Predicting Dissolved Inorganic Carbon in Photoautotrophic

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Predicting Dissolved Inorganic Carbon in Photoautotrophic Microalgae Culture via the Nitrogen Source Binh T. Nguyen* and Bruce E. Rittmann Swette Center for Environmental Biotechnology, Biodesign Institute, Arizona State University, P.O. Box 875701, Tempe, Arizona 85287, United States S Supporting Information *

ABSTRACT: Dissolved inorganic carbon (DIC) and pH are key factors that control the growth rate of microalgae growing photoautotrophically. Being able to quantify how DIC and pH independently affect growth kinetics requires a means to control each parameter independently. In this study, we used the Proton Condition (PC) to develop means to control pH and DIC independently. Using the PC, we found that different N sources systematically affect the alkalinity and the DIC in distinct ways. With pH controlled at a fixed level by CO2 addition, using nitrate as the N source increased the alkalinity and DIC concentration in proportion to the increase in biomass concentration. In contrast, using ammonium caused the alkalinity and DIC to decline, while using ammonium nitrate left the DIC nearly unchanged. Experiments with a model photoautotroph cyanobacterium, Synechocystis sp. PCC 6803, in batch experiments with modified BG-11 media and a pH-stat confirmed all of the DIC predictions of the PC-based model. Thus, this study provides a mechanistic basis for managing the DIC for photoautotrophic cultures through the N source. In particular, using ammonium nitrate makes it possible to control DIC and pH independently in a pHstat.

1. INTRODUCTION Microalgae, the collective name for eukaryotic microalgae and prokaryotic cyanobacteria, can capture sunlight energy and fix carbon dioxide at a rate 5 to 20 times higher than that of the terrestrial plants, do not require arable land, and are amenable to genetic modification.1−3 Thus, microalgae have promise to become a major source of renewable energy and chemical feedstock.1,4 Microalgae take up dissolved inorganic carbon (DIC) for synthesis, and carbon makes up around 50% of the dry weight of microalgae.5 Due to the essential role of DIC, its effects on growth kinetics have been studied extensively in mass culture.6−9 The choice of growth medium and nutrient components can influence the DIC concentration that occurs for a given pH. Typical growth media, such as BG-11 and f/2,10,11 contain sodium nitrate as the nitrogen source. Although a few microalgae can fix nitrogen gas,12 ammonium is the only widely relevant alternative to nitrate as the nitrogen source. Utilizing an ammonium-rich waste stream, such as that in many wastewaters, to grow microalgae appears to be win−win situation: simultaneously removing nutrient pollution and producing useful biomass.13,14 Previous studies identified clear trends of alkalinity when microbes synthesize biomass with different sources of nitrogen,15,16 suggesting that the choice of nitrogen for large-scale culturing is important,17 and attempted to correlate DIC with the alkalinity.18 Despite these generally well-established trends, © XXXX American Chemical Society

how DIC controls biomass growth has not been systematically quantified. Having a mechanistically sound and quantitative means to stabilize DIC as photoautotrophs synthesize biomass is the first step for more fundamental studies, such as growth kinetics of microalgae on DIC,6,18,19 as well as for practical management of microalgae production systems. Here, we systematically evaluate how the nitrogen source affects the DIC concentration during the growth of microalgae with pH fixed by at a constant level by means of automated CO2 supply. First, we apply the Proton Condition (PC) to track the change in proton-deficient and excess species when using different nitrogen sources: nitrate alone, ammonium alone, and a combination of nitrate and ammonium. Our analysis confirms that the DIC concentration systematically and strongly depends on the nitrogen source. Second, we evaluate the PC-based predictions by carrying out experiments with the cyanobacterium Synechocystis sp. PCC 6803. The experimental results with a fixed pH confirm that nitrate increases the DIC proportionally to biomass synthesis, ammonium reduces DIC in proportion to biomass synthesis, and ammonium nitrate holds the DIC in a narrow range. Received: April 6, 2015 Revised: July 25, 2015 Accepted: July 28, 2015

A

DOI: 10.1021/acs.est.5b01727 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

