Microalgae Cultivation in Pilot Scale for Biomass Production Using Exhaust Gas from Thermal Power Plants

Abstract

Exhaust gases from thermal power plants have the highest amount of carbon dioxide (CO₂), presenting an environmental problem related to a severe impact on ecosystems. Extensively, the reduction of CO₂ from thermal power plants has been considered with the aid of microalgae as a cost-effective, sustainable solution, and efficient biological means for recycling of CO₂. Microalgae can efficiently uptake CO₂ and nutrients resulting in high generation of biomass and which can be processed into different valuable products. In this study, we have taken Nephroselmis sp. KGE8, Acutodesmus obliquus KGE 17 and Acutodesmus obliquus KGE32 microalgae, which are isolated from acid mine drainage and cultivated in a photobiological incubator on a batch scale, and also confirmed that continuous culture was possible on pilot scale for biofuel production. We also evaluated the continuous culture productivity of each cultivate-harvest cycle in the pilot scale. The biomass of the cultivated microalgae was also evaluated for its availability.

1. Introduction

Nowadays, an increase of CO₂ levels in the atmosphere is extensively recognized as a major contributor for global warming. Recent reports are highlighted that atmosphere contains CO₂ level of 450 ppm [1]. The atmospheric CO₂ can be trapped by green plants via photosynthesis. However, terrestrial plants are estimated to reduce only 3–6% of global CO₂ emissions, which is significant given the slow growth rates of plants. On the other hand, microalgae can grow much faster than terrestrial plants, and their CO₂ reduction efficacy was 10–50 times higher than plants [2,3]. The variety of microalgae cultivated in comfortable environmental condition to produce comparably 15–300 times higher energy sources than plants, which also reduce the land area for cultivation and continuously increase the yield per unit area [2,4,5]. Microalgae can biologically store CO₂ through photosynthesis in the form organic compounds and then use microalgal biomass as a feedstock for renewable energy after CO₂ fixation [6]. Moreover, microalgae have been documented as source of valuable biomaterials such as fertilizers, live feed, medicines, and other value-added products.

The large-scale microalgae culture system was divided into two systems, namely the open and closed systems. In the case of the open system, it is difficult to control the amount of light intensity and it may vary depend upon the local time, and also difficult to maintain the temperature. The closed system, which is a device to overcome these limitations, is able to control the light intensity, external influence, and temperature, though the operation cost and the manufacturing cost are high when compared to the open system. Especially, the closed system microalgal growth rate is 1.5–4 times higher than the open system [7]. The high growth rate of microalgae has a large impact on CO₂ capture and may lead to an increase in biomass production. According to the various research condition, closed system may be designed as airlift column, horizontal tube, stirred tank, and flat panel photobioreactor (PBR) [7,8].

Obviously, industrial exhaust gases contain 10–20% of CO₂ with trace amounts of SO_x and NO_x. The selection of microalgae plays a vital role in CO₂ reduction efficacy and represents a significantly cost-effective route for biomass production. The desirable qualities of microalgae comprise high growth and CO₂ consumption rates, also patience towards trace constituents of exhaust flue gases such as SO_x and NO_x and production of valuable products. Maeda et al. (1996) used Chlorella sp. T-1 as a potential microalga for the biological removal of exhausted CO₂ from coal-fired thermal power plants. Aslam et al. (2017) have identified that mixed microalgae societies like Desmodesmus spp. can slowly grow in 100% unfiltered exhausted gas from coal combustion with phosphate buffering condition [9]. Kassim and Meng (2017) studied biofixation of CO₂ by two microalgae species such as Chlorella sp. and Tetraselmis suecica with various CO₂ concentration [10]. Even though the above said studies have been carried out in exhausted gas which adversely affects the microalgal growth. To the best of our knowledge, no study has yet reported on the actual injection of exhaust gas, and there is a lack of research on biomass tendency when continuously injecting the gas into large-scale bioreactor.

Hence, the objective of this study is to evaluate the feasibility of microalgae species like Nephroselmis sp. KGE8, Acutodesmus obliquus KGE 17 and Acutodesmus obliquus KGE32, which were cultivated in a laboratory with the supplementation of power plant exhaust gas. Then, evaluate the growth potential of the microalgae in the semi-continuous photobioreactor (PBR) operating with the exhaust gas injection, and evaluate the microalgae productivity at each cultivate-harvest cycle. Finally, we also assessed the feasibility of biodiesel, lipid and C16-18-FAME contents in recovered microalgae.

