Abstract
British Phycological APPLIED PHYCOLOGY Society 2023, VOL. 4, NO. 1, 34–43 Understanding and using algae https://doi.org/10.1080/26388081.2022.2140073 Replacing expensive synthetic media with banana stem compost extract medium for production of Chlorella vulgaris Kulwa Mtaki, Margareth S. Kyewalyanga and Matern S. P. Mtolera Institute of Marine Sciences, University of Dar es Salaam, Zanzibar, Tanzania ABSTRACT ARTICLE HISTORY Received 10 August 2021 Microalgal cultivation by small scale aquaculture farmers is limited by the high cost of synthetic Accepted 16 October 2022 culture media. The current study was conducted to investigate use of banana stem compost extract (BSCE) as an alternative medium for cultivation of the microalga Chlorella vulgaris. KEYWORDS C. vulgaris was batch cultured for 24 days in the laboratory using synthetic Bold Basal Medium Alternative culture media; (BBM) as a control and BSCE at concentrations of 2%, 5% and 10% (by volume) as the treatments. banana stem; Bold Basal Algal growth was evaluated by measuring dry cell weight and specific growth rate (SGR) during the Media (BBM); Chlorella experimental period. Chemical composition was analysed following standard analytical methods. vulgaris; circular economy; nutritional value Variations in growth trends among culture media were attributed to variations in nutrient con- centration and lack of acclimatization period. Some macro- and micronutrients in BSCE-cultivated C. vulgaris were higher than or similar to those observed in BBM-cultivated algae. The macronu- trients differed among BSCE treatments. It was concluded that BSCE can be used as the culture medium, providing similar nutritional value and supporting similar growth performance to syn- thetic media. However, selection of BSCE concentration should be based on macronutrients and take into account the intended use of cultivated microalgae. Introduction production is frequently uses synthetic media such as Microalgae are a diverse group of eukaryotic photosyn- BG 11, Zarrouk and Bold Basal media (BBM) which are thetic microorganisms that can produce amino acids, pro- very expensive and account for up to 50% of microalgal tein, minerals, vitamins, antioxidants and other bioactive production costs (Michael, Kyewalyanga, & Lugomela, substances (Mtaki, Kyewalyanga, & Mtolera, 2020; 2019; Mtaki, Kyewalyanga, & Mtolera, 2021; Xia & Sathasivam, Radhakrishnan, Hashem, & Abdallah, 2019). Murphy, 2016). These synthetic media are not easily Their cultivation has received growing interest around the accessible to small scale aquaculture farmers in devel- world due to their high growth rate, high nutritional value, oping countries (Michael, Kyewalyanga, & Lugomela, carbon dioxide capture, ability to grow in different culture 2019). It is therefore imperative to replace expensive media and their use of different nutrition modes e.g., auto- synthetic media with cheap and locally available and/or heterotrophic (Metsoviti, Papapolymerou, media. Here we show how this can be produced from Karapanagiotidis, & Katsoulas, 2019; Ramaraj, Tsai, & banana stem by-products so as to recycle nutrients and Chen, 2015). Microalgae are used in multiple industries, improve farmers’ livelihoods. Banana plants (Musa for instance in aquaculture Chlorella vulgaris is utilized as spp.) could be used as as cheap and widely available live feed for larval and/or juvenile of some crustaceans and resource needed to make culturing medium for micro- finfish, some bivalve molluscs (oysters, scallops, clams and algal production. mussels) and enrichment of zooplankton e.g., Artemia spp Banana pseudo-stems are cut down after the fruit is (Koyande et al., 2019; Rizwan, Mujtaba, Memon, Lee, & harvested and are usually left in the field, creating an Rashid, 