2. APPLYING THE PROTON CONDITION To relate the effects of the N source to the alkalinity and DIC concentration, we employed the proton condition (PC), a special mass balance that tracks proton-deficient and -excess species.20,21 Unlike an electroneutrality condition, the PC specifically tracks the change in proton status of all components that participate in acid−base reactions by using the change in total base (or alkalinity) in the system. Thus, the PC focuses only on species relevant to acid−base reactions, and it also eliminates problems of rounding errors when changes in acid− base status are small compared to the concentrations of all cations and anions. To use the PC, we first select reference species: in this case, water (H2O), carbonic acid (H2CO3), and phosphoric acid (H3PO4). The PC contains all other species that have more or fewer protons than the reference level. Also included in the PC is the total amount of base or alkalinity that is added to the system from any source; we designate this value as b (eq/L). The PC for a general case is as follows:

makes a major difference in how photosynthesis affects the alkalinity, or b. We first present the balanced, stoichiometric reaction for biomass synthesis R1 by photosynthesis when the N source is NO3−, as in the standard BG-11 growth medium. 5CO2 + NO−3 + 0.05H3PO4 + 3.925H 2O → C5H 7O2 NP0.05 + 7.0625O2 + OH−

When one mole of new biomass is generated according to eq R1, one mole of alkalinity (OH−) is also generated. Hence, the total alkalinity in the system is b = b0 + Δb, where b0 = the starting alkalinity (eq/L) and Δb = the increase in alkalinity, which also equals the increase in the molar concentration of biomass R1. The concentration of phosphate declines due to uptake for biomass synthesis, making CP,Δb = CP,0 − 0.013Δb, where 0.013Δb accounts for phosphorus taken up into biomass based on C5H7O2NP0.05. Then, the PC becomes the following: [H+]Δb + b = [OH−]Δb + (α1,C, Δb + 2α2,C, Δb)CC, Δb

[H+] + b = [OH−] + (α1,C + 2α2,C)CC + (α1,P + 2α2,P + 3α3,P)C P

+ (α1,P, Δb + 2α2,P, Δb + 3α3,P, Δb)CP, Δb

b0 = [OH−]0 + (α1,C,0 + 2α2,C,0)CC,0 + (α1,P,0 + 2α2,P,0 + 3α3,P,0)C P,0 − [H+]0

(E3)

where the subscript 0 reflects the condition before photosynthesis begins. Rearranging E2 to solve for the concentration of inorganic C based on biomass synthesis with NO3− as the N source gives E4:

[H+]Δb + b0 + Δb − (α1,P, Δb + 2α2,P, Δb + 3α3,P, Δb)C P, Δb − [OH−]Δb α1,C, Δb + 2α2,C, Δb

When the growth pH is fixed, the ionization fractions for each component, [H+], and [OH−] are known and constant, e.g., α1,C,0 = α1,C,Δb. Substituting b0 from E3 into E4 and canceling terms lead to the following: CC, Δb = CC,0 +

(E2)

where b was defined above, and subscript Δb indicates after growth of Δb moles of biomass. The initial alkalinity of the cell-free medium (b0) can be computed from E1 for the initial conditions:

(E1)

where [H+] and [OH−] are the concentrations of hydrogen and hydroxyl ions in bulk liquid [mol/L); CC and CP are the concentrations of DIC and total soluble phosphate in bulk liquid [mol/L]; α1,C and α2,C are the ionization fractions of bicarbonate (HCO3−) and carbonate (CO32−); α1,P, α2,P, and α3,P are the ionization fractions of dihydrogen phosphate (H2PO4−), hydrogen phosphate (HPO42−), and phosphate (PO43−); and b is the total alkalinity (eq/L). Photoautotrophic synthesis fixes C from DIC, N from the N source, and P from phosphoric acid into biomass, represented as C5H7O2NP0.05, based on refs 5 and 22, in which N comprises 13.2% and P comprises 1.3% of the dry weight. The N source CC, Δb =

(R1)

CC, Δb = CC,0 −

Δb(1 − 0.013(α1,P + 2α2,P + 3α3,P)) α1,C + 2α2,C (E6)

The trends of DIC and alkalinity in E6 are opposite to those in E5. As biomass grows, alkalinity and Ci decline. Equations E5 and E6 illustrate why it is impossible to keep pH and DIC constant when either nitrate or ammonium is the nitrogen source and the biomass concentration is increasing. In principle, this situation can be alleviated if ammonium nitrate (NH4NO3) is the N source. The following reaction occurs if NO3− and NH4+ are utilized equally for synthesis:

Δb(1 + 0.013(α1,P + 2α2,P + 3α3,P)) α1,C + 2α2,C (E5)

Equation E5 indicates that the concentration of DIC increases with biomass growth and alkalinity generation (i.e., Δb), and the proportionality is set by the pH, which determines the ionization factors. When ammonium (NH4+) is the N source, the stoichiometric reaction is as follows:

5CO2 +

5CO2 + NH+4 + 0.05H3PO4 + 1.925H 2O → C5H 7O2 NP0.05 + 5.0625O2 + H+

(E4)

