Rationally leveraging mixotrophic growth of microalgae in different photobioreactor configurations for reducing the carbon footprint of an algal biorefinery: a techno-economic perspective

Abstract

Mixotrophy, the ability of an organism to simultaneously assimilate carbon dioxide (CO₂) and organic carbon, is of prime importance to algal biorefineries because it boosts algal biomass productivities, associated-CO₂ capture rates and intercellular lipid content; along with wastewater remediation. In the present study, the effect of light, CO₂ and glucose, in various permutations, on the growth of Chlorella vulgaris, cultivated in flat panel and bubble column photobioreactors, was investigated. The average specific growth rate of Chlorella vulgaris in light-sufficient conditions when supplied with (−glucose, −CO₂); (−glucose, +CO₂); (+glucose, −CO₂) and (+glucose, +CO₂) were 0.048 h⁻¹, 0.075 h⁻¹, 0.080 h⁻¹ and 0.084 h⁻¹, respectively in flat panel reactors. The corresponding specific growth rates in bubble column reactors were 0.042 h⁻¹, 0.070 h⁻¹, 0.082 h⁻¹ and 0.083 h⁻¹. Chlorella vulgaris was found to require light for assimilation of glucose because it did not grow in the dark under glucose-sufficient conditions. Furthermore, carbon balance revealed that the mixotrophic cultivation of Chlorella vulgaris had a positive carbon footprint (pseudo-mixotroph). Literature review revealed that the carbon footprint of mixotrophic algal cultivation may be negative (true-mixotroph) or positive depending upon the predominant mode of nutrition and the cultivation conditions. The novelty of the present study lies in the formulation of a strategy to identify true-and-pseudo-mixotrophs and their subsequent utilization for design of low-cost, minimal-resource and high-throughput cultivation strategies for biomass generation and CO₂ sequestration in algal biorefineries.

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Introduction

Photosynthetic microalgae have recently gained prominence as a global-and-green solutions for food, feed, biofuel and carbon dioxide (CO₂) capture, in addition to high-value nutraceutical and therapeutic applications.¹ The chief bottleneck in the advancement of algal technologies is the slow reaction-kinetics of the Rubisco enzyme that is responsible for CO₂ fixation, which in-turn, leads to low biomass and product generation rates.² A mixotrophic mode of algal metabolism (simultaneous utilization of organic and inorganic carbon sources) was considered to be a possible solution for the aforementioned problems.^3–10 Heterotrophy was expected to initiate rapid cell growth while the algae would sequester CO₂ using photoautotrophy. Thus, the research in mixotrophic cultivation of algae gained momentum due to its application in simultaneous waste-water remediation with CO₂ capture.^1,6,11

The carbon skeleton of algae, cultivated via mixotrophy, is acquired from both organic and inorganic sources. A critical lacuna in literature is that mixotrophic growth of algae is assumed to occur upon supplying the algae with organic carbon source in presence of light. While the assumption held valid in some cases,^3–6 elementary carbon balance (assuming algae has 50% carbon by weight¹²) revealed that many cultivation processes which claimed to be mixotrophic were in fact, emitting CO₂.^7–10 The lack of CO₂ capture in the “assumed” mixotrophic processes illustrates the need for formulation of a strategy to identify CO₂-capturing mixotrophic algae and their CO₂ capture potential.

Therefore, in the present study, the individual amount of carbon stored in Chlorella vulgaris from glucose (organic) and CO₂ (inorganic) was calculated. Furthermore, the effect of cellular reaction dynamics, during photoautotrophy, heterotrophy and mixotrophy, on the elemental composition of algae was investigated. The study was made independent of photobioreactor geometry by cultivating Chlorella vulgaris in flat panel reactors (FPR) and bubble column reactors (BCR). Finally, a possible strategy to exploit the innate CO₂ capture potential of Chlorella vulgaris during mixotrophic cultivation, for use in an algal biorefinery, was explored.