2018). It has been suggested that large-scale pro- agri-waste problem and environmental nuisance duction of C. vulgaris could be used as the means to (Gumisiriza, Hawumba, Okure, & Hensel, 2017; improve aquaculture production and address global food Gupta, Baranwal, Saxena, & Reddy, 2019). Banana shortages (Abdullah et al., 2019; Chang, Nichols, & pseudo-stems are reported to contain 58% carbohy- Blackburn, 2013). drate, and high levels of nutrients including calcium Cultivation costs of C. vulgaris are too high for com- and phosphorus (Aziz et al., 2011; Ho, Noor Aziah, & mercial applications in developing countries: mass Bhat, 2012; Mtaki, Kyewalyanga, & Mtolera, 2020). CONTACT Kulwa Mtaki mtakikulwa@yahoo.com © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. APPLIED PHYCOLOGY 35 These nutrients make banana stems an ideal candidate BSCE media at 2%, 5% and 10% concentrations as the for a cheap alternative medium for microalgal cultiva- treatments. The microalgae were batch cultured in 2 l tion. However, there are limited studies on the use of Erlenmeyer flasks (in triplicate per treatment) using 800 banana by-products or their derivatives as medium for ml of the respective culture medium and 200 ml of 6 −1 microalgal cultivation. The current study aimed to eval- C. vulgaris with an initial count of 0.3 x 10 cells ml . uate application of banana stem compost extract (BSCE) The experiment was conducted in a controlled environ- as a potential replacement for synthetic media in ment with pH set at 9.0 and maintained throughout by C. vulgaris cultivation. The study also aimed to estimate adding appropriate quantities of 5 M sodium hydroxide the financial cost of preparing BSCE medium compared and 1 M hydrochloric acid. Temperature was maintained to the synthetic medium BBM. The hypothesis tested at 28 ± 1°C; compressed air was provided; light intensity −2 −1 was that replacing BBM with BSCE culturing medium was set at 5000 ± 10 lux (c. 500 µmol m s ) with 16:8 would result in similar growth and biochemical compo- photoperiods (light:dark cycle) monitored using a vertex sition of C. vulgaris while reducing operational cost. VXLM-636 light metre and automatic timer respectively. Microalgae were evaluated for growth performance every three days throughout the experimental period and were Materials and methods analysed for chemical composition at the end of the experiment. Microalgal culture media preparation The banana stems (Musa x paradisiaca) were harvested Microalgal growth evaluation from a local farm in Dar es Salaam, Tanzania. The stems’ outer layers (epidermis) were removed manually by peeling Growth of C. vulgaris was determined by measuring dry and the inner part was taken to the Botany laboratory at cell weight and expressed as specific growth rate (SGR). −1 University of Dar es Salaam, Tanzania for media prepara- The algal dry weight (biomass concentration, g l ) was tion. The stems were laid on the ground, covered with dry measured by taking 10 ml from the culture flask and grasses, and left for two weeks to allow decomposition. The filtering it using pre-weighed Whatman GF/C filters. compost was washed using running tap water, cut into The filters were washed using distilled water to remove small pieces and blended. Blended stem materials were adsorbed salt and oven-dried at 105°C until constant filtered using 44 µm mesh nano-filter and extracts were weight was achieved. The microalgal dry weight was centrifuged (Hettich Zentrifugen 168 Universal 1200, calculated as the difference between the weight of oven- Germany) at 978.02 x g for 10 minutes. The collected com- dried filters containing microalgae and the empty filter. post supernatant was sterilized at 121°C for 15 min in an SGR was measured as an increase in biomass over