2. Materials and Methods

2.1. Conditions of Microalgae Cultivation in Batch Scale

Microalgae species were derived from acid mine drainage which include Nephroselmis sp. KGE8, Acutodesmus obliquus KGE 17, and Acutodesmus obliquus KGE32. The batch type cultivation was performed in 140 mL serum bottle with 100 mL of Bold’s Basal Medium (BBM) [11] which contained strains with optical density of 0.010 in UV spectroscopy 680 nm region.

The exhaust gas from the Y coal-fired thermal power plant (Gangwon-do, South Korea) was used as a carbon source for microalgae growth. The collected gas was filtered through a 0.2 μm filter and then supplied at a flow rate of 0.5 L min⁻¹ for 1 h to complete saturation of the medium. Composition of the exhaust gas details appeared in

Table 1. Y Power plant gas contents and concentrations located in Gangwon-do.

Gas Contents	Initial Gas Concentration
CO2 (v/v %)	14.9 (±0.2)
NOx (ppmv)	220.2 (±10.5)
SOx (ppmv)	32 (±5)
CO (ppmv)	1549 (±242)
O2 (%)	5.47 (±0.03)

. The microalgae cultivation was conducted at 25 °C with 120 µmol photon m⁻²s⁻¹ of light intensity, and the content was agitated in incubator shaker (Witeg, Wisecube WIS-ML, Germany) at 120 rpm to prevent agglomeration for 7 days.

2.2. Cultivation of Microalgae in Photobioreactor (PBR)

The culture system used in this study was a multistage photobioreactor (PBR), each PBR has capacity of 2000 L and it is shown in Figure 1. Initially, exhausted gas was injected through PBR 1 and then subsequently passed through the PBR 2, -3 and -4 respectively. Finally, unutilized gas was ejected from the PBR 5, the concentration of CO₂ in each stage was measured and attached in

Table 2. Injection CO₂ concentration in each stage.

Stage	CO2 (v/v %)	NOx (ppmv)	CO (ppmv)	O2 (v/v %)
PBR1	14.90 (±0.18)	220.2 (±10.5)	1548.5 (±242)	5.47 (±0.03)
PBR2	8.08 (±0.52)	124.4 (±21.8)	941.1 (±41.9)	12.42 (±0.93)
PBR3	6.72 (±0.73)	99.3 (±16.0)	703.3 (±51.4))	13.89 (±0.99)
PBR4	4.99 (±0.52)	72.3 (±12.1)	537.2 (±21.6)	15.81 (±0.86)
PBR5	3.87 (±0.52)	51.9 (±9.3)	421.1 (±35.2)	17.03 (±0.95)
Out	3.11 (±0.60)	51.8 (±8.18)	337.0 (±5.1)	16.86 (±1.41)

. The initial and final exhaust gas concentration was analyzed by Testo 350 K emission analyzer (Testo, Germany). According to Blair et al. (2014) red light emitting diode (LED) was installed at each stage for maximize light absorption [12]. The first stage of the pilot scale, the growth rate of each stage of Nephroselmis sp. KGE 8 was determined and grown for 20 days. The second cultivation was performed for 22 days, from the 18 days when the specific growth rate (μ_max) started to increase, the possibility of continuous cultivation was evaluated by harvest and regrowth. On day 18, the microalgae were recovered and diluted and re-cultured for 16 days and supplemented with BBM to prevent nutrition loss. The interval between collection and incubation was 2 days, and cultures were collected and cultivated three times.

2.3. Analysis of Microalgal Growth

The growth rate of microalgae cultivated in pilot plant was obtained by analysis of OD₆₈₀, from spectrophotometer (Hach DR/2800, Loveland, CO, USA) which values were converted to dry cell weight (DCW) concentration (g L⁻¹). DCW of Nephroselmis sp. KGE 8 was calculated by:

Dry weight (g L⁻¹) = 0.3997 × OD₆₈₀ − 0.0471 (R² = 0.9871)

(1)

Further, the specific growth rate (SGR) was calculated by Equation (2):

μ = (lnX₂ − ln X₁)/(t₂ − t₁)

(2)

where X₁ and X₂ are the mass of initial and final weight of microalgae, respectively, which is used to calculate DCW in this study, and t₁ and t₂ are the initial and final incubation times respectively.

2.4. Algal Harvest

In pilot scale, the algal harvest was performed by sludge pump, and collected to storage tank. The algae of each stage were harvested, and the harvested algae were precipitated and recovered by separating the supernatant and algae.