Materials and methods

Algal species and inoculums development

Chlorella vulgaris was gifted by Dr Tapan Chakrabarti, National Environmental Engineering Research Institute, Nagpur, India.¹³ 3N-Bold's basal medium¹⁴ was used for the inoculum development and experiments. The algal culture stock was repeatedly sub-cultured in 150 mL Erlenmeyer's flasks (Borosil®) in an orbital shaker (Labtech, Korea) at 40 μmol photons m⁻² s⁻¹, continuous illumination, 25 °C, and 120 rpm. Inoculums (5% v/v) were developed in 0.5 L bubble column reactors, maintained at 30 ± 2 °C, until the algal density was 1 ± 0.1 g L⁻¹ (mid-logarithmic growth phase). The initial pH of the inoculum and experimental growth media were set at 7 ± 0.1. Analytical grade chemicals, which were procured from Merck©, India, were used in the present study.

Photobioreactor design

The design parameters of the 2 L-FPR (Fig. 1a) and the 0.5 L-BCR (Fig. 1b), used in the present study, are elaborated in Subramanian et al.¹³ and Yadav et al.,¹⁵ respectively.

Fig. 1 Schematic of (a) flat panel reactor and (b) bubble column reactor. All dimensions are excluding wall thickness.

Experimental design

The green microalga, Chlorella vulgaris was cultivated under different experimental conditions where light, CO₂ and glucose were supplied in various combinations. The details about the various combinations are presented in Table 1. Samples (10 mL for FPR, 5 mL for BCR) were collected at 8 h intervals for all the experiments. The experiments were run until 90% of sodium nitrate was consumed. All experiments were performed in triplicates.

Table 1 Experimental design for cultivation of Chlorella vulgaris

Experimental parameters	Expt. 1	Expt. 2	Expt. 3	Expt. 4	Expt. 5	Expt. 6	Expt. 7	Expt. 8
a Supply of experimental parameter is indicated with ‘+’ and absence of the same is indicated by ‘−’.
Light	+^a	+	+	+	−	−	−	−
CO2	−	+	−	+	−	+	−	+
Glucose	−	−	+	+	−	−	+	+

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Photobioreactor operations

The FPR were sterilized using 2% sodium hypochlorite solution¹³ whereas BCR were sterilized by autoclaving at 121 °C for 15 min.¹⁵ The experimental setup is elaborated in Fig. 2. The aeration from the air compressor (Seeboy, power = 3 W) and CO₂ supply (from CO₂ cylinder) were measured using pre-calibrated air and CO₂ rotameters (Indus), respectively. The air–CO₂ mixtures were filter-sterilized using 0.22 μm membrane (Whatman). The initial media pH was 7.1 ± 0.1 in all the experiments.

Fig. 2 Experimental set-up to study mixotrophy in Chlorella vulgaris.

As per the experimental requirements (Table 1), FPR were continuously illuminated from both sides (incident light intensity = 200 μmol photons m⁻² s⁻¹) via cool white fluorescent tube lights (Philips, 36 W). The light intensity was measured using a luxmeter (HTC LX-102). Similarly, 2.5 g L⁻¹ of glucose and 3% CO₂ (v/v) were supplied as per the experimental requirements for glucose or CO₂ supplementation. Photobioreactor temperature was maintained between 33 ± 1 °C by air conditioning. Aeration rate was fixed at 0.375 vvm.¹³ The working volume during the experiments was 2 L.

Similarly, light intensity of 200 μmol photons m⁻² s⁻¹ was supplied continuously on single side of BCR. The glucose and CO₂ concentrations in air supply along with photobioreactor temperature were same as used in FPR experiments. Aeration rates for BCR was fixed at 0.5 vvm.¹⁵ The working volume of BCR experiments were 0.5 L.