time, autoclave (SANYO, MLS-3750). The supernatant was and was determined using Equation 1 below. diluted to 2%, 5% or 10% BSCE using sterilized (121°C Specific Growth Rate ¼ lnðW =W Þ=ðT T Þ (1) 2 1 2 1 for 15 min) and filtered (20 µm filter mesh) tap water. BBM medium was prepared as per standard operating procedure (Connon, 2007). Chemical analysis All culture media were chemically analysed for their nutrient composition at the beginning of the experi- Sample preparation and protein analysis ment. The media were analysed for ammonium- The C. vulgaris biomass was harvested after the individual nitrogen, phosphorus and nitrate-nitrogen using indo- treatments had reached the stationary growth phase which phenol blue, ascorbic acid and cadmium reduction was day 18, 21, 21 and 24 for 2% BSCE, 5% BSCE, BBM and methods. Calcium, magnesium, zinc, manganese, 10% BSCE respectively, by centrifugation at 978.02 x g for boron, potassium and molybdate were determined 10 min. The centrifuged mass was washed using distilled calorimetrically using an Atomic Absorption water and air-dried in a darkroom for about a week. The Spectrophotometer (AA240 Varian, USA). dried biomass was ground to powder and used for all biomass chemical composition analyses. The crude protein content in C. vulgaris biomass was determined using the Experimental design Kjeldahl standard method (Barbano, Clark, Dunham, & A culture of C. vulgaris isolated from fish pond water of pH Flemin, 1990). 8.5 (for details see Mtaki, Kyewalyanga, & Mtolera, 2021) was grown in standard BBM medium for seven days prior Total lipids to the start of the experiment. Thereafter, C. vulgaris was Total lipid extraction followed Bligh & Dyer’s (1959) cultured for 24 days using BBM medium as control and procedures, whereby sampled C. vulgaris (~5 g) was 36 K. MTAKI ET AL. mixed with chloroform, methanol and water in a 1:2:0.8 shaken vigorously for 15 min. The mixture was centrifuged ratio. The mixture was homogenized using a Soxhlet for 10 min and filtered using Whatman No. 1 filter paper. apparatus for 2 min, then chloroform and water were The sample was put into a rotary evaporator (Gmbh & Co. added to give a solvent ratio of 2:2:1.8. The solvent was KG, Germany) at 40°C and set under reduced pressure so filtered using Whatman filter paper No. 44 to remove as to remove ethanol and obtain clear extracts. Vitamin biomass residues. The filtered solvents were separated A (as beta-carotene) was determined by vigorously stirring into two layers, chloroform (bottom phase) and aqueous 100 mg of dried extract with 10 ml of acetone-hexane methanol (top phase), using a separating funnel and the mixture (4:6) for 1 minute and filtered using Whatman volume of the chloroform layer was recorded. The No.4 filter paper. The filtrate absorbance was measured at chloroform layer was collected, pipetted and weighed 453, 505 and 663 µm, and the obtained values were used to using a pre-weighed, clean oven-dried evaporation dish. calculate beta carotene in Equation 3. The aliquot was oven-dried at 40°C for 60 minutes, Beta carotene mg 100 mg cooled in a desiccator and weighed. The percentage ¼ 0:216 A 0:304 A þ 0:452 A (3) 663 505 453 lipids in C. vulgaris biomass was analysed gravimetri- cally, based upon starting and end mass. Vitamins B1 (thiamine), B2 (riboflavin), B3 (niacin), B6 (pyridoxine) and C (ascorbic acid) were determined using Total carbohydrates thiamine hydrochloride, riboflavin, nicotinamide, pyri- Total carbohydrates were quantified using Allen’s doxine hydrochloride and ascorbic acid stock solutions (1989) method whereby 5 g of sampled biomass was respectively as described by Rajput, Kumar, Kumar, & mixed with 30 ml of distilled water in a 100 ml conical Res (2011). Individual stock solutions were prepared by