Achieving pH control in microalgal cultures through fed-batch addition of stoichiometrically-balanced growth media
© Scherholz and Curtis; licensee BioMed Central Ltd. 2013
Received: 13 August 2012
Accepted: 25 February 2013
Published: 7 May 2013
Lack of accounting for proton uptake and secretion has confounded interpretation of the stoichiometry of photosynthetic growth of algae. This is also problematic for achieving growth of microalgae to high cell concentrations which is necessary to improve productivity and the economic feasibility of commercial-scale chemical production systems. Since microalgae are capable of consuming both nitrate and ammonium, this represents an opportunity to balance culture pH based on a nitrogen feeding strategy that does not utilize gas-phase CO2 buffering. Stoichiometry suggests that approximately 36 weight%N-NH4+ (balance nitrogen as NO3-) would minimize the proton imbalance and permit high-density photoautotrophic growth as it does in higher plant tissue culture. However, algal media almost exclusively utilize nitrate, and ammonium is often viewed as ‘toxic’ to algae.
The microalgae Chlorella vulgaris and Chlamydomonas reinhardtii exclusively utilize ammonium when both ammonium and nitrate are provided during growth on excess CO2. The resulting proton imbalance from preferential ammonium utilization causes the pH to drop too low to sustain further growth when ammonium was only 9% of the total nitrogen (0.027 gN-NH4+/L). However, providing smaller amounts of ammonium sequentially in the presence of nitrate maintained the pH of a Chlorella vulgaris culture for improved growth on 0.3 gN/L to 5 gDW/L under 5% CO2 gas-phase supplementation. Bioreactor pH dynamics are shown to be predictable based on simple nitrogen assimilation as long as there is sufficient CO2 availability.
This work provides both a media formulation and a feeding strategy with a focus on nitrogen metabolism and regulation to support high-density algal culture without buffering. The instability in culture pH that is observed in microalgal cultures in the absence of buffers can be overcome through alternating utilization of ammonium and nitrate. Despite the highly regulated array of nitrogen transporters, providing a nitrogen source with a balanced degree of reduction minimizes pH fluctuations. Understanding and accommodating the behavior of nitrogen utilization in microalgae is key to avoiding ‘culture crash’ and reliance on gas phase CO2 buffering, which becomes both ineffective and cost-prohibitive for commercial-scale algal culture.
Algae-based biofuels have been gaining attention as a potential production platform for renewable fuel and biochemicals. Algal systems offer advantages over terrestrial plant sources, such as higher productivity, increased oils, avoidance of food-for-fuel, and the potential for using both wastewater and saltwater [1, 2]. However, an improved understanding of nutrient utilization in algal cultures is needed to develop the high density culturing methods that are required to achieve economic feasibility of these systems. Increasing reactor productivity through application of high-density cell concentration reduces downstream harvesting costs. The work in this article aims to achieve this goal by maintaining culture pH through the use of stoichiometrically- balanced growth media.
High density algal culture requires the development of media which provides high inorganic salt concentrations while avoiding the accumulation of inhibitory levels of counter-ions . Metabolic flux analysis has been used in an effort to characterize relative rates of consumption in cellular processes, but is only as effective as our limited understanding of algal metabolism . Another approach to formulate growth media is through an examination of the biomass composition . This method is effective for optimization of key inorganic components, but does not address the coupling of nitrogen utilization with the energy balance as reflected in the oxygen and hydrogen components of the biomass. A better understanding of the overall stoichiometry and energetics of growth including water splitting to provide reducing power is required to achieve the goal of ultra-high density algal cultivation.
Algebraic expression of stoichiometric coefficients for common nitrogen sources
The nature of ϕi for different nitrogen sources and its relation to energetics will be the focus of subsequent study. In this paper our objective is to design media to facilitate pH control (ϕi ≈ 0) using a combination of nitrogen sources with different degrees of reduction. The issue of pH control is implicitly appreciated in typical culture methods for algae. Ammonium is rarely used for growth because of its associated ‘toxicity’
While elevated CO2 provides for buffering of pH, this excess CO2 presents severe limitations to achieving high yield of CO2 use in a photosynthetic system. The unused CO2 that is not taken up by cells is ultimately released into the atmosphere. Providing elevated CO2 is expensive, unsafe, not sustainable, and difficult to implement in large-scale algal culture systems. To improve both the economic feasibility of commercial scale systems and reduce greenhouse gas emissions, it is desirable to maximize CO2 yield, which will require reducing the gaseous CO2 supplementation level. Therefore, typical pH control achieved through nitrate metabolism and elevated CO2 is not feasible. Buffers and acid/base addition are alternative pH control methods for bench-scale reactors, but result in accumulation of counter ions which can contribute to ‘culture crash’ when operating continuously at ultra-high density (unpublished observation). As a result, we have developed a new pH control strategy based on the stoichiometry of growth to allow for maintaining pH at the commercial-scale.