1 1 NO−3 + NH+4 + 0.05H3PO4 + 2.425H 2O 2 2

→ C5H 7O2 NP0.05 + 6.0625O2 (R2)

(R3)

The PC remains E2, and substitution yields the following:

The PC with NH4+ remains E2, but the total alkalinity is b = b0 − Δb due to the mole of hydrogen ion produced with each mole of biomass synthesized R2. Following the same steps as with NO3−, the PC can be solved for CC,Δb for NH4+:

CC, Δb = CC,0 + B

0.013Δb(α1,P + 2α2,P + 3α3,P) α1,C + 2α2,C

(E7)

DOI: 10.1021/acs.est.5b01727 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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The PBR was a ∼1.5-L (working volume) glass jar that served as the main reactor and a gas-flow meter with stop valve and connected to a CO2 compressed tank. CO2 gas was delivered to the bulk liquid by a 1/4″ plastic tube inserting through the reactor’s lid. To increase gas transfer, the tube was connected to a 1/16″ tip that was placed right above the stir bar. Input and exit gas flows were filter through 1-μm Pall Bacterial Filter to prevent contaminant to and from the culture. Biomass was mixed by one 5 cm magnetic stir bar at a constant rate of 200 rpm. Samples were taken twice a day through a silicon tube inserted through the reactor’s lid. Roughly 25 mL of liquid was pulled out using a syringe and contained in 50 mL polypropylene centrifuge tube (VWR). Aliquots were assayed for biomass density by spectrometry. During experiments, the culture temperature was maintained at 30 °C by automated air-cooling. The incident light intensity was 320 μE/m2.s from T5 fluorescent bulbs (Hydrofarm, U.S.). The target pH was set by a web-based interface before each run and maintained at ±0.05 pH units. The software could record and store the pH and temperature for up to 7 days. In each set of experiments, we replicated selected runs to evaluate reproductivity of results. The bars on each data points represent analytical standard deviations of at least triplicate measures. We tested effects of pH and nitrogen sources in several ways. For standard BG-11, we experimented with fixed pH values of 6.5, 7.5, 8.5, 9.5, and 10.0. To evaluate effect of starting alkalinity, we augmented an additional 1.5, 3.5, or 5.0 mM bicarbonate with pH of 8.5 and with only 5.0 mM bicarbonate added for pH values of 7.5 and 9.5. With ammonium chloride as N source, we augmented the modified BG-11 with different starting alkalinity by adding bicarbonate at 3.0, 4.8, and 6.0 mM, all runs with a fixed pH of 8.5. Finally, we used ammonium nitrate as N source and augmented starting alkalinity at 0.2, 2.0, 2.6, and 5.0 mM of bicarbonate and a fixed pH of 8.5. To ensure ammonium and nitrate are taken up equally, we added ammonium nitrate 0.5 N (pH = 8.5) stepwise based biomass increments expected between sampling times. 3.4. Analyses. Optical Density (OD) was measured by a UV−vis BioSpec-mini spectrometer at 730 nm (Shimadzu Corp., Japan). As needed, a sample was diluted with deionized ion (DI) water to obtain an OD less than 0.8. The spectrometer was autozeroed with DI water before each measurement. The OD reading was converted to biomass dry weight using an empirical conversion factor of 254 mg dry weight biomass/L per of unit of OD.24 A mole-per-liter concentration of biomass was calculated from dry weight using an empirical biomass formula of C5H7O2NP0.05. DIC was analyzed using a TOC-VCPH analyzer with an autosampling tray (Shimadzu Corp., Japan). Culture samples were filtered through a 0.20-μm polyvinylidene fluoride (PVDF) syringe filter (Whatman GPX), and the filtrate was collected into glass Falcon tubes and immediately sealed by parafilm to prevent any carbon or water loss. To analyze DIC, a 50-μL aliquot was injected into the IC chamber, which contained 25% H3PO4 by weight (IC reagent, pH ≈ 0.3) so that all inorganic carbonic species were converted to CO2. The CO2 was purged, using ultrapure air, to a nondispersive infrared (NDIR) gas analyzer. The DIC concentration then was determined by a calibration using a 50/50 molar ratio of sodium hydrogen carbonate/sodium carbonate standards (Nacalai Tesque, Inc., Japan). This instrument can inject each

The alkalinity and DIC depend mainly on the starting alkalinity for a fixed pH. The consumption of phosphate as the P source leads to only a small increase in DIC. This means that it ought to be possible to maintain nearly constant pH and DIC as biomass grows photoautotrophically. This outcome is particularly valuable for studying how DIC and pH independently affect growth kinetics of photoautotrophic microalgae.