flask and boiled at 100°C for two hours. The boiled dissolving a known weight of standard vitamin in a known sample was allowed to cool to room temperature and volume of distilled water and the working solution was filtered through a Whatman filter paper No. 44. A clear prepared by dissolving a known weight of each C. vulgaris sample solution (aliquot) was put into a test tube and extract in a known volume of distilled water. The stock anthrone reagent prepared with boiled hot water was solutions, working solutions and blank were used to deter- added. The mixed solution was allowed to cool, and its mine vitamin B1 at 430 µm, vitamin B2 at 444 µm, vitamin absorbance was measured at 625 µm. The percentage B3 at 450 µm, vitamin B6 at 650 µm and vitamin C at 450 soluble carbohydrate was calculated based on the for- µm using a UV/visible spectrophotometer (Jenway 6305). mula given in Equation 2 below. CðmgÞ x extraction volumeðmlÞ Soluble carbohydrateð%Þ ¼ Estimation of microalgal media cost 10 x aliquotðmlÞ x sample weightðgÞ (2) The cost of making 1 l of the medium was calculated. The costs of BBM medium included price of each reagent, Where C = mg of glucose in the sample aliquot obtained taxation and consumed electricity. The price of BBM from a calibration graph of the standard. reagents were obtained from https://www.alibaba.com and https://www.sigmaaldrich.com. Value added tax was Mineral composition set at 18% of the reagent price as indicated by Tanzania The sampled biomass was digested using nitric perchlo- Revenue Authority while electricity cost was calculated ric acid to determine mineral composition as described from the electricity used to prepare medium. The product by Jones (1984). The sampled C. vulgaris (0.5 g) was was then multiplied by the price of one unit of electricity placed in a beaker followed by a mixture of 5 ml con- which was 350 TSh (~0.15 USD) per kWh in Tanzania. The centrated nitric acid (70% HNO ) and 1 ml perchloric estimation of BSCE media cost considered collection, acid (72% HClO ). The solution was heated at 120°C transport and processing (sterilization and blending) until brown fumes disappeared which indicated com- costs only since banana stem was obtained free in the plete digestion of organic matter. The solution was then current study. cooled and distilled water added to make a volume of 100 ml. The Ca, Mg, Fe, Mn, Zn and Cu concentrations of the digested solution were determined using Atomic Statistical analysis Absorption Spectrophotometer (AA240 Varian, USA). Collected data were analysed using R software (version Vitamins 4.0.3), and data were checked for normality using the Vitamin extraction was by mixing 0.5 g of sampled bio- Shapiro-Wilk test and homogeneity of variances using mass with 100 ml 95% ethanol into a conical flask that was Levene’s tests. One-way analysis of variance (ANOVA) APPLIED PHYCOLOGY 37 −1 and Kruskal Wallis were used to analyse differences in than in 10% BSCE (0.15 μ day ) while 5% BSCE (0.17 μ −1 parameters measured among culture media for nor- day ) was not significantly different from either of the mally and not normally distributed data respectively. two. It cost more to prepare 1 l of BBM medium com- Tukey’s (ANOVA) and Dunn (Kruskal Wallis) post pared to the BSCE treatments (Table 1). hoc tests were used for mean pairwise comparison among culture media. Results were presented as mean Chemical analysis ± SE (standard error of the mean) and difference was considered significant when p ≤ 0.05. There were variations in nutritional values of culture −1 media (Table 2). BBM had higher nitrate (45.5 mg l ) −1 but lower ammonium and potassium (21 mg l ) than Results other culture media. The 10% BSCE had higher nitrate, potassium, phosphorus and ammonium than Growth parameters and