Defining a balanced NH4+/NO3-media
Excluding storage compounds such as lipids and carbohydrates, the composition of biomass is nearly constant across organisms. The stoichiometry of biomass growth has often relied on the detailed compositional analysis of E. coli, CH1.776 N0.165O0.495[20, 25], which corresponds to 9.6% nitrogen by mass (10.4 g biomass/gN). This biomass composition gives a 30% ammonium composition (Δ = 0.3) in our mixed nitrogen source media design (Equation 6). The biomass composition of Chlorella has been reported as CH1.73 N0.067O0.327 (4.7% N by mass) and for Chlamydomonas as CH1.82 N0.103O0.594 (5.8% N by mass) [26–28], which both give Δ = 29%. This value of ammonium-nitrogen is consistent with the composition of MS media  for plant tissue culture (ΔMS = 0.355), which have been arrived at empirically for heterotrophic growth without pH control ϕ MS ≈ 0.
For a fuel molecule, the carbon to oxygen ratio will be high such that r « z and nitrogen will not be present (q = 0). Since the degree of reduction of a fuel product is greater than that of the biomass, there will be an increased demand for a reduced nitrogen source (Δfuel > Δbiomass) with the added energetic advantage of feeding more ammonium relative to nitrate [30, 31]. Therefore, the nitrogen ratio that will achieve a proton balance will be dependent upon the level and composition of products formed. An ammonium level of 36% of the total nitrogen (36%N-NH4+) was chosen as the base media composition for pH-balanced algal growth. In the results below, the observation of differential nitrogen uptake will be shown to dramatically affect the dynamics of pH. Therefore, further refinement of the proton balance involves controlling the dynamics of nitrogen availability in addition to the stoichiometric composition. The work that follows demonstrates how fed-batch addition of our stoichiometric media can be used to overcome the pH instability that results from the inability of microalgal cultures to selectively consume either ammonium or nitrate.
Results and discussion
Proton imbalance in microalgal cultures is caused by preferential ammonium uptake
Towards achieving pH control using nitrogen feeding, proton secretion in conjunction with ammonium metabolism can be calculated from the observed pH drop and growth data. The change in proton concentration during ammonium uptake increased from 0.015 to 0.035 mol H+/mol N-NH4+ as the nitrogen provided from ammonium increased from 6% to 9% when buffering is neglected (inset of Figure 3B). This large change in apparent ϕ/ψ (Equation 3) is influenced by the buffering capacity of the media including the bicarbonate equilibrium (Equation 4). The buffering of the algal cultures is shown to change dramatically throughout a batch culture as illustrated in Additional file 3: Figure S1.
This substantial change in the buffering capacity of the media during growth precludes accurate assessments of proton secretion (ϕ) from pH measurements and will be deferred to future studies with instrumentation designed for monitoring the proton balance more accurately. Nonetheless, it is clear that the proton imbalance must be considered in the overall mass balance. The role of carbonate buffering at higher pH is also evident as the same final pH was achieved in all cultures. More CO2 can absorb into the culture at higher pH as a result of the CO2↔HCO3-↔CO32- ‘carbonate’ equilibrium. This represents an additional bioreactor design constraint because the bicarbonate buffering not only masks changes in the proton concentration due to nitrogen metabolism, but also alters pH as a function of bioreactor CO2 transport rates and biological uptake rates. It is important to note that proton efflux in stoichiometric terms is very different from the simplistic local charge balance of 1:1 molar exchange for a transporter. Proton exchange per mole of nitrogen assimilated (ϕ/ψ) reflects the incorporation of hydrogen into biomass and allows for net charge balance by alternative cations. Understanding the combined role of nitrogen stoichiometry and CO2 dynamics is an important step towards implementing media-based control of pH that is needed for a large-scale algal process that does not rely on buffering and is a prerequisite to accurately closing the mass balance on algal biomass growth.
