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 [1,2,3]. 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 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 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 [8,9,10]. Many species of filamentous fungi are known for their ability to self-pelletize, 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 algae-fungi 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, the lipid content of fungi used to harvest oleaginous algae may be important (quantitatively and qualitatively) when the purpose of harvesting is biodiesel production. In addition, the ability of fungi to degrade cellulosic residues and use them for growth is an important ecological and economic advantage. Hence, the current work aimed to isolate lipid-rich cellulolytic fungi capable of causing flocculation harvesting of the oleaginous microalga Chlorella sp. as biodiesel feedstock. Such lipid-rich cellulolytic fungi represent a sustainable exploitation of the abundant agricultural residues in the production of bioflocculant for different microalgae to be used in biofuel production. The work also was aimed to investigate the influence of different conditions in order to optimize the harvesting efficiency of the targeted microalga. 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.

2 Materials and methods

2.1 Samples collection

Nine soil samples have been collected as fungi isolation sources from different locations in the Nile Delta region, in Northern Egypt. Among obtained samples, one sample was from animal manure-enriched soil and one from soil contaminated by urban solid wastes (from Menoufia governorate). The rest of the samples were collected from the rhizosphere of different agricultural crops in the governorates of Gharbia, Sharkia, Qalyubia, and Kafr El-Sheikh. The collected soil samples were transported in sterile plastic bags to laboratory under proper aseptic environment and kept in a refrigerator (at 4 °C) until use.

2.2 Isolation of lipid-rich cellulolytic fungi

The isolation strategy targeted the isolation of lipid-rich cellulolytic fungi with the ability to cause flocculation and sedimentation (harvesting) of the targeted microalgae. Different fungal isolates were obtained from soil samples by direct isolation according to [18, 19] with minor modifications. In brief, 10 g of individual samples was suspended in sterile tab water (90 mL) and shacked (140 rpm for 30 min) at room temperature, and left static for another 10 min. Tenfold serial dilutions were then prepared in sterilized tap water, and 100µL of the 5th dilution were spread on agar plates of carboxymethylcellulose (CMC) medium (CMC, 10 gL−1; peptone, 5 gL−1; KH2PO4, 1 gL−1; MgSO4·7H2O, 0.5 gL−1; Rose Bengal, 0.033 gL−1; and streptomycin, 0.03 gL−1; pH 5.5–6.0). The inoculated plates were incubated at 30 °C for 5–7 days to allow the growth of cellulolytic fungi in colonies. Colonies that displayed distinction morphological appearance were repeatedly cultured on the same medium until obtaining pure fungal isolates. The purified isolates were cultivated on slants of potato dextrose agar, kept at 4 °C, and re-cultured on proper intervals [20].

Because one of the objectives of the study is the production of biodiesel, so the lipid percentage of biomass in the fungi used for harvesting is important in the whole process. Hence, the obtained cellulolytic fungal isolates were screened for their lipid contents according to Bligh and dyer method described by [21] with some modifications. In brief, fungal isolates were cultivated in liquid CMC medium for 5 days on rotary orbital shaker (30 °C and 140 rpm). After growth, produced fungal biomass was collected, dried, weighted, and grounded. The lipid content of fungal biomass was extracted (from 0.5 g dry biomass) with chloroform–methanol (2:1, v/v) in capped glass tubes and then methanol and water were added to cause the separation of chloroform layer (the final solvent ratio became chloroform:methanol:water of 1:1:0.9). The chloroform/lipid layer was pippeted in new tube and washed with 20 mL of 5% NaCl solution to remove impurities. The purified chloroform/lipid was transferred into pre-weighted vials, evaporated to dryness, and the weight of the crude lipid obtained from each sample was measured gravimetrically. The lipid content was calculated and represented as a percentage of the dry weight of fungal biomass.

2.3 Microalga and cultivation conditions

The oleaginous freshwater microalga Chlorella sp. used in this study (as model biofuel microalga) was previously isolated and stored in the Department of Agricultural Microbiology, National Research Centre, and Egypt. The bold basal medium was used in this study for the cultivation of the targeted microalga [22]. Unless otherwise stated, cultivation was carried out in 5-L conical glass flasks aerated with air bubbles for 2 weeks under constant white fluorescent illumination.