media cost the other concentrations. Phosphorus was higher in −1 There were variations in growth trends (Fig 1) among 10% BSCE (18.2 mg l ) than in BBM medium (13.3 −1 culture media whereby C. vulgaris cultured in high mg l ). The culture media significantly affected (p < BSCE concentrations (5% and 10%) had a slow initial 0.05) crude protein (CP), carbohydrate and lipids con- growth rate. Exponential growth phases were delayed in tents of C. vulgaris biomass (Table 3). The BBM- 5% and 10% BSCE compared to other culture media. cultured C. vulgaris had higher CP (45%) than algae Moreover, 10% BSCE took longer to reach stationary cultured in 2% (21.4%) and 5% BSCE (24.5%). CP phase (day 24) compared to BBM (day 21), 5% BSCE values did not differ between BBM and 10% BSCE (day 21) and 2% BSCE (day 18). There were no statis- (34.8%). The carbohydrate content in C. vulgaris bio- tical differences in specific growth rate (SGR) between mass did not vary among treatments, however, it was control (BBM) and treatments (BSCE). However, the relatively higher in 10% BSCE (36.4%) than in BBM difference was significant (p = 0.046) among treatments (20.8%, p < 0.05). Lipid contents did not differ −1 whereby SGR was higher in 2% BSCE (0.18 μ day ) between treatments and controls, however, there 2.0 BBM 1.8 2% 1.6 5% 1.4 10% 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 3 6 9 12 15 18 21 24 Cultivation days Figure 1. Mean dry cell weight of Chlorella vulgaris cultivated using Bold’s Basal Medium (BBM) (asterisks), and media formulated with 2% (filled triangle), 5% (empty circle), or 10% (empty triangle) volume/volume additions of Banana Stem Compost Extract (BSCE) (n = 3, error bars = standard error). D ry cell w eigh t (g /L ) 38 K. MTAKI ET AL. Table 1. Production cost in US$ (converted from Tanzanian Shillings) per unit (1 l) of Bold Basal Media (BBM) and Banana Stem Compost Extract (BSCE) culture media. Microalgal growth media Costs BBM 2% BSCE 5% BSCE 10% BSCE Reagents 0.14 0 0 0 Value Added Tax (18%) 0.02 0 0 0 Stem collection 0 0.01 0.01 0.03 Electricity consumption 0.01 0.01 0.01 0.01 Total costs (US$) 0.17 0.02 0.03 0.04 Table 2. Chemical composition of Bold Basal Media (BBM) and Banana Stem Compost Extract (BSCE). –1 Concentration (mg l ) Parameter BBM 2% BSCE 5% BSCE 10% BSCE NO - 45.6 2.3 ± 0.003 5.7 ± 0.007 11.4 ± 0.015 NH - 1.2 ± 0.01 2.9 ± 0.03 5.9 ± 0.05 P 13.3 3.6 ± 0.02 9.1 ± 0.04 18.2 ± 0.09 K 21.0 126 ± 0.3 315 ± 0.7 630 ± 1.5 Na 20.6 11 ± 0.2 27.5 ± 0.6 55 ± 1.2 2+ Ca 1.7 61.7 ± 1.3 154.1 ± 3.3 308.3 ± 6.6 2+ Zn 0.008 28.4 ± 0.9 70. 9 ± 2.3 141.8 ± 4.6 Mn 0.002 6.1 ± 0.2 15.2 ± 0.5 30.3 ± 0.9 2+ Mg 1.8 67.4 ± 0.837 168.6 ± 2.1 337.2 ± 4.2 2+ Cu 0.002 5. 3 ± 0.1 13.3 ± 0.3 26.6 ± 0.6 2+ Fe 0.2 16.9 ± 0.3 42.1 ± 0.8 84.3 ± 1.5 2+ −4 Co 4 x 10 - - - 3+ −1 B 2 x 10 - - - 4+ −3 Mo 2 x 10 - - - Table 3. Chemical composition of Chlorella vulgaris biomass cultured in Bold Basal Media (BBM) and Banana Stem Compost Extract (BSCE). Growth medium BBM 2% BSCE 5% BSCE 10% BSCE Macronutrients (%) a b b a,b Protein 45.0 ± 0.1 21.4 ± 0.4 24.5 ± 0.3 34.8 ± 0.2 a a,b a,b b Carbohydrate 20.8 ± 0.2 29.1 ± 0.2 29.8 ± 0.3 36.4 ± 0.3 a,b a a b Lipids 15.5 ± 1.4 30.8 ± 0.9 28.1 ± 0.1 13.9 ± 0.9 Minerals (mg 100 g 1) a b a a Ca 95.3 ± 0.3 67.1 ± 0.9 111.2 ± 0.5 117.1 ± 0.8 a b b a Fe 24.3 ± 0.1 21.6 ± 0.04 21.4 ± 0.6 26.5 ± 0.2 a b b a Mg 165.5 ± 1.0 134.0 ± 0.9 147.7 ± 0.3 182.4 ± 0.6 a b b b Na 888.0 ± 1.0 932.6 ± 1.4 953.0 ± 0.4 923.0 ± 0.9 a a,b b b K 1113.6 ± 0.6 1156.7 ± 0.8 1219.7 ± 1.2 1235.2 ± 2.5 a b a a Zn 2.2 ± 0.02 1.0 ± 0.02 3.0 ± 0.17 3.1 ± 0.01 a b b a Mn 1.4 ± 0.05 1.2 ± 0.01 1.2 ± 0.02 1.6 ± 0.01 Vitamins (mg 100 g 1) a a,b a,b b A 27.6 ± 0.5 26.3 ± 1.8 28.0 ± 0.2 31.0 ± 0.9 a b b c C 5.6 ± 0.13 6.6 ± 0.03 6.4 ± 0.03 7.6 ± 0.16 a b b a,b B1 