3. MATERIALS AND METHODS 3.1. Inoculum Preparation. We used a cyanobacterium, wild-type Synechocystis sp. PCC 6803 (hereafter called Synechocystis), as the model photoautotroph. We obtained this strain as pure colonies on agar plates provided by the laboratory of Dr. Willem F. J. Vermaas, School of Life Sciences, Arizona State University (Tempe, AZ). Colonies were transferred to BG-11 liquid medium and incubated for 3−5 days at 30 °C and ∼80 μE/m2.s to grow sufficient biomass for experiments. 3.2. Growth Medium. We used standard BG-11 medium,23 which is recommended for Synechocystis. Each liter of BG-11 contains 17.6 mM NaNO3, 0.23 mM K2HPO4, 0.30 mM MgSO4, 0.24 mM CaCl2, 0.01 mM citric acid, 0.021 mM ferric ammonium citrate, 0.0027 mM Na2EDTA, 0.19 mM Na2CO3, and trace metals.10 To evaluate different source of nitrogen, we modified standard BG-11 by substituting other nitrogen sources for sodium nitrate, but keeping all other constituents the same. We prepared separate stocks of ammonium nitrate and ammonium chloride (0.5 N) and added to the culture during experiment. The medium was steam sterilized with a liquid cycle (121 °C and 15 min) in a Tabletop Sterilizer (SterileMax, Thermo Scientific, IA). 3.3. Experimental Setup. We built a pH-stat that automatically maintains the pH at the set value and installed the pH-stat in a photobioreactor (PBR); Figure 1 is a schematic

Figure 1. Schematic diagram for the PBR equipped with a pH-stat.

of the set up. The pH-stat included a pH probe connected with a pH controller (Neptune Systems, CA) that activates a solenoid valve (Milwaukee Instruments, NC) to feed pure CO2 gas to the reactor when the pH exceeds the set point. The pH probe was submerged in the Synechocystis culture to provide instantaneous readings to the pH controller and allow continuous data recording. C

DOI: 10.1021/acs.est.5b01727 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Figure 2. Left panel: DIC concentration as Synechocystis grew in standard BG-11 (containing only NO3−), starting with ∼0.2 mM carbonate. Right panel: DIC trends as Synechocystis grew in the standard BG-11 augmented with 5 mM of bicarbonate (i.e., making the starting alkalinity 5.5 mequiv/ L (pH = 8.5), circles with growth pH = 7.5, 8.5 and 9.5) and BG-11 contained 1.5 and 3.5 mM of C with growth pH = 8.5. In both panels, the lines are the predictions based on eq E5, and the symbols are the experimental data. The bars present two standard deviation of DIC measurement, or the 95% confidence level.

Figure 3. Left panel, DIC trends as Synechocystis grew in BG-11 modified by substituting sodium nitrate with ammonium chloride. Right panel, DIC trends as Synechocystis grew in BG-11 modified by substituted sodium nitrate with ammonium nitrate. For both panels, the pH was 8.5. The lines are predictions from eqs E6 for the left panel and E7 for the right panel, and the symbols are the experimental data. The numbers on the top rows give the starting concentration of DIC achieved by adding, one time, HCO3− before inoculation. Runs ending in (A) and (B) were replicates. The bars represent two standard deviation of DIC measurement.

CO2. Another possibility is the hydroxyl ion generated from photosynthesis is consumed by functional groups in microalgal culture such as simple carboxylic (pKa = 4−6).25 Those possibilities needed to be investigated further. The right panel in Figure 2 shows the experimental and modeling trends with the addition of 5.0 mM bicarbonate at the beginning of the experiments with a fixed pH of 7.5, 8.5, and 9.5. For the fixed pH of 8.5, two runs with additional 1.5 and 3.5 mM bicarbonate are presented as well. From eq E3, the higher starting alkalinity should result in a higher DIC concentration for the same pH. Compared to the left panel, the slope of each line is unchanged for the same pH, but the curve is offset exactly by the increase in b0 as predicted by eq E5. The left panel of Figure 3 shows the changes in DIC when ammonium was the sole N source. As predicted by eq E6, using ammonium reduced alkalinity and DIC as biomass grew when the pH was controlled by CO2 addition. The decline with biomass growth was the same for all initial DIC concentrations (3.0 to 6.0 mM C of added bicarbonate), which made b0 be 3.4 to 6.4 mequiv/L of alkalinity. Therefore, the starting alkalinity is especially important to maintain sufficient DIC for photosynthesis when ammonium is the N source. The right panel of Figure 3 shows the DIC trends with ammonium nitrate as the N source. As expected from eq E7, the DIC changed only slightly from the starting level. The small increases in DIC were due to the uptake of phosphate P for

sample up to 20 times. We used from 5 to 10 injections. Vials with acidified deionized water (18.2 MΩ, pH = 2.5) were included at beginning, at the end, and between every 4 to 6 samples.