Differential cellular regulation of nitrogen metabolism facilitates novel approach to pH control in microalgae
To test this behavior, a photoautotrophic culture of Chlamydomonas reinhardtii actively growing on nitrate was subjected to a pulse ammonium feed and displayed rapid pH drop (See Additional file 4: Figure S2). This suggested that there is sufficient expression of ammonium transporters during nitrate utilization so that the incremental addition of ammonium can be used for periodic reduction of pH . This approach will thereby provide a means to implement the proposed stoichiometrically-balanced nitrogen feed (36 % N-NH4+and 64 % N-NO3-).
Incremental addition of stoichiometrically-balanced media provides favorable pH for sustained growth of Chlorella vulgaris
During the lighted hours, the pH was maintained between 7.0 and 7.8 as shown in Figure 6A. Within this pH range, bicarbonate is the dominant dissolved inorganic carbon species, which is believed to be the preferred carbon source for photosynthesis in Chlorella vulgaris[14, 39]. After the first four ammonium nitrate additions, a pH drop was followed by a pH rise as ammonium was preferentially consumed followed by nitrate assimilation. It is interesting to note that the pH response following the last feeding (red arrow in Figure 6A) did not display the characteristic recovery of pH as nitrate levels reached 0.10 gNO3-/L (1.7 mM N), which would still be within the range of the low affinity transport system that operates above 1.1 mM N . This unexpected response to ammonium addition suggests that other cellular regulation might be encountered that affect the pH control strategy.
The rapid rise in pH that occurs during the dark culture hours resulted because the supplemental CO2 was turned off ‘at night’ and the inorganic carbon species shift back to equilibrium with ambient CO2 (0.039%). Note that the mean gas residence time within the enclosed trickle film reactor bag enclosure was estimated at 6-min so that it takes about half an hour to change the gas composition. This dark period increase in pH illustrates the significant effect of CO2 transport and the resulting bicarbonate buffering system on the culture pH that result from 5% CO2 gas-phase supplementation. A long-term goal of our research program is to achieve high-density growth without CO2 buffering, which will require an understanding of the relationship between nitrogen regulation, carbon availability, and pH dynamics.
The accumulation of algal biomass during the 32-hr photobioreactor run with fed-batch nitrogen addition is shown in Figure 6B. This trickle film bioreactor run reached 5 gDW/L, which was a substantially higher density than observed in prior batch experiments with comparable feeding (0.3 gN/L). A possible explanation for the improved biomass yield is that maintenance of a more uniform pH during growth allowed the cells to more effectively utilize the energy available in the reduced nitrogen source. During the 24 hours of lighting, the culture grew at a specific growth rate of 0.088/hr (doubling time = 7.88 hr). The observed exponential growth suggests that the culture had not become limited by light, CO2 transport or inorganic nutrients during this period. The change in proton secretion during ammonium uptake (ϕ/ψ) after an ammonium nitrate pulse was smaller at higher cell densities (Figure 6B inset), which is consistent with the previously observed increase in buffering capacity at higher culture density (Additional file 3: Figure S1).
pH Control based on nitrogen feed can be implemented during nitrogen-limited growth of Chlamydomonas reinhardtii
Under nitrogen-depleted conditions, the pH dropped, recovered and became constant as shown in more detail in Additional file 5: Figure S3. This suggests a nearly balanced proton secretion and uptake (ϕ/ψ) during the respective phases of ammonium and nitrate assimilation as illustrated by the Figure 7B inset. The observed change in pH with nearly identical magnitude but with opposite signs as ammonium and nitrate are consumed following NH4NO3 addition is consistent with charge balance of proton flux during nitrogen ion uptake. This simplistic view of pH change could greatly simplify pH control and might in part result from small rapid nutrient additions as well as buffering. However, it must be remembered that the CO2 and cell density-dependent buffering are significantly contributing to this pH response and will require more detailed study where buffering is minimized. Nonetheless, these short-term pH responses must be superimposed on the longer time-scale mass balance where the final redox state of nitrogen within the cell as well as the overall cation/anion uptake must be satisfied. Our near-term goals are to incorporate these models into an adaptive control strategy that will incorporate more comprehensive modeling of growth and pH dynamics, as it is dependent on the variable growth conditions that algae will experience in outdoor environmental conditions. Towards achieving this goal, a final experiment is presented for growth conditions under ambient (air) CO2 growth conditions.
