FORMATION OF VALUE-ADDED PRODUCTS FROM MICROALGAE GROWN IN HYDROLYSATE OF WASTE BIOMASS

Abstract

Energy crisis and worldwide attention on seeking new and renewable resources have drawn considerable attention among researchers to develop value-added products from biomass. Previously, other source of biomass was the prime feedstock for developing such products. Generally, these biomasses were pre-treated and hydrolysed chemically or enzymatically or both to produce hydrolysate rich in monomeric sugars. Hydrolysate thus produced can be exploited further to produce numerous types of value- added products ranging from renewable energy to novel biomaterials. There are several specialised microorganisms ranging from yeast, algae, and bacteria that can produce numerous chemicals, which can be used substantially in our daily life. Different algal species also have the capability to produce several specialised chemicals including lipids when they are grown in the synthetic media. Hence, to develop such chemicals, one needs to find specialised microorganisms and cheap growth media in which such microorganisms can be grown intensively. Therefore, this study was focused on investigating the diversity of microalgal species present in nearby waterbodies and finding ways to isolate such microalgal species. After investigating the diversity and isolating microalgal species, this study focused on developing hydrolysate media from locally available waste biomass. Later, such hydrolysate media was used to grow algal strain isolated in this study for lipid and carbohydrate production. Water samples were collected from local wastewater treatment ponds treating domestic wastewater located in Uttarakhand, India. Several algal species were found in these water samples. Various elements and organic compounds, found in wastewater, may affect the composition and proliferation of algal species. Several media and their modifications were employed to understand the role of these elements and compounds in the species composition and proliferation. Euglena sp., Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorococcum sp., several diatoms, and cyanobacteria were found in the wastewater samples. In stagnant and flowing municipal wastewater, species like Euglena, Chlorococcum, various cyanobacteria, and Volvox were predominant. In bold basal medium, Scenedesmus sp. and various diatom species were observed along ii with Chlorococcum sp. In the presence of acetate, which is present in anaerobically digested effluent, Selenastrum species were abundant. In Knop’s medium, Chlamydomonas reinhardtii and Chlorella sp. were dominant. Over a period, from mid-summer to winters, water samples were collected from the waste stabilisation pond and analysed using second generation sequencing to determine the molecular diversity of algae and bacteria in the wastewater treatment pond. Metagenomic analysis of wastewater revealed that during the winter season, eukaryotic phylum i.e. Tetradesmus wisconsinensis (21.23%) and Actinastrum hantzchii (11.48%) were the dominant species. However, during the summer season, Prokaryotic phylum like Proteobacteria (53.80%) and Cyanobacteria (8%) were dominant. Some cyanobacterial species accumulated near the high-intensity light region. Hence, subsequent phototaxis experiments were performed using various light colours. Under the white light, most of the species showed positive phototaxis. However, under the blue and red light, all the algal species present in the sample showed negative phototaxis. Under the green light, Chlorococcum and Chlamydomonas showed positive phototaxis and Euglena showed negative phototaxis. However, under the pink light, neither negative nor positive phototaxis was observed. Hence, the obtained results provided new insights into the effects of the studied parameters on the species' diversity and proliferation. Further, an attempt was undertaken to isolate algal strains using different growth media from the wastewater sample. Two different algal strains (isolated using BG-11 and Knop’s media at pH-9) were used in this study for the accumulation of lipid. Both the strains were genetically similar to Chlorella sorokiniana as per the 18s rRNA sequencing method. Therefore, in the present study, the microalgal strains were represented as Chlorella sorokiniana 1 for the algae isolated using BG-11 medium and Chlorella sorokiniana 2 for the algae isolated using Knop’s medium. It was observed that algal biomass, Colocasia esculenta (Taro) leaves and water hyacinth are available as waste biomass in the vicinity of our campus as well as in other areas in India. These biomasses can be used as attractive renewable and sustainable resources for sugars and nutrients. Hydrolysis of dried algal biomass and Colocasia esculenta (Taro) leaves were investigated using 1%, 2% and 5% solutions of ferric-chloride, nitric acid and acetic acid for the reaction times of 30 and 60 minutes at 121 °C and 103.4 kPa (15 psi). 