Bioflocculation harvesting of oleaginous microalga Chlorella sp. using novel lipid-rich cellulolytic fungus Aspergillus terreus (MD1) for biodiesel production

Biomass Conversion and Biorefinery https://doi.org/10.1007/s13399-023-04822-5 ORIGINAL ARTICLE Bioflocculation harvesting of oleaginous microalga Chlorella sp. using novel lipid‑rich cellulolytic fungus Aspergillus terreus (MD1) for biodiesel production Hala I. Ayad1 · Ibrahim A. Matter2 · Mohamed M. Gharieb1 · Osama M. Darwesh2 Received: 9 June 2023 / Revised: 11 August 2023 / Accepted: 27 August 2023 © The Author(s) 2023 Abstract The isolation of lipid-rich cellulolytic fungi was targeted to be investigated as bioflocculant agents for microalgae harvesting. The fungal isolate coded MD1 was selected based on its lipid content, cellulolytic activity, and its harvesting efficiency for the freshwater oleaginous microalga Chlorella sp. The selected fungus which was molecularly identified as Aspergillus terreus has been applied as bioflocculant after solid state cultivation on pre-treated rice straw (as abundant agro-cellulosic waste). Optimization of harvesting efficiency of Chlorella microalga using A. terreus/rice straw biomass as the “bioflocculant” was investigated. The optimization conditions included microalga/bioflocculant ratio, microalgal age, contact time between the bioflocculant and the microalga, pH of microalgal culture at harvesting time, and cell density of microalgal culture. The obtained results revealed that the harvesting efficiency could reach 97.6% due to 24 h as contact time at 30% flocculant/ microalga ratio and pH 7. While after 2 h contact time, 93.3% harvesting efficiency could be obtained using the same bioflocculant:microalga ratio at pH 6. The lipid extracted from harvested Chlorella/A. terreus mixture was applied to produce biodiesel (fatty acid methyl ester) after methylation. The resulted biodiesel contains high percentage (67.2%) of C18:1,2 unsaturated fatty acids which is considered a suitable fraction for biodiesel production. Obtained results revealed the suitability of the novel A. terreus strain as sustainable bioflocculation agent to harvest microalga(e) for biofuel production. Keywords Bioflocculation · Microalgae · Biodiesel · Cellulolytic fungi · A. terreus 1 Introduction The need for microalgal biomass to produce biofuels like biodiesel is growing, especially in light of the clear adverse impacts of excessive fossil fuel usage. However, there are still several technological and cost-related obstacles to the fully commercial production of microalgae [1–3]. A major challenge is harvesting microalgae, especially with their small cell size and negative charges on their surfaces that keep them suspended in their relatively dilute cultures. According to the species and growing circumstances of the microalgae, the cost of harvesting microalgae can often be * Ibrahim A. Matter ; 1 Botany Department, Faculty of Science, Menoufia University, Shibin Al Kawm, Egypt 2 Agricultural Microbiology Department, National Research Centre, 33 EL‑Buhouth St., Dokki 12622, Cairo, Egypt as high as 60% of the total production cost. Therefore, a high-efficiency and economical harvesting method must be created in order to commercialize microalgae-based technologies [4, 5]. There are several harvesting methods for microalgae that include sedimentation (by centrifugation or gravity), mechanical screening, membrane filtration, air flotation, and flocculation methods. It is worth to mention that, no one harvesting method can be regarded as technically or financially viable for all microalga species and/or purposes. Flocculation is considered one of the most widely used harvesting techniques for various large and commercial scale microalgae applications due to its relatively low cost and efficiency with many species. Flocculation means the accumulation of fine/unstable particles through “surface charge neutralization,” “electrostatic patching,” and/or “bridging” due to the addition of “flocculating materials” in the form of sedimentary flocs [6]. Flocculating materials (flocculants) can be physical particles, organic or inorganic chemicals, or biological materials (bioflocculants). Bioflocculants are a good alternative to inorganic and chemically synthesized 13 Vol.:(0123456789) Biomass Conversion and Biorefinery flocculants for microalgal harvest because of their biodegradable and nontoxic qualities [7]. Bio-flocculants can be bacteria, filamentous fungi, yeasts, or certain types of self-flocculating microalgae as well as “their exudate-rich culture supernatants” [5]. Filamentous fungi are among the most important microorganisms used in microalgae bioflocculation harvesting [8–10]. Many species of filamentous fungi are known for their ability to selfpelletize, allowing algal cells to be captured by fungal pellets and adsorbed onto their surfaces. The application of such fungi as bio-flocculating agents for microalgae harvesting can be through co-culturing the fungus with the microalga(e) or by adding ready-grown fungal pellets (or biomass) to the target microalgal culture [11]. There are many factors that could affect the interaction between the microalgae and fungi which causes the algaefungi coagulation/flocculation such as the electrostatic interaction, hydrophobic interaction, and specific components on cell wall. The negatively charged functional groups on algal cells can be protonated and deprotonated depending on the surrounding pH to create charges and potentials on the surfaces of algal cells [12]. Moreover, filamentous fungi are known to secrete diverse organic acids (e.g., citric acid, gluconic acid, and acetic acid) into the culture medium [13, 14]. Mainly, due to the acidic conditions in fungal media, the surface functional (carboxylic and amine) groups of mycelia remain protonated, leading to the net positive charges of the fungal hyphae [9, 14]. Therefore, charge neutralization can be established and harvesting happens when positively charged fungus adequately interact with negatively charged algae cells, whereas the hydrophobic interaction between the hydrophobic and hydrophilic parts of the outer cell wall proteins can lead to the formation of an amphipathic membrane, and thus can help fungi attach to other microbial surfaces [14, 15]. Based on this amphipathic property, the hydrophobins from filamentous fungi can be utilized to immobilize suspended algae cells on surfaces via adhesive force. The hydrophobic parts of microalgae can contact with filamentous fungi to initiate hydrophobic interactions; meanwhile, the amphipathic film from fungal hydrophobins may regulate the surface property of algae cells, subsequently making it easier to form co-pellets [16]. Moreover, specific components on fungal cell wall (glucans, lipids, chitin, polysaccharides, and proteins) contribute to interactions with the external environment and adhesion to other microbial cells such as microalgae [17]. On the other hand, the mass of fungi used for harvesting may represent a significant portion of the final biomass when harvesting algae. Therefore, (...truncated)