Carbon limitations reveal additional regulatory mechanisms on nitrogen metabolism in Chlamydomonasthat lead to unpredictable pH dynamics
The pH was maintained between 6 and 8.5 during exponential growth (0–29 hours) with the expected pH decline and recovery following each NH4NO3 media addition that exhibited a more rapid response as the culture density increased (Figure 8B). The same regulatory mechanisms for nitrogen metabolism previously presented in Figures 5 and 6 are apparent in air-grown cultures as long as the carbon concentrating mechanism can maintain excess intracellular carbon. The inset of Figure 8B shows the decline in apparent pH change for growth on ammonium (ϕ/ψ) as anticipated, justifying the increase in NH4NO3 dosage at higher densities prior to carbon limitation. Following carbon limitation the pH dynamics became unpredictable and the pH ranged from 5 to 9. When aqueous carbon is limiting, the carbon concentration mechanism (CCM) can no longer provide sufficient CO2 to RuBisCO to support its maximum turnover rate. Feedback regulation from CCM to the nitrogen assimilation pathways acts to avoid intercellular accumulation of exogenously supplied or nitrate-derived ammonium and coordinate with 2-oxogluatarate availability for glutamate formation [33, 44]. This altered nitrogen assimilation under carbon limitation adds complexity to the pH control strategy because the presence of ammonium is no longer sufficient to completely inhibit nitrate uptake . A more detailed assessment of metabolic fluxes will clearly be required beyond simple on/off control that is possible when carbon is in excess. There is a tremendous value in further understanding carbon limited nitrogen assimilation due to its implications in both natural and engineered algal growth systems.
Regulation of nitrogen assimilation in algae has important implications to achieving stoichiometrically-balanced media and associated control of culture pH
Although the concept of achieving pH control through stoichiometrically-balanced ammonium and nitrate is shown to have validity, this cannot be implemented with a simple batch media formulation. The preferential uptake of ammonium ions over nitrate results in a pH drop proportional to the ammonium provided. Our observation of preferential uptake of ammonium for a wide range of algae and cyanobacteria is consistent with the literature . The implication of pH-independent preferential ammonium uptake is that algae will literally kill themselves by consuming ammonium ions even if nitrate is available to prevent media acidification. This result is in contrast to higher plants, which have the ability to utilize nitrate and ammonia in a balanced matter so that pH does not reach toxic levels. This is very notably observed in plant cell suspension culture where cultures can be grown to extremely high densities (> 50 gDW/L) on a mixture of ammonium and nitrate .
Physiologically it makes sense that cells within tissue must be capable of controlled metabolism of nitrogen so that the pH is maintained. In contrast, unicellular algae would not typically be grown at sufficiently high concentrations where metabolism would dramatically impact the surrounding water. In addition, ammonium ions are typically far less abundant in an environmental context (in part due to their favorable energetic utility). As a result, a high-density algal photobioreactor system creates an unnatural environment that microalgae have not evolved to accommodate and the metabolic control that does exist is problematic. The incremental addition of a fed-batch strategy for pH control is only a viable solution if the response to media addition is predictable. Current efforts are combining models of CO2 transport, CO2 equilibrium, and nitrogen assimilation as the basis of an adaptive control strategy for algal photobioreactors. Part of this experimentation seeks to explicitly evaluate the proton balance through measurement of ϕi (Equation 3) and allow for more explicit validation of the stoichiometrically-balanced media formulation approach presented in this research. A particularly important extension of this work is to confirm that pH control can be achieved for cyanobacteria since they are a platform for genetic engineering of biochemicals. Cyanobacteria have far more simplistic and less redundant NH4/NO3 assimilation pathway which may complicate the dynamic response to mixed nitrogen media additions; nitrogen-fixing cyanobacteria present an additional challenge that may preclude this pH strategy all-together.
The scope of this work relates to understanding algal nitrogen metabolism in the context of achieving pH control in high-density algal photobioreactors . Although a balance of protons during culture growth has received little attention, it has profoundly affected the development of algal media and growth conditions by selection of elevated CO2 and nitrate or urea, which avoid this problem. In terms of utilizing algal culture at a commercial outdoor scale of thousands of acres, pH control must be achieved for a widely dispersed culture that experiences highly variable day-to-day growth conditions. A balanced media to achieve pH control is a scalable alternative to expensive buffering, feedback acid/base control, or CO2 enrichment (which severely limits CO2 conversion efficiency). These observations are particularly important for proposed life cycle analyses that presume the ability to utilize wastewater in which ammonium is a dominant form of nitrogen. The work is also relevant to natural systems that experience agricultural runoff.
