proportional to the nitrogen assimilation. As shown in below equations, with slightly
different fluctuation under different CO2 concentration, the stoichiometric relation
between consumed proton and assimilated nitrate is close to 1 (proton consumption),
while the stoichiometric relation between consumed proton and assimilated ammonium is
close to -1 (proton generation). (Figure 3 & 4)
OH ,
H
This result is unexpectedly simple and seems as trivial as the requirement of charge
balance. However, considering the widely observed phenomena of such nitrogen
dominated proton imbalance, the underlying reason has to be a fundamental biochemical
4
or chemical reactions that are common to most organisms, therefore usually would be
simple.
Even so, due to the fact that there are so many factors contributing to the pH
change, plus the fluctuation caused by intracellular nitrogen pool and associated variation
of biomass composition, it was desirable to independently verify the 1:1 nitorgen proton
exchange ratio. This was accomplished with another series of experiments with
alternative nitrogen sources) by incrimental feeding of nitric acid since the generated
hydroxides are predicted to directly counter the proton in nitric acid. Ironically, the final
pH would became slightly higher than that of the beginning, after the cultivation with
nitric acid. Consistant with prediction, the cultivation of urea, NH4OH, and NH4NO3 all
showed similar result (Figure 3). Compared to the cultivation on KNO3 or NH4Cl which
experienced large fluctuations in pH, the strong acid / base are mostly balanced. By
combining the three statements, one could tell that the above measured 1:1 ratio is
generally correct while small “residue” also exists. Additionally, this deviation is likely to
be caused by the contribution of imbalanced proton from non-nitrogen metabolism, as a
minor factor.
Based on the observation, we purpose our tentative explanation of the underlying
biochemical process. As it is known that the assimilation of nitrate is a combination of
the nitrate reduction into ammonium and the assimilation of ammonium, the whole
process could also be explained with two sub-processes. The overall result of the first
sub-process, the ammonium assimilation, creates a net proton while feeding ammonium
into protein biosynthesis. The second sub-process, the nitrate reduction, generates two net
hydroxides instead. Therefore, an overall generation of one proton is introduced with
ammonium as nitrogen source, while one hydroxide is generated with nitrate as nitrogen
source.
5
1.20
0
1.08
0.90
1.00
0
0.80
0
0.60
0
H+/N (molar)
0.40
0
0.29
0.19
0.20
0
0.12
0.07
0.00
0
-0.20
0
-0.40
0
-0.60
0
-0.80
0
-1.00
0
-1.20
0
-1.40
0
-1.09
-1.11
KNO3
NH4Cl
HNO3
NH4OH
Urea
NH4NO3
0.6% CO2
1.08
-1.09
0.19
0.07
0.29
0.12
5% CO2
2
0.90
-1.11
Figure 3. Net
N consumpttion of protonn during cultivvation of Chloorella vulgariis using
diffferent nitrogeen source. Thhis graph incluudes the cultivation on NH
H4Cl and KNO
O3 for (a) 5%
CO
O2, and (b) 0.6
6% CO2, and HNO3, NH4OH,
O Urea, NH
H4NO3 for 0.66% CO2. Error bars on
KN
NO3 and NH4Cl are based on the stand error
e
of regression. 5% CO
O2 gas growthh condition is
abaandoned for the
t other part of experimennts due to its high
h
data variiance.
6
Figure 4. Measured stoichiometric coefficient for different nitrogen sources under two
CO2 gas concentrations. A) Cultivation with KNO3 under 5% CO2; B) Cultivation with KNO3
under 0.6% CO2; C) Cultivation with NH4Cl under 0.6% CO2; D) Cultivation with NH4Cl
under 5% CO2.
CONCLUSIONS
The primary goal of this initial work was to accomplished to characterize the the
quantitative relation between the proton imbalance that is associated with assimilated
nitrogen. This work has laid the basis for a feed-forward metabolic pH control strategy.
Tentative theoretical explanation is also given to broaden the capability of prediction.
Starting from current stage, we are going to build a feed forward pH control system by
switching nitrogen source. Our ultimate goal is to build a continuous algae biofuel
production system by combining it with our online cell density sensor, as well as tricklefilm bed reactor that has been developed for untra-high density algae growth .
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