2.4 Screening the fungal isolates for microalgae harvesting

The selected lipid-rich cellulolytic fungi have been screened as bio-flocculating agents to harvest the microalga Chlorella sp. The evaluation was performed in two stages: the first was based on the direct utilization of fungal pellets in microalga harvesting, while the second selection stage was based on the solid-state cultivation of the most potent cellulolytic fungi on rice straw (as common lignocellulosic waste) and utilization of this mixture as bioflocculation agents for Chlorella microalga.

For pellet-assisted harvesting, the mycelium pellets of the selected cellulolytic fungi have been examined for their bioflocculation capabilities of Chlorella sp. The influence of fungal cultivation medium on the bioflocculation efficiency was including also in this experiment. The fungi were individually cultivated on liquid malt extract medium and CMC in an orbital rotary shaker (140 rpm) at ambient temperature to allow the formation of fungal pellets. The incubation period in case of malt extract culture was 3 days while CMC-based cultures were incubated for 7 days. After incubation, the resulted fungal pellets were collected by filtration (using a 200-mesh sieve) and washed with sterilized deionized water to remove the excess of cultivation medium. A volume of 15-mL fungal pellets (from each fungus separately) was transferred to a 100-mL screw-caped glass bottle containing 50 mL of the microalgal culture. The pH of tested microalga culture was adjusted at the value of 7 just before the harvesting experiment. The fungal pellets/microalga mixture was continuously mixed at 140 rpm on a rotary shaker for 1 and 2 h at ambient temperature. The mixtures were left to stand for 10 min to allow the precipitate of flocculated microalgal cells and the microalga-pellets aggregates. Samples (5 mL) for measuring harvesting efficiency were taken from settled bottles at a constant distant (from the middle point of the mixture). A control treatment of Chlorella microalga has been applied without using any fungal addition for comparison. The harvesting efficiency (%) was calculated by comparing the “optical density” (OD) measured spectrophotometrically at 680 nm of control treatment and the collected samples according to the following equation [1]:

$$\mathrm{FE}\left(\%\right)=\frac{\left(\mathrm{ODi }-\mathrm{ ODf}\right)}{\mathrm{ ODi}}\times 100$$

where FE refers to flocculation efficiency at time t, ODi refers to initial optical density, and ODf refers to final optical density at 680 nm. Additional calculation for HE was performed by measuring the chlorophyll content (according to [21] of samples compared to the control treatment).

The second screening was based on the uses of rice straw-based fungal growth as biological flocculants. In brief, the rice straw was washed, cut into 1–2-cm pieces, dried, and ground into a fine powder then subjected to alkaline pre-treatment before it is used for fungi cultivation [23]. The dried and grounded rice straw was immersed in 1 M sodium hydroxide solutions and placed for 10 min in an autoclaved at 121 °C. After cooling, the rice straw-alkaline solution was subjected to filtration with filter paper (Whatman No. 1), and the rice straw was washed several times with sterile water until neutralization [24]. Finally, the powder rice straw was dried at 60 °C overnight before being used as a carbon source for fungi cultivation in addition to untreated rice straw as a control. Fungal cultivation on the pre-treated rice straw was performed as solid state on 250-mL conical flasks as follows: 10 g of the pre-treated rice straw and 15 mL of mineral medium were autoclaved and inoculated with three fungal discs (from 7-day-old agar plates), and incubated at ambient temperature for 15 days (without shaking). The whole fungal-rice straw cultures (10 g rice straw and fungal growth) was suspended with 50 mL DW and used as “the bioflocculant” for Chlorella harvesting trials. Similar to harvesting experiment with microalgal pellets, a volume of 15 mL of fungal-rice straw mixture (the bioflocculant) was used to harvest a volume of 50 mL Chlorella culture. FE of different fungi-rice straw bioflocculants was calculated as previously described compared with microalga culture harvested by the pre-treated rice straw without fungal growth.

2.5 Optimization of Chlorella harvesting using fungal-rice straw as bioflocculant

The most potent lipid-rich cellulolytic fungus that exhibited efficiency in harvesting Chlorella microalga was applied as a bioflocculant agent after being grown on pre-treated rice straw. Harvesting optimization of the targeted microalga using the selected fungus was optimized. Optimization factors included microalgal cultural density, pH value, fungal/microalgal ratio, microalgae age, and contact time. The bioflocculant was prepared by cultivation of the selected fungus on alkaline treated rice straw at solid-state condition and suspended in DW (10 g/50 mL) according to steps described in the previous section and used in further harvesting experiments.

The influence of cultural density of microalga has been investigated at three levels of optical densities, i.e., OD680nm at 1, 2, and 3. To perform this experiment, the optical density of a 2-week old culture of Chlorella sp. was adjusted at the specific values either by centrifugation or dilution by cell-free cultural filtrate of the microalga culture.