An analysis of basic photosynthetic reactions strongly suggests that current algal growth media are not stoichiometrically-balanced.
The benefit of elevated CO2 for algal growth is likely as much for pH control as it is for enhanced CO2 availability.
Media which contains ~36% of the total nitrogen in the form of ammonium ions is close to achieving a stoichiometric balance, which would avoid excess proton secretion or uptake.
If a stoichiometrically-balanced media is provided in batch culture, the preferential uptake of ammonium ions will result in a drop of pH to inhibitory/lethal levels.
Incremental addition of ammonium and nitrate ions can be used to control pH as long as the carbon availability is not severely limited and a substantial improvement in biomass yield on nitrogen can be observed.
The switch to preferential use of ammonium ions will take place in excess nitrate, or nitrogen-limited culture conditions.
Achieving pH control through metabolic use of oxidized and reduced nitrogen sources in large scale photobioreactors will require models of CO2 transport, CO2 equilibrium and nitrogen assimilation.
These observations of nitrogen assimilation in microalgae appear to be very general and make sense in terms of the physiology and environmental conditions under which these organisms typically grow; therefore, the approach to achieving pH control is anticipated to be true of both monocultures and natural algal consortia. The potential influence of microbial consortia within a non-aseptic algal culture system is an additional consideration that requires further study.
The algal strain Chlorella vulgaris was obtained from the UTEX culture collection (#2714) and algal strain Chlamydomonas reinhardtii cc-1690 was obtained from the Chlamydomonas Resource Center (http://www.Chlamy.org).
The following is the basal balanced WFAM-3g growth medium which contains sufficient nitrogen to support 3 grams dry weight per liter (gDW/L) based on 10% nitrogen by mass (0.3 gN/L at 36%N-NH4+ and 64%N-NO3-): 0.6 gKNO3, 0.61 gNH4NO3, 1-mL phosphates solution, 1-mL micronutrient stock solution, 0.024 gFe-EDTA · 2H2O, 0.121 gMgSO4 · 7H2O, 0.0486 gMgCl2, and 0.132 gCaCl2 · 2H2O in 1-L Milli-Q water. The phosphate solution contained 115 gK2HPO4 and 44.9 gKH2PO4 in 1-L Milli-Q water with the pH adjusted to 6.8. The micronutrient stock solution contained 1.83 gH3BO3, 0.54 gMnCl2 · 4H2O, 0.066 gZnSO4 · 7H2O, 0.031 gNa2MoO4 · 2H2O, 0.030 gCoCl2 · 6H2O, and 0.0075 gCuSO4 · 5H2O in 1-L Milli-Q water.
Optical density (OD) was measured using cuvettes with 1-cm path length in a Beckman Coutler DU 520 spectrophotometer at a wavelength of 550-nm (OD550) to avoid pigment absorption and maximize light-scattering contribution, referenced with tap water . To measure the dry weight (DW), 1-mL of well-mixed culture was added to a pre-tared 1.7-mL Eppendorf tube measured using an analytical balance to 0.00001 accuracy. The cells were pelleted in a microfuge (14,000 RPM, 10-min). The supernatant was removed without disturbing the pellet and the cells were rinsed. The Eppendorf tubes were stored in a −20°C freezer and transferred to a −80°C freezer for at least 30-min with lids open immediately before freeze drying. Samples were dried in a Labconco freeze dryer (−70C coil) run for 24–36 hours depending on the number of samples. Samples were re-measured to 0.00001 using an analytical balance to determine the final weight of the tube and cell pellet.
Online pH was measured using Cole-Parmer pH electrodes with double-junction BNC connectors interfaced to a LI-COR LI-1400 datalogger. Offline pH samples were measured using a Metler Toledo SevenEasy pH Meter S20. Samples were degassed on a gyratory shaker for 45-min to ensure dissolved inorganic carbon was in equilibrium with air to minimize the variability in offline pH readings due to partial degassing of samples during transport between reactor and pH meter.
