Flocculation and Cell Rupture in C. Vulgaris

Research Article

Ann Agric Crop Sci. 2023; 8(3): 1135.

Flocculation and Cell Rupture in C. Vulgaris

Khandual S¹*; Santiago-Mateo H¹; Maldonado-Ortiz AJ¹; Bonilla-Ahumada FJ¹; Kumar JS²

1Department of Industrial Biotechnology, The Center for Research and Assistance in Technology and Design of the State of Jalisco A.C (CIATEJ), México

2Department of Civil & Environmental Engineering, Old Dominion University, USA

*Corresponding author: Khandual S Department of Industrial Biotechnology, The Center for Research and Assistance in Technology and Design of the State of Jalisco A.C (CIATEJ), Normalistas 800 Ave, Colinas de La Normal, 44270 Guadalajara, Jalisco, México Email: mita@ciatej.mx

Received: April 27, 2023 Accepted: May 26, 2023 Published: June 02, 2023

Abstract

Among all carotenoids, the human body can easily convert β-carotene into retinol, which is a major ingredient in dietary supplements. In the United States, beta-carotene (95% pure) is permitted as a colorant under 21 CFR 73.95 and 21 CFR 166.110. In this study, we are reporting the evaluation of the content of lipids, β-carotene and lutein in the native strain of Chlorella vulgaris CIB 46 (from CIBNOR), Mexico during growth period to define the optimal harvest days for the specific products and future industrial use. This strain has the potential to produce lutein, β-carotene, lipids that can be used for functional foods. Chlorella vulgaris CIB 46 strain showed considerably high biomass productivity reaching almost 2.4g/L biomass on day 21 and reaching 3.0g/L on Day 24 in photobioreactors. Chlorella vulgaris presented a production of carotenoids up to 3.45mg/g at day 21. The Thin-Layer Chromatography (TLC) showed that they contain high β-carotene and lutein in their carotenoid fraction based on the intensity of the band. Our study results showed that Chlorella vulgaris-CIB46 is a potential strain to produce main carotenes such as β-carotene and lutein, they are 0.35% of dry biomass. We obtained a notable amount of lipids that is 37% in Chlorella vulgaris CIB 46 measured by sulpho-valiline method of extraction with enzymatic pretreatment with enzyme Celluclast at 2%. Lipid content was high at 15 days of culture whereas we found β-carotene was higher at 15th and 21st day.

Key words: Biomass; Carotenoids; Thin-layer chromatography; Beta-carotene; Lutein

Introduction

Currently microalgae importance increased many folds for its use as feed for aquatic and terrestrial animals for the nutritional value and includes use as colorant in aquaculture, and high-protein or polyunsaturated fatty acid supplement in human diets. The food, pharmaceutical and cosmetic markets have an enormous growing trend for microalgae-based products [1]. Furthermore, the large number of existing species of microalgae constitutes a large biodiversity, which has a scope for potential commercial exploitation for many novel products besides vitamins, pigments, and polyunsaturated fatty acids [2,3]. The key factor for their eventual economic feasibility is the possibility of good selected species, estimating their biomass production rate and metabolites producing algae to use it for industrial scale production [4,5]. The most popular microalgae source of carotenoids are Chlorella, Chlamydomonas, Dunaliella, Muriellopsis and Haematococcus sp. all of which belong to the Chlorophyceae family [1]. They tend to accumulate carotenoids and thus offering alternatives to chemical synthesis [6]. Among all natural sources Dunaliella is the most studied and commercially popular strain used for beta-carotene production, the highest content of 9-cis β-carotene [7,8] reaching up to 100g/kgDW, [9-11]. But it required high salt concentration and biomass production is less in comparison to other algal strains. Although many microalgae can produce carotenoids, most of them have not been reported with scientific data regarding pigment yield and their bioprocess for efficient extraction.

Here we intended to work on a Chorella vulgaris CIB46 strain from Mexico with the processes like cell wall breakage efficiency and harvesting to evaluate the feasibility of the strain to explore its carotenoid and lipid production for future use at an industrial scale. Using fresh water native algae as an alternative source of beta-carotene and lutein production with cheaper culture media has an advantage over saltwater algae Dunalliela salina. It gives an idea regarding alternative source of Beta-carotene and Lutein production from Chlorella vulgaris with autoflocculation and specific mechanical or enzymatic pretreatment processes method which does not includes toxic chemical use.

Materials and Methods

Algal Culture by Airlift Bioreactor

In this work algal culture done by aeriation with 200mL/min in an airlift bioreactor, without pH control at the room temperature. We observe growth and biomass by optical density at 680nm at 3 days interval up to 21 days. We evaluated biomass for extraction of lutein and lipids during the day 12-21 days. Culture used for the dry biomass, centrifuged at 4000rpm for 15 min at 25°C and kept them in the oven at 60°C for 48 hours.

Methods of Extraction and Estimation of Lipids

For lipid extraction, we used the method of Byreddy et al. [12] with wet biomass. We carry out a wash with 25mL of distilled water (dH2O) resuspending the biomass and shaking slightly manually. After centrifuging at 4,000rpm for 10 min at 25°C, the water was discarded, 5mL of KOH 5% in methanol (CH3-OH) was added to the biomass, stirred homogeneously and left to stand for 15 min to remove pigments. Then the biomass collected after centrifuge and cell wall breakage done by sonication for 10-15 minutes. We did the extraction with 3 different solvent combinations to compare lipid extraction efficiencies to determine the right solvent to use. Water was added and centrifuged to separate organic phase and then proceed to lipid quantification by sulfophospho-vanillin method.