The pH value of microalgal culture differs according to growth phase, type of microalgae, and type of cultivation medium as well as cultivation conditions. For this reason, it is important to study if the cultural pH influences the efficiency of the targeted microalga using the specific bioflocculant. Hence, the microalgal flocculation efficiency using the prepared fungal/rice straw bioflocculant was investigated over different pH values that commonly occurs in microalgal cultures, i.e., 6, 7, 8, and 9 [25]. To perform this experiment, the cultural pH (after 2 weeks cultivation) was adjusted to the specific pH values using either HCl or NaOH solutions (1N). After adjusting the cultural pH to the specific value, the bioflocculant was added and the microalgal flocculation efficiency was measured as previously mentioned.

Different bioflocculant:microalgal ratio as a volume was investigated to specify the ideal ration for efficient bioflocculation and harvesting process [11]. In this regard, the bioflocculant volume was added to the targeted microalgal culture at 10, 20, and 30% v/v ratios.

The effect of microalgal cultural age on FE using the applied fungi/rice straw bioflocculant was investigated. Microalgal cultures at 1, 2, and 3 weeks old were collected and used for flocculation harvesting experiments.

In order to get the best results, it is necessary to know when to harvest microalgae, since harvest time affects the process [26]. Therefore, different contact times were investigated to determine the efficiency of microalgal flocculation using the prepared fungal/rice straw bioflocculant, i.e., 1, 2, and 24 h. All flocculation steps and calculations were performed as previously described.

2.6 Identification of the selected fungal isolates

The identification of selected fungal isolate has been performed at two levels; the first was done based on the morphological microscopic characteristics (using Olympus cx41 microscope, Japan) up to the genus level. Meanwhile, the second level of identification was based on the molecular identification of the fungal species through sequencing of the internal transcribed spacer (ITS) region. Briefly, the total DNA of harvested fungal mycelium (cultivation in PDB medium for 3 days) was extracted using CTAB protocol [27]. The polymerase chain reaction (PCR) was performed to the extracted DNA in the presence of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers. The contiguous DNA sequence was compared with data from the reference and type strains available in public GenBank databases using the BLAST program (National Centre for Biotechnology Information) (http://www.ncbi.nlm.nih.Gov/BLAST). The obtained sequences were aligned using Jukes Cantor Model [28] and used to identify the fungal species. The obtained sequences were deposited in the GenBank to record an accession number.

2.7 Determination of chlorophyll contents

The chlorophyll content was determined according to the method described by Eida et al. [29] with minor modifications. In brief, 1 mL of the microalgal mixture (with or without fungal bioflocculant) was subjected to centrifugation for 10 min at 10000 rpm (K3 Series, Centurion Scientific, UK). The collected biomass was re-suspended in 1 mL of DMSO and sonicated for 15 min in an ultrasonic water bath at maximum power. The biomass/DMSO mixture was vortexed for 5 min before centrifugation, and the supernatant extract (contains chlorophyll) was collected in a new graduated tube. Second extraction steps were performed by adding another 1 mL of DMSO to the extraction tubes, vortexing, and centrifugation to collect most of the chlorophyll contents. After the second extraction, the mixture was centrifuged again (to spin down the debris) and the supernatant/chlorophyll has been compiled with the previously extracted part. At the end, the total volume was adjusted to 2 mL using DMSO, and the extract was spectrophotometrically measured at 675 nm. The chlorophyll content was calculated and used to estimate the harvesting efficiency of dark-colored microalga cultures due to the presence of rice straw/fungus mixture.

2.8 Biodiesel production and GC analysis of lipid profile

The oleaginous microalga Chlorella sp. (> 30% lipid of dry biomass) [30] was harvested via the fungal bioflocculant under the optimum conditions. The lipid extracted from microalga/fungus bioflocculant flocks mixture was extracted according to the previously described Bligh and dyer modified method [21]. Extracted lipid was used as biodiesel (fatty acid methyl ester (FAME)) feedstock according to the method of [31] with minor modifications. Briefly, around 100 mg of the lipid extracted from the biomass was dissolved in toluene (0.2 mL) and placed in glass test tubes with screw cap. The mixture was mixed with 1.8 mL methanolic HCl (8% w/v) and heated for 1 h in a water bath at 100 °C. The tubes were allowed to cool to room temperature, then 1 mL of hexane was added and vortexed for 5 min to extract the produced FAMEs. One milliliter of water was added to the FAMEs/hexane tubes, vortexed, and centrifuged at 2000 rpm to enable hexane layer separation.