2.5. Analysis of Lipid and C16–C18 Fatty Acid Methyl Ester (FAME)

The modified Bilgh and Dyer method (Ji et al. (2016)) was used to analyze the lipids and fatty acids in the harvested microalgae [13]. Fatty acids were identified by the modified Lepage and Roy method from Yun et al. (2015), which convert fatty acid into fatty acid methyl esters through esterification and is analyzed by Gas chromatography with a flame ionization detector (GC-FID) using HP-INNOWax capillary column (Agilent Technologies, USA) [14].

3. Results and Discussion

3.1. Growable Microalgae in Exhaust Gas Condition

Batch scale experiment results shows that both Nephroselmis sp. KGE 8 and Acutodesmus obliquus KGE 17 have lag phase up to two days and showed exponential growth phase until fifth day (Figure 2). At this moment, Nephroselmis sp. KGE 8 exhibited the maximum growth when compared to the Acutodesmus obliquus KGE 17. Another microalga like Acutodesmus obliquus KGE 32 exhibits the lag phase until three days, and the exponential growth phase was until five days, and it has the stationary growth phase. The growth rates of microalgae with a supply of exhaust gas are presented in

Table 3. Specific growth rate and lipid contents of various microalgal strains cultivated at different carbon dioxide concentration.

Species	Carbon Dioxide Concentration (%)	Incubation Condition	Medium	Lipid Contents (%)	Specific Growth Rate (μmax, d−1)
Scenedesmus obliquus KGE 9 a	14.1	Batch	BBM	22.8	1.00
Chlorella pyrenoidosaSJTU-2b	10.0	Batch	BG11	24.2	0.78
Acutodesmus obliquus KGE 30 c	14.1	Batch	BBM	17.5	1.09
Acutodesmus obliquus KGE 32 d	14.1	Batch	BBM	-	1.08
Acutodesmus obliquus KGE 17 d	14.1	Batch	BBM	-	1.37
Nephroselmis sp. KGE 8 d	14.1	Batch	BBM	59.4	1.41
0 Nephroselmis sp. KGE 8 d	14.1	Pilot scale	BBM	60.9	0.26

^a Ji et al. (2017) [19]; ^b D. Tang et al. (2011) [20]; ^c HS Yun et al. (2016) [14]; ^d This study.

. Ji et al. (2017) and Yun et al. (2016) evaluated the potential for biofuel production according to changes of CO₂ concentration in exhaust gas. Compare with these previous studies, algae production was faster. Also Tang et al. (2011) was focused on growth potential in high concentration of CO₂ and effective concentration of CO₂, growth rate and lipid contents was lower than this study. Nephroselmis sp. KGE 8 have the OD₆₈₀ value of 1.341 and the maximum specific growth rate (μ_max) was 1.41 d⁻¹ between 3–4 days of culture. Conversely, Acutodesmus obliquus KGE 32 and Acutodesmus obliquus KGE 17 possess the OD₆₈₀ values of 0.970 and 0.553 and μ_max were 1.08 d⁻¹ and 1.37 d⁻¹ respectively. The microalgae, which applied in this study, showed higher specific grow rates (1.08 to 1.37 d⁻¹) than previous study (Table 3). Continuous and excessive exposure of NO_x and SO_x gases to cells could leads to inhibition of microalgae growth rate [15,16]. Praveenkumar et al. (2014a) reported that algal FAME content and productivity increased from 129 to 168 mg fame/g cells and from 59 to 118 mg fame/L d, respectively, in coal-fired flue-gas inlet condition [17]. They also conclude that stress conditions could lead to improve algal lipid productivity [18].

3.2. Pilot Scale Cultivation

The pilot scale cultivation result discloses that, lag phase period of Nephroselmis sp. KGE 8 was increased from 2 days to 10 days when compared with batch scale results due to the stress present in the exhaust gas (Figure 3). Same trend was also observed in previous study by Borowitzka et al. (2018) and mentioned that adaptation by stress due to CO₂ [21].

Further, the microalgae growth was not similar with the batch scale (Figure 3). Due to the different character of coal and also the generated exhaust gas from the thermal power plant does not contain constant amount of CO₂, it may lead to irregular growth of microalgae in the pilot scale. Cheng et al. (2019) also reported that biomass yields were not constant for every cycle, even gas-adapted microalgae were injected with a constant concentration of mixed gas [22]. These results indicated that Nephroselmis KGE 8 is a microalga species that could adaptively grow, even when the exhaust gas was continuously injected.