1.8 ± 0.01 1.3 ± 0.01 1.5 ± 0.01 1.6 ± 0.02 a a,b b b B2 2.1 ± 0.07 2.3 ± 0.11 2.6 ± 0.17 2.8 ± 0.05 a a,b b b B3 8.3 ± 0.07 9.2 ± 0.03 11.1 ± 0.2 12.4 ± 0.15 a b a,b a,b B6 0.12 ± 0.002 0.33 ± 0.751 0.27 ± 0.004 0.28 ± 0.006 Means in the same row with different letters were significantly different (p < 0.05). −1 were differences among treatments whereby it was were lowest in 2% BSCE (67.1 mg 100 g and 1.0 mg −1 lower in 10% BSCE (13.9%) than in 2% (30.8%) and 100 g respectively), while Mn was highest in 10% −1 5% BSCE (28.1%). BSCE (1.6 mg 100 g ). The 10% BSCE media had −1 The Ca, Zn and Mn contents of C. vulgaris bio- the highest Mg (182.4 mg 100 g ) and Fe (26.5 mg −1 mass did not differ significantly (p > 0.05) between 100 g ) contents (p < 0.05) among active treatments. treatments and controls but the differences among The K content did not vary among treatments but it −1 treatments were significant (see Table 3). Ca and Zn was lower in BBM (1113.6 mg 100 g ) than in 5% APPLIED PHYCOLOGY 39 −1 BSCE (1219.7 mg 100 g ) and 10% BSCE (1235.2 mg changes carbon partitioning which increases lipids −1 100 g ). Culture media had a significant effect (p < and carbohydrate concentration while reducing pro- 0.05) on Vitamin A, C, B1, B2, B3 and B6 contents. tein content (Cointet et al., 2019; Metsoviti, Vitamins A, C, B2 and B3 were significantly higher Papapolymerou, Karapanagiotidis, & Katsoulas, in treatments than control. There were no variations 2019; Wang, Xiong, Hui, & Zeng, 2012). Organic in Vitamins A, B1, B2, B3 and B6 contents among carbon was not measured in BSCE media due to treatments but Vitamins C was significantly higher in limited resources, but it should be analysed in future −1 10% BSCE (7.6 mg 100 g ) compared to 2% (6.6 mg studies to determine the potential for mixotrophy. It −1 −1 100 g ) and 5% (6.4 mg 100 g ) BSCE. The BBM- is likely that variation in observed lipids and carbo- cultured C. vulgaris had significantly (p < 0.05) hydrate among treatments relates to C:N ratios, with −1 higher Vitamin B1 (1.8 mg 100 g ) than 2% (1.3 nitrogen levels increasing with BSCE concentration. −1 −1 mg 100 g ) and 5% (1.5 mg 100 g ) BSCE. These results could be useful in selecting the appro- priate BSCE concentration and manipulation of C:N ratios in culture media to favour heterotrophy by Discussion increasing organic carbon when the end product is lipids or carbohydrate. Nutritional composition Future studies should also investigate effects of using Similar crude protein (CP) values in BBM and 10% BSCE levels of above 10% on protein, lipids and carbo- BSCE (Table 3) could be explained by the high nitrate hydrates values. Carbohydrate extracted from microal- supply in both culture media (Table 2). Nitrogen is an gal biomass could find uses in the bioethanol and essential nutrient for protein synthesis and there is biohydrogen industries (Behera et al., 2019). a positive relationship between CP values in microalgal Moreover, high phosphorus contents observed in −1 biomass and nitrogen concentration in the culture BSCE (3.6–18.2 mg l ) could also make banana stem media (Hodaifa, Sánchez, Martínez, & Órpez, 2013; Ji a cheap phosphorus source for cultivation of other −1 et al., 2014; Li et al., 2020; Zhang et al., 2018). The CP crops. Some trace elements (Zn, 28.4–141.8 mg l ) values observed in BBM and 10% BSCE were within were higher in the BSCE media (Table 2), but not in ranges reported for C. vulgaris cultured using synthetic biomass of BSCE-cultured C. vulgaris (Table 3). Zn −1 media, kitchen waste or monosodium glutamate waste- concentrations (1.0–3.1 mg 100 g ) in BSCE cultured water, i.e., 36–51% (Ji et al., 2014; Prabakaran et al., C. vulgaris were within WHO/FAO recommended 2019). The similarity in CP values for BBM and 10% amounts for human and livestock consumption as was BSCE showed the potential of using banana stem com- noted by Elbagermi, Edwards & Alajtal (2012). The post to produce high quality protein products which can differences in Zn between culture media and cultured be used in aquaculture production (Barros, Gonçalves, biomass is because only a small proportion of the nutri- Simões, & Pires, 2015; Koller, Muhr, & Braunegg, 2014; ent is transferred between trophic levels and the rest Sathasivam, Radhakrishnan, Hashem, & AbdAallah, could have remained in the culture media. 