The FAME/hexane layer was transferred to new tube, purified through a membrane filter, and the produced FAME was analyzed using gas chromatography (GC) to investigate the suitability of its fatty acid profile as fuel. The FAMEs of the extracted lipid were analyzed by a gas chromatography system (Hewlett Packard, HP 6890 series, USA) equipped with an Alltech BPX70 Capillary Column (60 m × 320 μm × 0.25 μm) coated with 70% poly silphenyl-siloxane supplied with flame ionizing detector. Fatty acid profile was expressed as a percentage of the total fatty acids identified in the extracted lipid.

2.9 Statistical analysis

All experiments were performed in triplicates and the results were presented as average ± standard error. The averages of data for each experiment were statistically analyzed using analysis of variance by using IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp., and means were compared by using Duncan Multiple Range Test at 0.05% probability.

3 Results and discussion

3.1 Isolation of lipid-rich cellulolytic fungi

The present work aimed to use fungi as a bioflocculant to harvest microalgae as biofuel feedstock. The lipid content of flocculants (fungi) can be critical if they make up a sizeable fraction of the final biomass, much as the lipid content of target microalgae is crucial for the synthesis of biodiesel. Additionally, a variety of fungi can be grown using agricultural cellulosic wastes as a promising, affordable, and sustainable feedstock (cellulolytic fungi). Hence, isolation of lipid-rich cellulolytic fungi was targeted to be investigated as bioflocculant agents for microalgae harvesting. To this end, nine soil samples collected from the rhizosphere of different agricultural crops were used as fungi isolation sources. After serial dilution and direct isolation on CMC plates, a total of 50 fungal colonies were selected. The selection of colonies was based on their good growth taking into account the morphological diversity of the colonies as well as the diversity at the sample source. The selected colonies were screened (after purification on malt age plates) for growth on liquid CMC medium. Out of the screened isolates, only 38 fungal isolates exhibited a good pellet growth on the liquid CMC medium. The ability of such fungal isolates to utilize CMC as the sole carbon source of the liquid medium demonstrated its ability to degrade cellulose and considered as “cellulolytic fungi” [32]. The selected cellulolytic fungi were further screened for their lipid contents, where the results (as in Fig. 1) revealed that the lipid contents of all fungi were ranged between 3.75 and 14.69%. However, the three isolates, i.e., H25, H27, and H35, significantly recorded the highest lipid contents as they exceed 10% of their dry biomass. And twenty-nine isolates, i.e., H1, H2, H3, H4, H5, H6, H8, H9, H10, H11, H12, H13, H14, H16, H19, H20, H21, H 22, H23, H24, H26, H28, H29, H30, H31, H32, H33, H34, and H37, recorded percentage of lipid between 6 and 10%, while the isolates H7, H15, H17, H18, H36, and H38 recorded a significant low lipid percentage (less than 6%). According to [33], there was diversity in the percentage of lipids present in different fungi, and it ranged between 1.5 and 14.1%; for example, Volvariella volvacea had 1.5–4.5% while Alternaria sp. had 14.1%.

Fig. 1
figure 1

Lipid contents of fungal isolates (% based on dry biomass)

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3.2 Screening for bioflocculation capability

All fungal isolates were screened for their ability to cause flocculation and sedimentation of Chlorella microalga to be used as biodiesel feedstock. Microalgae harvesting from culture broth are energy intensive and expensive owing to their small size in addition to the low cell density [34]. Hence, it is urgent to develop alternative harvesting technologies to enhance the efficiency of harvesting systems. An efficient and low-cost bioflocculation strategy was developed and evaluated for harvesting relatively diluted microalgal cells in culture broth using filamentous fungi. The evaluation was performed in two stages; the first was based on utilization of the filamentous fungal pellets, while the second one relied on growing cellulolytic fungi on rice straw (as a common lignocellulosic waste) and utilizing the mixture of fungi/partially decomposed rice straw as bioflocculating agent for the freshwater microalga Chlorella sp.