In continuous culture potential evaluation experiment, Nephroselmis sp. KGE 8 reached the exponential growth phase at 17 days after initiated the cultivation (Figure 4). According to Tan et al. (2018), the productivity of microalgae tended to decrease with increasing amount of cultivation [23]. The growth of Nephroselmis sp. KGE 8 was different in each stage. PBR 2 show a microalgae concentration of 0.6002 g L⁻¹ for the first time and 0.4932 g L⁻¹ for the second cultivation. Also the microalgae concentration in PBR 3, PBR 4, and PBR 5 was decreased from 0.5644 g L⁻¹ to 0.4955g L⁻¹, 0.5343 g L⁻¹ to 0.4722 g L⁻¹, and 0.4421 g L⁻¹ to 0.4116 g L⁻¹ respectively. In contrast, the microalgae growth in PBR 1 has increased from 0.4996 g L⁻¹ to 0.5710 g L⁻¹, unlike other stages. Biomass productivity is affected by growth factors, and according to Sun et al. (2018), the growth factors like high temperature and large N source will increase biomass growth [24]. When NO_x is dissolved in water, it tends to form nitrite, which can help to grow the biomass [25].

3.3. Lipid and Fatty Acid Productivity

We have compared the biomass productivity of Nephroselmis sp. KGE 8 in both batch and pilot scale. In batch scale, the biomass productivity was found to be 0.696 g L⁻¹ d⁻¹, also contain 59.4% and 95.1% of lipid and C16–C18 FAME respectively. In contrast, the biomass productivity, lipid, and C16-C18 FAME contents were decreased to 0.163 g L⁻¹ d⁻¹, 39.4%, and 77.8% respectively in pilot scale experiments (

Table 4. Biomass productivity, C16-C18 ratio, and lipid content of Nephroselmis sp. KGE8 at laboratory scale and pilot scale.

Strain	Volumetric Productivity of Biomass at μmax (g L−1 day−1)	C16–C18 Ratio (wt %)	Lipid Content (wt %)
KGE8 cultivated in Laboratory	0.696	95.1	59.4
KGE8 Cultivated in Pilot scale	0.163	77.8	60.9

). Park et al. (2013) reported that the maximum lipid content of the Nephroselmis sp. KGE 8 species was 38.8% [26].

The average lipid content of harvested microalgae in PBR 1 was 41.24%, which was higher than that of other stages. Arief et al. (2009) reported that the content of lipid in microalgae increased with increasing CO₂ concentration. The fatty acid content of the recovered Nephroselmis sp. KGE 8 illustrated in Figure 5. The average of C16 to C18 FAME contents in recovered microalgae at each harvest cycle illustrated Figure 5A. The fatty acid content was 74.38% (w/w), and the highest fatty acid content showed the highest fatty acid content as 87.29% in PBR 5. However, as the number of continuous cultures increased, the fatty acid content of PBR 1 also increased, while that of PBR 2 to PBR 5 tended to decrease (Figure 5B). In particular, PBR 5, which showed the highest fatty acid content at the initial stage, showed a sharp decrease in fatty acid content as the number of times increased. Sharmaet al. (2012), and Nayaket al. (2018) reported an increase in Oleic acid (C18: 1) in the fatty acids of cultured algae in coal combustion gases [27,28].

4. Conclusions

Batch scale studies reveal that Nephroselmis sp. KGE 8 showed the best growth under exhaust gas conditions. Nephroselmis sp. KGE 8 showed growth potential (0.696 g L⁻¹) in the semi-continuous PBR operation with the exhaust gas injection. The lipid content and C16-C18 FAME content were 39.4% and 77.8% in PBR1, respectively. The microalgae productivity of five reactors showed range from 0.4116 g L⁻¹ to 0.5468 g L⁻¹ at each cultivate-harvest cycles. PBR 1 showed highest microalgae productivity during PBR operation.

When exhaust gas is directly injected, changes in NO_x and temperature condition accelerate the microbial energy conversion. Singh et al. (2014) reported that some algal species obtained maximum biomass in 15% CO₂ [29]. Based on the result, it was concluded that direct injection of exhaust gas is the most suitable condition for utilization of energy source of microalgae. This microalgal cultivation system could be a suitable process for the massive cultivation of microalgae with exhaust gas from power plants.