2019). Additionally, variations in CP among culture Contents of minerals and vitamins (A, B1 and B3) media in this study are attributed to different nitrogen reported in this study were within ranges reported else- concentrations in the media as C. vulgaris cultures were where for C. vulgaris (Andrade, Andrade, Dias, & harvested when they reached stationary growth phase. Nascimento, 2018; Panahi et al., 2012; Prabakaran Lipid concentration declined with an increase in BSCE et al., 2019; Tokuşoglu & Üunal, 2003). The results concentration while carbohydrate content seemed to indicated that replacing BBM with BSCE did not affect increase with an increase in BSCE concentration most of the micronutrient concentrations. The (Table 3). C. vulgaris produced can be used as the source of micro- C. vulgaris increases lipids and carbohydrate con- minerals for humans and livestock so as to ensure nor- centration under environmental stress since polysac- mal body functioning (Gatlin, 2003; Marsan, Conrad, charides and lipids are used as an energy source Stutts, Parker, & Deeds, 2018; Paul & Mukhopadhyay, when there is nitrogen deficiency (Cho et al., 2015; 2016). The biochemical composition of C. vulgaris from Cointet et al., 2019; Illman, Scragg, & Shales, 2000; different media were report here accounted for only 80– Wang, Xiong, Hui, & Zeng, 2012). However, it 83% of total biomass (Table 3), because other compo- should be noted that C. vulgaris are mixotrophic nents such as chlorophyll, carotenoids and antioxidants (both auto- and heterotrophic) hence carbon to were not analysed as they were of limited interest in the nitrogen ratio (C:N) in culturing media can influence current study. Moreover, the current study did not biomass chemical composition. Increasing C:N determine nutrient uptake or cultivation rate among 40 K. MTAKI ET AL. microalgae as our primary objective was to establish if cultivation cycles per year could make 2% BSCE C. vulgaris can grow successfully on BSCE media. more productive in terms of annual yield. The SGR Future studies should investigate further the composi- reported in the current study was higher than the −1 tion of BSCE media post-cultivation so as to determine 0.06 μ day reported for microalgae cultured in carrying capacity for effective utilization of BSCE and hydroponic systems (Supraja, Behera, & customization of the medium. Balasubramanian, 2020). The differences in SGR between the current and previous studies could be due to differences in the strain of C. vulgaris (uni- Chlorella vulgaris growth and cost of culture dentified in this study), media composition and media nutrient concentration. Since we did not carry out Observed differences in growth rates (Fig 1) among any molecular characterization (e.g., DNA barcod- culture media could be attributed to several factors. ing) of our strain, future experiments should address C. vulgaris was cultured in BBM before the start of this limitation and assess potential use of BSCE for the experiment and there was no acclimation period cultivation of specified C. vulgaris strains. However, prior to culture in BSCE. This could have initially similar SGR in control and treatments showed that favoured algal growth in BBM