Oxygen evolution during glucose-and-light sufficient conditions was verified by cultivating Chlorella vulgaris using 3N-Bold's basal medium (supplemented with 2.5 g L⁻¹ of glucose) in nitrogen-sparged and airtight Erlenmeyer's flasks. The experimental conditions included continuous illumination of 40 μmol photons m⁻² s⁻¹, culture temperature of 25 °C, and agitation of 120 rpm in the orbital shaker.

The utility of Chlorella vulgaris for mixotrophy-based algal biorefinery was demonstrated in FPR using 3N-Bold's basal medium supplemented with 0.25 g L⁻¹ of glucose. External CO₂ supplementation (3% v/v) was provided after 16 h since the commencement of cultivation. Other FPR operating conditions were maintained as mentioned previously.

Measurements and calculations

The parameters measured and analyzed during the experiments included:

Algal biomass densities. Algal biomass concentration (X) was estimated gravimetrically and expressed in g L⁻¹. The optical density (OD) of the biomass sample was also measured 750 nm using a UV-Visible spectrophotometer (Chemito).¹⁶ The calibration curve between OD and X is given by eqn (1).

OD₇₅₀ = 2.26 × X, R² = 0.9815 (1)

Glucose and sodium nitrate concentrations. Sodium nitrate and glucose concentrations were estimated using colorimetric method¹⁷ and dinitrosalicylic acid assay,¹⁸ respectively. The initial and final glucose concentrations are depicted by G_i (g L⁻¹) and G_f (g L⁻¹), respectively.

Average specific growth rate. The average specific growth rate (μ_avg) of Chlorella vulgaris was determined using the formula¹⁹where, X_i and X_f are the initial and final biomass densities at time t_i (h) and t_f (h), respectively. t_i = 0.

Elemental analysis and ash analysis of algae. The percentage carbon content (C) of Chlorella vulgaris (ash-free basis) was measured using a Vario MACRO Cube elemental analyzer (ElementarAnalysensysteme GmbH, Germany). The percentage ash content (A) of algae was determined using the protocol given in ASTM D3172-89.

Determination of photoautotrophy and heterotrophy. The amount of carbon stored in algal biomass (C_total, units: g L⁻¹), the amount of carbon present in consumed glucose (C_hetero, units: g L⁻¹) and the amount of carbon that was obtained by the microalga from CO₂ (C_auto, units: g L⁻¹) were calculated using eqn (3)–(5).where, n_c = number of carbon atoms in one molecule of g, MW_c = atomic weight of carbon (g gmol⁻¹) and MW_OC = molecular weight of organic carbon source (g gmol⁻¹). The n_c, MW_c and MW_OC for glucose are 6, 12 g gmol⁻¹ and 180 g gmol⁻¹, respectively.

C_auto = C_total − C_hetero (5)

Results and discussion

Mixotrophic growth of algae is presumed to occur when the microorganisms are exposed to light and organic carbon. Furthermore, it is also assumed that, algal growth rates during mixotrophic cultivation are greater than those during heterotrophic cultivation because of CO₂ fixation. Finally, mixotrophy is believed to enhance the CO₂ sequestration rate of algae as compared to photoautotrophic cultivation of the same.^1,3–11 The aim of the present study is to rigorously test the aforementioned suppositions and test the utility of Chlorella vulgaris for CO₂ capture in an algal biorefinery concept. In order to ensure exponential growth of algae in the experiments; the incident light intensity, CO₂ concentration in air supply, initial glucose concentrations, photobioreactor temperature, media pH and initial sodium nitrate concentrations were standardized in initial experiments (data not shown).