4. RESULTS AND DISCUSSION All runs started with a low OD (∼0.1 to 0.2 absorbance unit), and the cells grew continuously in linear phases up to an OD of as high as 2.6 (∼5.8 mM equivalent of dry weight biomass). The culture appearance always was a healthy green, and no irregular growth or biomass loss was observed. Supporting Information Figures S1−S4 show all the data for the batch experiments. The accumulation of DIC during growth of the cyanobacteria in standard BG-11 (with NO3− as the N source) is shown in the left panel of Figure 2. The experimental results (symbols) confirm the trends predicted by eq E5 (lines): As biomass grew, the DIC increased proportionally when nitrate was the sole N source with a fixed growth pH. Furthermore, the pH affected the accumulation rate of DIC in ways that conform to the changes in the fraction ionization factors in eq E5: The increase was the greatest for the lowest pH value, which had the smallest values of α1 + 2α2 for the carbonate system. The experimental data fit the PC-based modeling well, except for the run with pH = 6.5, which had lower DIC concentration than predicted. A possible cause is that slow CO2 gas transfer at this relatively low pH prevented equilibrium of gas-phase CO2 with liquid-phase D

DOI: 10.1021/acs.est.5b01727 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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the experiments, interpretation of the data, and writing the manuscript.

synthesis; the alkalinity originally in phosphate species was redistributed to carbonate species, which required a slightly higher DIC concentration for the fixed pH. The results in Figure 2 and Figure 3 verify the value of using PC-based eqs eqs E5, E6, and E7 to explain and quantify the effects of the N source on alkalinity and DIC. The equations directly link the changes in DIC and alkalinity to the amount of biomass generated, the pH, and the starting concentration of alkalinity. Key to using the PC-based equations is knowing the starting concentration of alkalinity (b0), which is the sum of all sources of alkalinity, including carbonate and phosphate salts, hydroxide salts, and any other added bases. Also critical for correct use is that the pH at the beginning of the experiment is the same as the pH value held constant by addition of CO2 and that the stoichiometric formula for biomass is known. Perhaps the most important finding is that the choice of N source has a very strong impact on the alkalinity and DIC concentration when the pH is held constant. Using nitrate causes the DIC to rise in proportion to the amount of biomass synthesized, while using ammonium causes both concentrations to decline. The former can have profound impacts on the precipitation of carbonate minerals, such as CaCO3. The latter can lead to severe DIC limitation of the photoautotroph’s growth rate. In this study, we used the Proton Condition to predict and experimentally validate DIC trends for a cyanobacterium grown in a fixed pH and with different N sources. Using nitrate increased the DIC, while ammonium reduced the DIC; in both cases, the changes were proportional to new biomass synthesized. Because pH and DIC can have separate impacts on photoautotrophic growth kinetics, it is valuable to have a system in which DIC and pH can be controlled independently. Our results show that using ammonium nitrate at the N source makes it possible to control the DIC independently of the pH. Achieving this goal was a primary motivation that led us to develop the PC-based analysis, and we are exploiting it to carry out experiments to quantify how pH and DIC control the growth kinetics of Synechocystis. This PC-based method also is valuable for the practical operation of photoautotrophic systems used for biomass production.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported through a grant, EE0006100: “Managing the Microbial Ecology of a Cyanobacteria-Based Photosynthetic Factory Direct!,” from the U.S. Department of Energy. We thank Drs. Willem Vermaas and Shuqin Li in the School of Life Sciences at Arizona State University for providing Synechocystis sp. PCC 6803 wild type and modified strains. We thank Levi Straka on his technical advice.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b01727. Four figures, the experimental data for the biomass density over time corresponding to the four cases presented in the manuscript: using solely nitrate as the N source (Figures S1 and S2), using solely ammonium as the N source (Figure S3), and using combined ammonium and nitrate as the N source (Figure S4) (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1- 480-965-5847; fax: +1- 480-727-0889; e-mail: bin. [email protected] (B.T.N.). Author Contributions

B.T.N. contributed to conception and design of experiments; performing experiments; analysis and interpretation of the data; and writing of the article. B.E.R. contributed to conception of E

DOI: 10.1021/acs.est.5b01727 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.est.5b01727 Environ. Sci. Technol. XXXX, XXX, XXX−XXX