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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

jvw5246@psu.edu, bjg5192@psu.edu, raj5001@psu.edu, wrc2@psu.edu

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:

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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

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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.

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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

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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.

difCOKNaba

Figure 3. Nfferent nitrogeO2, and (b) 0.6NO3 and NH4andoned for t

0.6% C

5% CO2

-1.40

-1.20

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

H+/N

(mol

ar)

Net consumpten source. Th6% CO2, and Cl are based the other part

KNO3

O2 1.08 2 0.90

1.08

0

0

0

0

0

0

0

0

0

0

0

0

0

0

tion of protonhis graph inclu

HNO3, NH4Oon the stand eof experimen

NH4Cl

-1.09

-1.11

-1.09

0.90

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n during cultivudes the cultiOH, Urea, NHerror of regrents due to its h

HNO3

0.19

0.19

-1.11

vation of Chlovation on NH

H4NO3 for 0.6ssion. 5% COhigh data vari

NH4OH

0.07

0.07

orella vulgariH4Cl and KNO6% CO2. ErroO2 gas growthiance.

Urea

0.29

0.29

is using O3 for (a) 5% r bars on

h condition is

NH4NO3

0.12

0.12

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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 trickle-

film bed reactor that has been developed for untra-high density algae growth .

REFERENCES

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