Algal growth in photoautotrophic, heterotrophic and mixotrophic growth regimes

Chlorella vulgaris underwent cell division only in the presence of light (in FPR and BCR) and showed negligible growth in dark, even under glucose or CO₂ supplementation. The probable reason for the inability of Chlorella vulgaris to utilize glucose in dark could be the light-dependency of glucose uptake and metabolism in algae.²⁰ The growth of Chlorella vulgaris in FPR and BCR, under light-sufficient conditions, are elaborated in Fig. 3a and b, respectively. Chlorella vulgaris grew best in FPR upon glucose-and-CO₂ supplementation (μ_avg = 0.084 h⁻¹), followed by growth in presence of glucose but not CO₂ (μ_avg = 0.080 h⁻¹). The photoautotrophic growth of Chlorella vulgaris (glucose absent in growth media) was more pronounced in presence of CO₂ (μ_avg = 0.075 h⁻¹) than its absence (μ_avg = 0.048 h⁻¹). Similarly, the μ_avg of Chlorella vulgaris in BCR under the growth conditions of (−glucose, −CO₂); (−glucose, +CO₂); (+glucose, −CO₂) and (+glucose, +CO₂) were 0.042 h⁻¹, 0.067 h⁻¹, 0.082 h⁻¹ and 0.083 h⁻¹, respectively. The experimental results of the microalga, cultivated in FPR and BCR, are provided in Tables 2 and 3, respectively.

Fig. 3 Growth of Chlorella vulgaris when cultivated in illuminated (a) flat panel reactors (b) bubble column reactors. Experimental conditions are () = (−glucose, −CO₂); () = (−glucose, +CO₂); () = (+glucose, −CO₂) and () = (+glucose, +CO₂).

Table 2 Cultivation of Chlorella vulgaris in flat panel reactors under light-sufficient conditions

Experimental conditions	Xi (g L^−1)	Xf (g L^−1)	tf (h)	Residual nitrate (g L^−1)	Residual glucose (g L^−1)	μavg^a (h^−1)	A (%)	C (%)
a All standard deviations are <0.001 h^−1.
−CO2, −glucose	0.045 ± 0.002	1.41 ± 0.091	72	0.067 ± 0.023	NA	0.048	3.10 ± 0.1	49.83
+CO2, −glucose	0.054 ± 0.001	1.44 ± 0.102	44	0.068 ± 0.037	NA	0.075	2.80 ± 0.14	50.21
−CO2, +glucose	0.053 ± 0.002	1.53 ± 0.070	42	0.042 ± 0.014	0.022 ± 0.003	0.080	3.15 ± 0.03	50.36
+CO2, +glucose	0.051 ± 0.002	1.48 ± 0.032	40	0.046 ± 0.016	0.024 ± 0.007	0.084	2.96 ± 0.02	49.73

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Table 3 Cultivation of Chlorella vulgaris in bubble column reactors under light-sufficient conditions

Experimental conditions	Xi (g L^−1)	Xf (g L^−1)	tf (h)	Residual nitrate (g L^−1)	Residual glucose (g L^−1)	μavg^a (h^−1)	A (%)	C (%)
a All standard deviations are <0.001 h^−1.
−CO2, −glucose	0.052 ± 0.003	1.00 ± 0.042	72	0.246 ± 0.053	NA	0.042	3.23 ± 0.13	50.57
+CO2, −glucose	0.053 ± 0.001	1.511 ± 0.089	48	0.048 ± 0.037	NA	0.070	2.92 ± 0.11	50.96
−CO2, +glucose	0.053 ± 0.003	1.38 ± 0.025	40	0.063 ± 0.016	0.178 ± 0.013	0.082	2.87 ± 0.01	50.42
+CO2, +glucose	0.052 ± 0.002	1.46 ± 0.030	40	0.033 ± 0.016	0.094 ± 0.012	0.083	3.19 ± 0.08	51.70

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Chlorella vulgaris was exposed to larger amount of light in FPR than BCR because the illuminated surface area to volume ratio of FPR (0.9 cm⁻¹) was larger than that of BCR (0.444 cm⁻¹). This resulted in the photoautotrophic growth of Chlorella vulgaris to be faster in FPR than BCR. On the other hand, the growth of algae under glucose-and-light-sufficient conditions was identical in both the photobioreactors because glucose was uniformly distributed in the photobioreactors by aeration-induced mixing. This indicates that heterotrophy is the dominant mode of nutrition during cultivation of Chlorella vulgaris under glucose-and-light-sufficient conditions and the growth of Chlorella vulgaris could either be mixotrophic or diauxic (heterotrophic in presence of glucose and photoautotrophic in its absence). The lack of improvement in μ_avg upon CO₂ supplementation during glucose-and-light-sufficient conditions indicated that the growth regime of Chlorella vulgaris might be diauxic.