than in BSCE hence banana stem can be used to replace expensive syn- explaining the fast initial growth rate in BBM. thetic media for microalgal cultivation without However, C. vulgaris in 2% BSCE also had rapid initial affecting productivity. growth compared to algae in 5% and 10% BSCE which The BSCE had lower preparation cost (0.02–0.04 is attributed to its lower levels of ammonium (1.2 mg USD) compared to BBM (0.19 USD, Table 1) −1 −1 −1 l ) compared to 2.9 mg l and 5.9 mg l observed in because banana stem was free hence BSCE cost 5% and 10% BSCE respectively. High ammonium con- might vary depending on access to banana stems centration in culture media is toxic and reduces growth (free or purchased). There is a chance that banana rate of C. vulgaris (De Lourdes, Josefina, Ulises, & De stem could be assigned monetary value and become Jesús, 2017). Therefore, use of high BSCE concentra- very expensive due to an increase in its demand for tions would require an acclimation period of at least 6 culture medium. Also, environmental stress like days, after which a fast growth rate was noted in 5% drought, diseases e.g., black Sigatoka leaf disease, and 10% BSCE (Fig 1), or other means of reducing and pests such as banana weevils (Batte et al., ammonium levels in the media. Nonetheless, observed 2019; Ndayihanzamaso et al., 2020) could lower growth trends in this study were similar to those banana productivity and reduce the relevance of reported by Singh, Babcock, & Radway (2000), using BSCE as culture media. We remain optimistic Venckus, Kostkevičienė, & Bendikienė (2017) and despite the potential challenges that BSCE could be Abu-rezq et al. (2010) who noted similar growth pat- an ideal and cheap culture medium especially among tern in different culture media. small scale aquaculture producers in developing The C. vulgaris dry weight obtained in this study countries. Its relevance among small scale aquacul- −1 was within the range (0.67–4.23 g l ) reported for ture producers is due to integrated aquaculture prac- this species cultivated using aquaculture waste water tices i.e., keeping fish with horticultural crops like or effluent from sewage sludge (Cho et al., 2015; banana and national banana breeding programs aim- Mtaki, Kyewalyanga, & Mtolera, 2021). The high ing to improve production (Batte et al., 2019; biomass could provide large amount of raw materials Ndayihanzamaso et al., 2020). It was clearly estab- for livestock feed, in fuel production and in pharma- lished in this study that BSCE can be used to replace ceutical industries. The lower specific growth rate synthetic media although the study was unable to −1 (SGR) in 10% BSCE (0.15 μ day ) than in 2% determine the appropriate BSCE concentration for −1 BSCE (0.18 μ day ) was a result of the longer best results across all parameters in cultivated exponential growth phase in the former than the C. vulgaris. Lower SGR in 2% BSCE could give latter (day 24 versus day 18). The delay to achieving relatively high biomass per annum compared to exponential growth phase in 10% BSCE could indi- 10% BSCE. However, 2% BSCE had lower CP and cate that a longer acclimatization period is needed other micronutrients than those seen in BBM for this medium to achieve maximum biomass con- (Table 3). We recommend that the choice of BSCE centration. The higher organic carbon in this med- concentration should be based on targeted macro- ium facilitates mixotrophy which in turn increases nutrient (protein, lipids or carbohydrate) and the exponential growth rate. 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Journal
Applied Phycology
– Taylor & Francis
Published: Dec 31, 2023
Keywords: Alternative culture media; banana stem; Bold Basal Media (BBM); Chlorella vulgaris; circular economy; nutritional value