Solvents used:

a) Method 1. Chloroform–methanol 2:1

b) Method 2. Chloroform–methanol 1:1

c) Method 3. Ethanol-Hexane 2.5:1

Lipid Estimation by Spectrophotometry

The lipid estimation was carried out by the method of Mishra et al. [13] using Sulfo-Phospho-Vanillin (SPV). With this method it was possible to make the direct quantitative measurement of lipids within a culture. SPV reacts with lipids to produce a distinct pink color, and its intensity can be quantified using spectrophotometric methods for absorbance measurement at 530nm. We made a standard curve with vegetable oil for the interpretation of the amount of lipids extracted in microalgae.

The solvent was completely evaporated. We added 100μL of deionized water (diH2O) to each tube, then added 1mL of concentrated sulfuric acid (H2SO4 97-99%) and shaken with extreme care. The tube was placed in an oven at 100°C for 15 minutes, then removed from the oven and immediately placed in ice for 5 min. Then 1mL of (SPV) was added, shaken homogeneously and then the tubes were placed in an incubator at 36-37°C for 15 minutes. The tubes were shaken and measured in triplicate in an EON BioTek spectrophotometer at a wave length of 530nm.

Method of Extraction and Estimation of Carotenoids

Extraction using different solvents

First step we quantify lutein content by different types of solvents to consider which solvent is better suitable for this strain. We compare hexane, acetone, ethanol, methanol, and tetrahydrofuran. In the case of solvents, we found acetone and ethanol were with higher extraction yields of Lutein. Methanol was also good for extraction, but due to toxic nature to use in the food sector it was avoided. The quantification was with spectrometry method and TLC for quality analysis in extracts.

Carotenoid saponification and estimation

In the present study the method of Rajashree Hajare et al. [14] was used for carotenoid saponification and extraction. We extracted 200mL of Chlorella vulgaris culture to obtain wet biomass by centrifugation. The biomass samples were dried in an oven at 60°C for 48 hours. Grinded dry biomass added with 1mL of absolute ethanol, transferred to the glass tubes and sonicated for 30 min at 25°C. Then 20mL of absolute ethanol was added, placed in an oven at 50°C for 20 minutes and then removed to keep them at room temperature. Then 50mL KOH at 4% and 6mL of Hexane was added. Then the samples were left to stand overnight (18 hours) in the absence of light, centrifuged at 7000 rpm for 15 min to separate the hexane phase. Then proceeded for optical density measurement in a BIOTEK brand EON spectrophotometer at 448 and 446nm. The remaining sample was dissolved in the appropriate amount of solvent so that it could be mounted on TLC to see qualitative analysis.

To determine the concentration of lutein we use the following formula: Lut=[(A)(V)(Fd)]/(€xW). Where: Lut=Concentration of lutein (μg/g biomass), A=absorbance at 446 nm, V=volume of the extract in mL, Fd=Factor of dilution, €=Coefficient of the absorption (2589), W=dry biomass (g).

Enzymatic method

Acetate Buffer (ACB) was prepared and adjusted the pH to 5.5. For the rupture of the cell wall, the enzymatic hydrolysis of the cellulose was carried out, through treatments where the conditions were varied according to the supplier's instructions. Novozyme celluclast enzyme was used with acetate buffer at pH 5.5 with an incubation temperature of 55°C. With the same buffer, the Trichoderma cellulase enzyme (SIGMA) was used at pH 5 in 55°C incubation. The hydrolysis conditions were standardized with the same culture medium without adjusting the pH to determine if it was effective for cell degradation without consuming another type of buffer with different pH. We used 10mL of Chlorella vulgaris culture concentrate, transferred to Eppendorf 200μL of sample in duplicate, the simples were considered like: Control 0, Control C1 (thermal treatment), T1 (thermal-enzymatic treatment-in BBM medium), T2 (treatment thermal-enzymatic- in acetate buffer). For samples T1 and T2 (x4 of each sample), centrifugation is carried out at 2000rpm for 1 min, the supernatant liquid is discarded and 196μL of BBM medium are added for T1 and acetate buffer for T2. The samples were placed in the Thermo-shaker Brand: Benchmark, (samples C1, T1, T2) at 55°C at 300rpm for 3.5 hours. After 3.5 hours, a Neubauer cell count was performed to evaluate the percentage of live and dead cells with this treatment.

Results

Biomass Yield

This strain shown considerably higher biomass production reaching almost 2.4g/L biomass on day 21 and reaching 3.0g/L. Day 24 with airlift culture condition. Zheng et al., [15] reported that in the aerated culture of Chlorella vulgaris a biomass concentration of approximately 3.28g/L was obtained, and a lipid productivity of 35%. In our case we also found similar results with respect to biomass production with aeration.

Flocculation Study for Effective Harvest

Various reports have shown that Chlorella does not present auto-flocculation, however, in our case we did observe auto-flocculation in 12 hours of darkness with 82% flocculated bio mass. The settled biomass was remarkable, this implies good results to avoid the use of so many chemicals. We also compared flocculation efficiencies by using chemicals to compare previous reports.

The results shown with Ca (OH)2 treatment, 71% biomass was flocculated in 5 min and 82% biomass flocculated in 2 hours with 28% volume of solution added. With other reagents, we did not found flocculation immediately, but after 2 hours we found 57-81% flocculated biomass with spending 60% reagent by volume which is not recommendable (Table 1 & Figure 1). An alkaline treatment with Ca (OH)2 is recommended for flocculation and at the same time serves to break the cell wall for the extraction of biofuel and other compounds. Auto flocculation presented better results than the other flocculation treatments.