Optimization of CO2 biofixation rate by microalgae in a hybrid microfluidic differential carbonator using response surface methodology and desirability function
Introduction
The increase in atmospheric CO2 causes massive environmental damage (e.g. global warming, acid rains). To reduce CO2 in the atmosphere, many conventional methods such as injection into geological formations, deep oceans or utilization of absorbent materials are available [[1], [2], [3]]. However, these methods are costly, especially in terms of the capital and the running costs [4] and harmful to the environment, particularly on living organism, due to the formation of hydrate gases [5].
Biological method is one of the well-known environmentally sustainable methods for reducing the atmospheric CO2. Microalgae have been stated to have high CO2 fixation compared to terrestrial plants [6]. It was also found that microalgae can reduce the CO2 level during their growth through photosynthesis and sequester more than 80 % of CO2 from the growth medium [7,8]. At the same time, a substantial amount of biomass is produced which could be employed for production of biofuels, valuable products (e.g., organic acids, acetic acid, butyric acid), bio-fertilizers, and wastewater treatment [4,[9], [10], [11], [12]]. However, to maximize the CO2 biofixation, the growth of microalgae needs to be maximized. Therefore, optimum conditions must be provided for the growth of microalgae.
There are several hundred microalgae species reside in the nature. Chlorella vulgaris is one of the promising microalgae species for CO2 sequestering as well as biofuel feedstock [13]. Rapid growth, easy cultivation due to its high efficiency in capturing CO2 from the atmosphere, and high lipid content make them more effective for practical applications [[14], [15], [16]]. Keeping these into consideration, Chlorella vulgaris was selected as one of the potential microalgae species to investigate CO2 biofixation rate in this study. The growth of microalgae is generally affected by several key environmental factors such as the composition of culture medium, the amount of CO2 provided, the temperature, light intensity, pH, and others [13]. Generally, the ideal temperature for most of the microalgal species varies between 20 °C–35 °C. Chinnasamy et al. found that the growth temperature of Chlorella vulgaris fluctuates from 25 °C to 30 °C [17]. Microalgal chlorophylls, phycobilins, and carotenoids absorb the visible light. In addition, the CO2 (obtained from environment) is converted to an organic compound during photosynthetic process. However, too much light intensity may decrease microalgal growth due to photo-inhibition. The favorable range of light intensity is usually 1000–10000 lux [18]. Regarding the effect of the CO2 concentration on microalgae growth, it is found that the increasing the concentration of CO2 leads to better growth, until saturation is attained. However, such change is deeply dependent on the type of microalgae and its media. For instance, Chlorella vulgaris showed best biomass growth at CO2 concentration of 6% [19]. The media pH is usually decreased with inclusion of high concentration of CO2 due to formation of carbonic acid (H2CO3).
It must be noted that some of the aforementioned variables have been optimized in our previous studies using batch reactor systems [[20], [21], [22]]. It is well known that the mass transfer and other control operations are not so efficient and precise in batch reactor approaches. Therefore, the acquired optimal conditions may suboptimal instead of true optimal settings. The bottlenecks can be easily overcomed by using microfluidic-based photocatalytic microreactor systems instead of conventional batch reactor schemes. Microfluidic-based microreactor system doesn’t only guarantee better mass transfer and control on the operating conditions, but also maintains the lab-scale yield upon transition from pilot scale to industrial scale [23,24]. Statistical design of experiment (DoE) including Full Factorial Design (FFD) and Response Surface Methodology (RSM) can also be utilized for modelling and optimization [25]. The RSM is categorized into mainly Box-Behnken Design (BBD) and Central Composite Design (CCD). Face Centered-Central Composite Design (FC−CCD) is a special type of CCD. Generally, FFD is used for screening the essential factors while RSM with either BBD, CCD or FC−CCD is applied for optimization the parameters of the process. Overall, these approaches discuss the important independent factors, interactions among factors, and optimal conditions for maximizing the responses or outputs [[26], [27], [28], [29], [30]]. However, within BBD and CCD approaches, CCD has been used widely in the literature for parameters optimization due to its rotatability and high robustness features [20,22].
In this study, an in-house fabricated microreactor (a hybrid microfluidic-differential carbonator, μ-DC) was introduced to investigate microalgal growth as well as its CO2 biofixation capability. In this regard, three important independent factors such as light intensity, percentage of CO2 in air and volume ratio of microalgae to media were taken into consideration. Initially, a full factorial design (FFD) was utilized to identify the most significant factors with a minimum number of treatments. Once the most leading factors were obtained, optimum values of the independent factors affecting the process were estimated by using RSM with FC−CCD and desirability function based approaches. The desirability is categorized into individual (d) and composite (D). Individual desirability (d) explains the settings of single objective optimization while composite desirability (D) assesses the settings of multi-objective optimization as an overall. Generally, the values of desirability vary from 0–1, in which the value 1 is considered as an ideal. Overall, the application of the approaches (FFD and RSM-FC−CCD) enables the following: (i) identification of the most effective parameters, (ii) obtaining a mathematical model for each individual response or output (e.g., specific growth rate, cell counts, CO2 biofixation rate) that describes the relationships between input and output variables, and (iii) identification of the best conditions for all responses simultaneously based on multi-objective optimization. It is noteworthy that microfluidic-differential oxygenator (μ-DO) has been reported for characterization of bacteria and mammalian cells growth [31,32]. However, to the authors’ best knowledge, the development and application of microfluidic-differential carbonator (μ-DC) in microbial research (especially in the field of microalgae) has not been reported yet in the open literature.
Section snippets
Strain selection and growth media
The microalgae strain and the proper growth media are the most important factors to cultivate microalgae. The Chlorella vulgaris (obtained from National Mariculture Centre, Ministry of Municipalities and Urban Planning, Kingdom of Bahrain) was used in this work with f/2 media as a growth media [33]. Regarding media preparation, the seawater was filtered using a GF/F filter to make sure that the seawater is free from small waste particles. An appropriate amount of f/2 media was added to every
Screening the factors using 3k factorial design
In this regards, three independent factors such as the ratio of microalgae to media, light intensity, and % CO2 were utilized using a 33 full factorial design. A total of 81 experiments (including 3 replicates) were conducted and CO2 biofixation rates were calculated for each experiments. The values of experimental and predicted biofixation rates were documented in Table 3. In order to find the important factors among the three independent variables, main effect plot was generated using Minitab
Conclusion
In this study, an in-house fabricated hybrid microfluidic-differential carbonator (μ-DC) was applied for assessment of microalgal (Chlorella vulgaris sp.) growth as well as the rate of CO2 biofixation. The effect of CO2 concentration, microalgae to media ratio, and light intensity was analyzed and screened using FFD approach. Both microalgae to media ratio and % CO2 in air were found to be significant factors within the operating ranges. Further, analyses on the significant factors and their
Declaration of Competing Interest
As a corresponding author, I confirm that there is no conflict of interest for publication of this manuscript.
Acknowledgements
The authors would like to gratefully acknowledge Dr. Layla Hazim, Department of Biology, University of Bahrain for providing the f/2 media. The authors would also like to thank Department of Chemical Engineering, University of Bahrain.
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