3.2.1 Fungal pellet–assisted harvesting of Chlorella sp.

The selected 38 fungal isolates that exhibited cellulolytic activity on liquid CMC medium have been cultivated on liquid malt extract and CMC media to allow pellets to be formed. The formed fungal pellets of different isolates have been collected by filtration and applied for harvesting Chlorella microalga. Mixing microalga with the filamentous fungal pellets for 2 h resulted in the formation of microalga/pellet flocks. The formed floccus allowed to sedimentation and the efficiency of sedimentation/harvesting was evaluated and is presented in Fig. 2. While the same fungus produced varying harvesting rates due to its growing medium, different fungi’s pellets showed different harvesting efficiencies. The fungal isolate nos. H5, H6, H7, H10, H12, H20, H21, H22, H25, H27, H30, and H32 showed significant high microalgal harvesting efficiency when cultivated on either malt or CMC media. However, the percentages of harvesting efficiencies were higher in case of malt extract medium, as it ranged between 61 and 85% in the case of fungal cultivated in malt and between 54.9 and 60.4% for CMC medium. It is worth noting that the diameter of fungal pellets for the same fungus was smaller when grown in the CMC medium than that grown on malt medium. The efficiency of pellet-assisted flocculation may vary (for the same fungus and the same microalga) according to the diameter of the fungal pellets, the difference in charges on their surfaces, or differences in the extra-materials on their surfaces [5, 14].

Fig. 2
figure 2

Pellet-assisted harvesting efficiency of Chlorella sp. after 2 h using different fungal isolates cultivated on CMC or malt media

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The percentages of harvesting efficiencies (HE) (%) for Chlorella sp. using the pellets of isolated fungi are incompatible with that recorded for different fungi in literatures. For instance, [35] recorded microalgal removal percentages between 73 and 93% using pellets of different white-rot fungi after 24 h of contacting. Another study was conducted on A. fumigatus (pellets) as a bioflocculant, the percentage of Chlorella vulgaris harvesting reached 90% after 24 h [24].

3.2.2 Rice straw–based fungal growth as bioflocculant

Based on Chlorella microalga harvesting results using fungal pellets as flocculant as well as lipid and cellulolytic activity, 12 fungal isolates namely H5, H6, H7, H10, H12, H20, H21, H22, H25, H27, H30, and H32 were selected for further investigations. The aforementioned fungal isolates were cultivated as solid state on alkaline-pretreated rice straw (as a carbon source instead of CMC). After sufficient fungal growth, the rice straw/fungus biomass mixture was considered the “fungal flocculants” which was applied to harvest Chlorella sp. in the rest of the following experiments. The harvesting process took place as previously mentioned with 30% v/v bioflocculant/culture, and results of the harvesting process after 2 h were recorded as shown in Fig. 3. The HE results of Chlorella microalga were ranged between 78.9 and 95% without significant change between different isolates except isolate H22 which was significantly lower than all other isolates. It worth to mention that the highest harvesting efficiencies (95.2%) was occurred using H12 isolate; hence, it was used for further studies in this research.

Fig. 3
figure 3

Harvesting efficiency of Chlorella microalga after 2 h contacting with the different fungal flocculants (fungi grown on alkali-assisted pretreated rice straw)

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3.3 Identification of the most potent fungus for Chlorella harvesting

The morphological examination of the selected fungus (isolate H12) suggests that it belongs to the genus Aspergillus. The molecular identification to species level was performed by the amplification and sequencing of the ITS genes followed by comparing with the available sequences in GenBank using BLAST program (http://www.ncbi.nlm.nih.Gov/BLAST). Based on obtained sequence and GenBank data, the phylogenetic tree showed that the targeted strain was highly similar to the type strains of A. terreus (Fig. 4). Therefore, this isolate was identified as A. terreus isolate MD1 with accretion number OR425012. A. terreus is considered oleaginous fungus that could accumulate more than 20% of their dry weight lipid [36, 37]. The assortment of lipids found in the biomass of yeasts and fungi is excellent. These lipids are comparable to vegetable oils and may be used to make biodiesel, which can replace fossil fuels in internal combustion engines and electricity generation [38]. Moreover, this fungal species is considered an efficient cellulolytic fungus [39] as well as its ability for co-pelletizing harvesting of microalgae [5, 40].

Fig. 4
figure 4

Phylogenetic tree of A. terreus with the close related reference strains

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3.4 Harvesting optimization using A. terreus/rice straw bioflocculant

According to previously harvesting experiments of Chlorella microalga using different fungal isolates, A. terreus was selected for further harvesting optimization experiments. The harvesting of the green, single-celled microalga was optimized using A. terreus/rice straw mixture as the “bioflocculant.” Optimization conditions included the microalga/bioflocculant ratio, microalgal age, contact time between the bioflocculant and microalga, pH of microalga at harvesting time, and cell density of the microalga. Unless otherwise mentioned, the ratio of the bioflocculant to the microalgal culture was 30% (v/v), the pH of the culture was 7, and the age of the algae culture was 2 weeks.