Nitrate measurement by Ion selective electrode (ISE)
The Nico2000 Nitrate ISE (ELIT 8021) and liquid double junction reference electrode (ELIT 003) were used for offline measurement of exogenous nitrate. The ISE and reference electrode were pre-conditioned in 10 g NO3-/L standard for at least 30-min. The ion selective electrode was calibrated using three independently prepared NaNO3 standards at 10 gNO3-/L each serially diluted to 0.01 g/L. The ISE and reference electrode were left in the experimental samples until the electrical potential remained constant for 1-min. Due to the drift in electrical potential that occurred with extended use, the calibration standards and samples were measured the same day. Nitrogen-free media was used to determine the background contribution from interfering ions to adjust the baseline concentration down to 0 gNO3-/L. Fresh media was used as the positive control for each treatment.
Light cycle and temperature
All batch and fed-batch experiments were executed in a Conviron BDW120 walk-in incubator. High intensity lighting was supplied to cultures using Philips 400 W high-pressure sodium vapor and Philips 400 W metal-halide lamps. These lights were set to diurnal cycle on an 8-hr dark/16-hr light cycle to imitate sunlight. In the first and last hour of the photoperiod, the light intensity was stepped to 1/3 of the maximum; combined with lamp warm-up, this avoided morning photo-inhibition. The temperature within the incubator was maintained at 28°C during the day and dropped to 25°C during the dark hours – ramped linearly over a 1 hour period.
Preparation of reactor inocula
Cultures were grown in media with 0 or 4.5% nitrogen as ammonium at 0.3 gN/L and all non-nitrogen components consistent with the stoichiometric growth media under 5% CO2 (v/v) supplementation in shake flasks. Cells were harvested by centrifugation (2000-g for 5-min at 24°C ), washed in nitrogen-free media to remove extracellular nitrogen, centrifuged a second time, and re-suspended in nitrogen-free media.
Loop air-lift photobioreactor
The air-lift bioreactors with working volume of 1.5-L were constructed from translucent polyethylene plastic tubing using a W-605A 24-inch Single Impulse heat-sealer with 5 mm seal (Recycle = 1, Congealing = 4, Sealing = 4) to form the bag configuration. Details of the reactor dimensions and pictures are described elsewhere (Tuerk, 2011), which is available online . A ceramic sparger attached to plastic tubing was inserted into the reactor through a hole near the top of the bag to form the riser in the narrow side. A hole cut above the liquid level served as the inoculation and sample port. A Cole-Parmer pH electrode was inserted into the bag reactor in the airlift ‘downcomer’. The bag reactor was placed between two metal wire racks without blocking light to limit the thickness to approximately 0.75-in. Gas was sparged into the reactor at 0.31 VVM at 5% CO2 (v/v) in air. The average light flux to the culture was 252 μmol/m2/s over a total surface area of 0.11 m2, and was determined by holding a LI-COR PI-190 quantum (PAR) light sensor normal to the bag surface.
Trickle film photobioreactor
The trickle film photobioreactor with working volume of 500-mL was the basis of extensive algal photobioreactor development studies described elsewhere . The specific configuration used in the work reported here consisted of two screens (fiberglass window screen stock) enclosed in a clear plastic bag (2 mil 30-in. × 34-in.) filled with humidified gas at 5% CO2 (v/v) in air. A 1-L glass reservoir was sealed with a silicone stopper that was fitted with a gas port, the liquid return, sample port, and temperature probe. The culture was collected in this reservoir after flowing down the screen and pumped out the bottom through a sidearm using a Watson-Marlow peristaltic pump 601S at 0.5 to 1-L/min. The culture temperature was maintained at 25°C through a heat exchanger in the culture recycle loop. The average measured light flux to the screen was 282 μmol/m2/s with a screen area of 0.3 m2, and was measured by holding the light sensor normal to the surface of the screen within the bag enclosure.
The authors would like to acknowledge Waqas Khatri for execution of the first batch growth/pH experiment with Chlorella vulgaris; Amalie Tuerk for assistance with the design, development and operational assistance with the air-lift photobioreactor runs as well as online monitoring techniques; Lisa Grady, Waqas Khatri, Amalie Tuerk and Robert Hendrix for design improvements to the trickle-bed and general bioreactor assistance and Jackie Guo and John Michael for assistance with data acquisition. Work in this manuscript was supported by the NSF Collaborative Grant No. CBET-0828648 titled “Development of a Sustainable Production Platform for Renewable Petroleum Based Oils in Algae” including a research experience for undergraduate (REU) supplement; NSF Grant No. DBI-0215923 supported installation of the high-intensity lighted growth chamber. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.
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