In order to resolve the ambiguity; Chlorella vulgaris was cultivated using glucose-supplemented 3N-Bold's basal medium in nitrogen-sparged and airtight Erlenmeyer's flasks. The growth of the microalga (X_f = 0.82 ± 0.031 g L⁻¹) in the oxygen-deficient environment, along with consumption of glucose (G_f = 1.13 ± 0.019 g L⁻¹), ensured that the growth is mixotrophic since the oxygen required for glucose metabolism could only be obtained via photosynthesis. Similar results were observed using Micractinium inermum⁷ where the researchers observed that the amount of oxygen produced by photosynthesis was sufficient to fulfill the oxygen requirement for respiration. Thus, the mixotrophic growth regime of Chlorella vulgaris, under glucose-and-light-sufficient conditions, was validated.

Effect of growth conditions on elemental composition of algae

The ash and carbon content of algae in all the experiments (Tables 2 and 3) were relatively constant at approximately 3% and 50% (ash-free basis), respectively. Thus, the experimental conditions and carbon sources did not affect the fundamental elemental composition of Chlorella vulgaris. A possible reason for the observation could be that lipid content of the microalga was observed to be nearly constant in all experimental conditions (data not shown). Subsequently, the mixotrophic cultivation of Chlorella vulgaris captured lesser amount of CO₂ than photoautotrophic cultivation (Table 4). As explained in the successive sections, the assessment of elemental composition of algae is vital to study the CO₂ capture potential of algae and development of the corresponding cultivation strategy.

Table 4 Carbon footprint of Chlorella vulgaris in light-sufficient conditions

Photobioreactor	Experimental conditions	Ctotal (g L^−1)	Chetero (g L^−1)	Cauto (g L^−1)
Flat panel reactor	−CO2, −glucose	0.659	NA	0.659
	+CO2, −glucose	0.676	NA	0.676
	−CO2, +glucose	0.720	0.991	−0.271
	+CO2, +glucose	0.690	0.990	−0.300
Bubble column reactor	−CO2, −glucose	0.466	NA	0.466
	+CO2, −glucose	0.721	NA	0.721
	−CO2, +glucose	0.650	0.929	−0.279
	+CO2, +glucose	0.707	0.963	−0.255

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Carbon footprint of photoautotrophic, heterotrophic and mixotrophic growth regimes

Chlorella vulgaris captured CO₂ (C_auto > 0) under autotrophic conditions because the lack of organic carbon source ensured C_hetero = 0 (Table 4). The amount of CO₂ fixed by the microalga remained constant in FPR and increased in BCR, as the CO₂ supply in the inlet air was increased. Chlorella vulgaris in the FPR suffers from light limitation only at X_f ≥ 2.1 g L⁻¹.¹³ Hence, the final biomass densities of the microalga in the FPR did not change in the presence or absence of CO₂ supplementation. On the other hand, Chlorella vulgaris cultivated in the BCR experienced light limitation at low CO₂ concentrations because the average light penetration distance in the BCR was 3.53 cm (π × radius/2) against 1 cm (width/2) in FPR. The light limitation was offset by enhancing the reaction kinetics of the Rubisco enzyme at higher CO₂ supply rates. The C_auto values under autotrophic conditions (Table 4), in both the photobioreactors, reflect the changes to algal growth due to light limitation.