1% and 2% H2SO4 treatments were used as the reference. Solid: liquid ratio was kept at 1:10 for all the experiments. For algal biomass, 5% acetic acid treatment for 60 minutes was found to be optimum with total carbohydrate release of 44.2 mg/g biomass (solubilised iii monomers- 0.82 mg/g of dry biomass) and N, P solubilisation of 1.8 mg total nitrogen/g dry biomass and 7 mg total phosphorus/g dry biomass. Whereas, for Colocasia esculenta (Taro) leaves, the maximum carbohydrate yield of 95 mg/g biomass (solubilised monomers- 43.6 mg/g of biomass) and nutrient solubilisation of 5.02 mg total nitrogen/g biomass was obtained with 5% ferric chloride treatment for 60 minutes. The results obtained showed that various hydrolysing agents used in this study acted differently on different types of biomass. Acetic acid worked best in hydrolysing the algal biomass, and for the hydrolysis of Taro leaves, ferric chloride and nitric acid were most effective. Besides sugar-recovery, nutrients-recovery from the biomass, mainly algae containing a large amount of N, P should be taken into account, as the recovered nutrients play a significant role in using hydrolysate as a microbial growth medium. This study also investigates the possibility of using three different chemicals for the hydrolysis of water hyacinth biomass with different concentrations (1%, 2% and 5%) for 30 and 60 minutes at the following temperature and pressure 121 °C and 15 psi. 1% and 2% H2SO4 treatment were used as the reference. The maximum total carbohydrate (100 mg/g of dry biomass) and nutrients (total nitrogen- 9.7 mg/g of dry biomass and total phosphorus- 1.3 mg/g dry of biomass) were obtained with 2% mineral acid treatment for 60 minutes with solid: liquid ratio of 1:10. According to HPLC analysis, solubilised monomer content was estimated around 85.8 mg/g of biomass (hexoses- 58.7 mg/g of biomass and pentoses- 27.1 mg/g of biomass) with no furfural and 5-hydroxymethylfurfural formation. The economic assessment of these different hydrolysates also proves that mineral acid treatments were the best for the hydrolysis of water hyacinth biomass. Based on the ANOVA analysis, chemical and concentration were found to be the most significant factor in the hydrolysis of biomass as compared to the time parameter. The results obtained showed that the solubilisation of complex nutrients from water hyacinth biomass to simple sugars and nutrients could be an attractive way to produce synthetic media. Hydrolysate prepared from the chemical hydrolysis of water hyacinth biomass contains a high amount of solubilised carbohydrates and nutrients. This hydrolysate was utilised as a medium for the cultivation of two strains of Chlorella sorokiniana, isolated from municipal wastewater treatment plant using two different media, i.e. BG-11 and Knop’s medium. Different light intensities, light-dark cycle and various concentrations of external carbon source (monosaccharides and inorganic carbon) were used to optimise the microalgal iv growth. For the accumulation of lipids and carbohydrates, the microalgal strains were transferred to a nutrient amended medium (N-amended and P-amended). It was observed that the combined effect of glucose, inorganic carbon and 12:12h light-dark cycle proved to be the optimum parameters for high biomass productivity (~200 mg/L/day). For Chlorella sorokiniana 1 (isolated from BG-11 medium), maximum carbohydrate content (22%) was found in the P-amended medium (N= 0 mg/L, P: 3 mg/L), whereas high lipid content (17.3%) was estimated in N-amended medium (N= 5 mg/L, P=0 mg/L). However, for Chlorella sorokiniana 2 (isolated from the Knop’s medium), both lipid (17%) and carbohydrate accumulation (12.3%) were found maximum in the N- amended medium. Chlorella sorokiniana 2 showed high saturated lipid accumulation as compared to other strains. Kinetic modeling of the lipid profile revealed that the production rate of fatty acids and its various constituents were species-dependent under identical conditions.