Upon comparing the values of C_total and C_hetero, under glucose-and-light-sufficient conditions in FPR and BCR, it was discovered that Chlorella vulgaris had released CO₂ instead of assimilating it (C_auto < 0, Table 4). Thus, the CO₂ capture potential of an algal cultivation strategy can only be evaluated using carbon balance (using C_auto, C_hetero and C_total) and the assumption that all mixotrophic growth regimes enhance algal-based CO₂ sequestration is disproved. For the purpose of the present study, we define mixotrophic organisms with positive carbon footprint as pseudo-mixotrophs while those with negative carbon footprints are true-mixotrophs.

Comparative analysis with literature

The authors reviewed literature (which claimed mixotrophic algal cultivation) to identify true-and-pseudo-mixotrophs among algal species. The analysis (Table 5) assumed that the ash and carbon content of algae to be 0% and 50%, respectively, because the values were not available in literature. The assumptions ensured that the CO₂ capture potential of the organism, thus determined, would be the theoretical maximum value. Chlorella sorokiniana (PCH02),³ Scenedesmus obliquus,⁴ Chlorella sp. KR-1 (ref. 5) and mixed microalgae consortia⁶ were true-mixotrophs since they consumed organic carbon source along with CO₂ and the cultivation strategy has a negative carbon footprint (C_auto = 0.135–0.915 g L⁻¹). On the other hand, Micractinium inermum⁷ was a pseudo-mixotroph because the cultivation process had a positive carbon footprint (C_auto = −0.397 g L⁻¹) and the species was observed to produce oxygen along with glucose consumption. The growth shown by species such as Chlorella sp. (PCH 10),⁸ Chlorella sp. Y8-1,⁹ Nannochloropsis sp.¹⁰ and Chlorella sp.¹⁰ could either be pseudo-mixotrophic or diauxic in nature because the oxygen evolution under glucose-and-light-sufficient conditions was not studied. In either case, the cultivation would have a positive carbon footprint as noted in Table 5.

Table 5 Identification of true-mixotrophy and pseudo-mixotrophy using literature data

Sl. no.	Microorganism and organic carbon source	Gi, Gf (g L^−1, g L^−1)	Xi, Xf (g L^−1, g L^−1)	Ctotal^a (g L^−1)	Chetero (g L^−1)	Cauto (g L^−1)	Nature of organism
a Assuming 0% ash and 50% C by wt.b Xi is assumed to be 0.05 g L^−1 because data is not given.
1	Chlorella sorokiniana (PCH02),^3 glycerol	(2.3, 1.035)	(0.05, 2.46)	1.205	0.495	0.710	True-mixotroph
2	Scenedesmus obliquus,^4 D-lactose monohydrate	(5, 2.28)	(0.05, 3.6)	1.775	1.088	0.687	True-mixotroph
3	Chlorella sp. KR-1,^5 glucose	(3, 0.5)	(0.05^b, 2.32)	1.135	1.000	0.135	True-mixotroph
4	Mixed microalgae culture,^6 glucose	(0.5, 0.05)	(1.25, 3.44)	1.095	0.180	0.915	True-mixotroph
5	Micractinium inermum,^7 glucose	(1.2, 0)	(0.003, 0.086)	0.542	0.939	−0.397	Pseudo-mixotroph
6	Chlorella sp. (PCH 10),^8 glycerol	(2.3, 0.161)	(0.006, 0.558)	0.276	0.837	−0.561	Pseudo-mixotroph or diauxic growth
7	Chlorella sp. Y8-1,^9 glucose	(1, 0)	(0.15, 0.60)	0.225	0.400	−0.175	Pseudo-mixotroph or diauxic growth
8	Nannochloropsis sp.,^10 glucose	(2, 0)	(0.05^b, 1.2)	0.575	0.800	−0.225	Pseudo-mixotroph or diauxic growth
9	Chlorella sp.,^10 glucose	(2, 0)	(0.05^b, 1.45)	0.700	0.800	−0.100	Pseudo-mixotroph or diauxic growth

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It is important to differentiate pseudo-mixotrophic or diauxic nature of algae as because CO₂ supplementation in presence of light may boost algal growth rates of pseudo-mixotrophic algae, but not diauxic algae. Chlorella sp. Y8-1 (ref. 9) was supplied with 10% CO₂ in the air supply, although its mode of nutrition under glucose-and-light-sufficient conditions was unknown. Thus, there is a possibility that CO₂ was wasted during its cultivation.

The diauxic growth regime of algae is not considered in literature due to the observable difference in the μ_avg of algae cultivated in under heterotrophic and mixotrophic conditions. As seen in Table 6, the μ_avg of Chlorella sp. Y8-1,⁹ Nannochloropsis sp.¹⁰ and Chlorella sp.¹⁰ which were grown in (light + glucose) increased significantly by 60%, 58.33% and 25%, respectively, when compared against the μ_avg in dark heterotrophy. This increase in specific growth rate was generally attributed to CO₂ fixation during photosynthesis. However, another possible reason for the behavior could be a light-inducible hexose transport system in the organism.²¹ For example, the Chlorella vulgaris used in the present study, could only uptake and/or assimilate glucose in presence of light and did not fix CO₂ under mixotrophic conditions. Also, Chlorella sp. KR-1 (ref. 5) (true-mixotroph) was observed to consume 1.5 g L⁻¹ of glucose during the 18 h light cycle and 1 g L⁻¹ of glucose during the 6 h dark cycle. Thus, the findings of this study and literature are in clear contradiction with the assumption that, the boosts to mixotrophic growth rates of algae (when compared against heterotrophic growth rates) are solely due to CO₂ fixation.

Table 6 Comparative analysis of average algal specific growth rate using literature data for mixotrophic and heterotrophic (dark) conditions

Sl. no.	Microorganism and organic carbon source	μavg heterotrophy (h^−1)	μavg mixotrophy (h^−1)
1	Chlorella sp. Y8-1,^9 glucose	0.008	0.005
2	Nannochloropsis sp.,^10 glucose	0.019	0.012
3	Chlorella sp.,^10 glucose	0.020	0.016

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Utility of mixotrophy in process design of algal biorefineries

The design of an algal biorefinery requires maximum algal biomass production rates, minimum resource wastage and a negative carbon footprint. Chlorella vulgaris cultivated under glucose-sufficient and glucose-deficient conditions (in presence of light and 3% v/v CO₂ supplementation) showed similar growth at low algal biomass concentrations (X ≤ 0.2 g L⁻¹), as seen in Fig. 3a and b. In order to satisfy the objectives of the algal biorefinery concept, Chlorella vulgaris was cultivated in FPR with an initial supplementation of 0.25 g L⁻¹ glucose. External CO₂ supplementation (3% v/v) was only provided after glucose was completely consumed (at 16 h). The final biomass obtained was 1.48 ± 0.038 g L⁻¹ at t_f = 42 h. Thus, the revised strategy (mixotrophic growth phase followed by photoautotrophic growth phase) had a negative carbon footprint (C_auto = 0.593 g L⁻¹).

The revised strategy had similar biomass production rates, conserved 16 h of CO₂ supply and had increased its CO₂ capture potential by 0.893 g L⁻¹ when compared against pseudo-mixotrophic cultivation with CO₂ supplementation. This 300% increase in CO₂ capture occurred due to the small duration of mixotrophic phase at low biomass densities (16 h, 0.05 ≤ X ≤ 0.19 g L⁻¹) as compared to the duration of photoautotrophic phase at high algal biomass densities (26 h, 0.19 ≤ X ≤ 1.48 g L⁻¹). The CO₂ capture potential of the revised strategy reduced by 14% when compared against photoautotrophic cultivation with CO₂ supplementation. However, the loss was compensated by 19% increase in μ_avg and conservation of 22 h (16 h at start and 6 h at end) of CO₂ supply. Thus, the utility of pseudo-mixotrophic cultivation of Chlorella vulgaris for designing algal biorefinery processes was demonstrated. The revised strategy was also observed in literature during the pseudo-mixotrophic cultivation of Chlorella sp. Y8-1.⁹ The microalga consumed 0.92 g L⁻¹ of glucose to produce 0.126 g L⁻¹ of biomass, in light-sufficient conditions, thus releasing 1.12 g L⁻¹ of CO₂ in the process. The amount of CO₂ released reduced to 0.642 g L⁻¹, as the microalga switched to photoautotrophic mode towards the end of the cultivation period. The failure of the revised strategy for Chlorella sp. Y8-1 (ref. 9) can be attributed to carbon balance not being studied in the literature, along with insufficient nitrogen supply during the photoautotrophic cultivation phase.

Furthermore, pseudo-mixotrophs are known to reduce the lag time of algal cultivation¹⁰ and increasing biomass density via dark heterotrophy may reduce the photooxidative damage suffered by low concentrations of algae at high light intensities.²² Increasing algal biomass density via dark heterotrophy might have the additional advantage of boosting algal biomass productivity (and associated CO₂ capture) by increasing the duration the photoautotrophic phase per light cycle. Chlorella vulgaris could only reduce lag time of growth in the light cycle but not increase initial biomass density in dark. It is also evident that diauxic growth of algae would function like pseudo-mixotrophic growth of Chlorella vulgaris in the present study.

Despite the advantages of pseudo-mixotrophy, true-mixotrophs are most beneficial to algal biorefineries as they boost algal biomass productivities along with CO₂ capture. The final cell density of the mixed microalgae culture⁶ nearly doubled during mixotrophic growth as compared to photoautotrophic growth (which had reached stationary phase at lower cell density). Thus, true-mixotrophs may increase the photosynthetic and CO₂ capture efficiencies of the biorefinery when supplemented by organic carbon source. The advantages of reduction in lag time of cultivation and increasing initial biomass density via dark heterotrophy are also applicable for true-mixotrophs.

Conclusions

The rise in global warming and wastewater generation is the principal motivation for the development of cost-attractive and sustainable mixotrophy-based algal biorefinery technologies. The novelty of the study lies in the systematic determination of algal mixotrophy via simultaneous oxygen evolution, organic carbon consumption carbon mass balance. Chlorella vulgaris was thus categorized as a pseudo-mixotroph and its negative carbon footprint was offset by cultivating the microalga under mixotrophic conditions (at low biomass densities) followed by a photoautotrophic growth phase (at high biomass densities). Another inference that can be drawn is that, the cultivation strategy (including the mixotrophic culture itself) can be leveraged to bioremediate wastewaters from all kinds of sources. Thus, the present study systematically provides a strategy to identify mixotrophic algae, determine their CO₂ capture potential and finally design and develop a high-throughput algal biomass cultivation strategy for biomass generation, minimal resource utilization and CO₂ sequestration along with wastewater remediation. Therefore, the process economics are expected to significantly improve due to simultaneous mitigation of low-cost wastewater along with enhanced CO₂ fixation efficiencies resulting in increased biomass productivity. Hence, the authors expect the present study to play a vital role in the progress of contemporary scientific literature.

Acknowledgements

RS gratefully acknowledges the financial support received from DST, Govt. of India (Grant No. DST/IS-STAC/CO2-SR-160/13(G), Date: 17-07-2013). GS and GY are grateful to Council for Scientific and Industrial Research, Government of India for financial assistance via Senior Research Fellowship. The authors are grateful to Dr Tapan Chakrabarti of National Environmental Engineering Research Institute, Nagpur, India for giving Chlorella vulgaris as a gift.

Notes and references

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