3.4.1 Contact time

The contact time between the flocculant agent and the targeted microorganism is a critical factor that affects the flocculation and harvesting efficiency [20, 41]. Hence, this experiment was conducted to examine the effect of contact time between the “bioflocculant” and the targeted microalga “Chlorella sp.” on the bioflocculation harvesting efficiency. The freshwater microalga Chlorella sp. was subjected for harvesting using A. terreus grown on pretreated rice straw with contact time of 1, 2, and 24 h (at pH 7). The results illustrated in Fig. 5 revealed that the longer contact time between the bioflocculant and the microalga resulted in significant higher harvesting efficiencies. The highest harvesting efficiency (97.6%) was recorded due to 24 h contact time between the microalga and the bioflocculant, while a percentage of 82.7% was recorded for 2 h contact time. In a similar study, the harvesting efficiency of Chlorella sp. was increased from 45 to 90% due to increasing the contact time between the microalga and the pellets of Penicillium sp. from 24 to 48 h [26]. Although the harvest rate after 24 h is higher than 2 h, the economic factor must be taken into account when determining the appropriate contact time in semi-industrial and industrial algae harvesting operations. Although the detailed interaction mechanism between fungi and microalgae still needs to be further studied, the higher contact time seems to allow more adhesion and trapping of microalgal cells on the surface of flocculating fungi. However, the “hyphal surface adsorption and chemisorption from extracellular proteins and exopolysaccharides” seems to be the main reason for the flocculation of microalgae using fungi [42].

Fig. 5
figure 5

Harvesting efficiency of Chlorella culture due to different contact time with the A. terreus fungus/rice straw bioflocculant

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3.4.2 Microalgal pH

The cultural pH is an important factor that influences the processes of flocculation and stability of suspended cells [43]. Among the several studied pHs of Chlorella cultures, i.e., 5, 6, 7, 8, and 9, harvesting results obtained from pH 6 treatment were significantly the highest (Fig. 6). The percentage of harvesting was above 91% when harvested by A. terreus/pre-treated rice straw after 2 h. It worth to mention that the autoflocculation of Chlorella microalgal cells increased due to the increase of cultural pH value. However, the lowest auto-flocculation was recorded at pH 5 (less than 22%) while the maximum auto-flocculation of Chlorella after 2 h was 36.6% at pH 9. In a similar biofouling study of Chlorella sp. using fungal pellets of A. terreus, [14] recorded harvest efficiencies of less than 90% when pHs were between 5 and 7. The finding that acidic conditions support the highest bioflocculation of microalgae using fungal growth was also concluded by [44]. The acidic pH of the fungus’ medium causes the surface functional (carboxylic and amine) groups of mycelia to remain protonated, which results in the net positive charges of the fungus’ hyphae in the pellet-assisted mode [9, 14]. Other studies supported such observation using chitosan where the highest harvesting efficiency for Chlorella sp. and Scenedesmus sp. were obtained at pH 6.0 [4, 45].

Fig. 6
figure 6

Harvesting efficiencies of Chlorella sp. microalga at different pH values due to application of the fungal bioflocculant or due to the autoflocculation

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3.4.3 Effect of microalga age

The effect of microalgal cultural age on FE using the applied A. terreus/rice straw bioflocculant was investigated. Chlorella sp. cultures at 1, 2, and 3 weeks old were used for flocculation harvesting experiments. The lowest HE of Chlorella was observed for 1-week-old culture (45.8%); this percentage was significantly increased as 87.1 and 94.7% for 2- and 3-week-old cultures, respectively (Fig. 7). Although the cell concentration and cultural pH were adjusted to be similar in 1-, 2-, and 3-week-old algae cultures, older cells were able to be harvested and sediment by bio-clustering than fresh cells. Similarly, the autoflocculation was increased due to the aging of the microalgae cells from around 10% at 1-week-old culture to 36.9% in 3-week-old culture.

Fig. 7
figure 7

Effect the age of Chlorella sp. on FE using the applied fungi/rice straw bioflocculant

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Autoflocculation increased in aging microalgal cultures due to the increased secretion of extracellular polymeric substances (EPS) that can lead to bridging of cells together. The EPS attached to the surface of algal cells and could cause changes in the outer charges of microalgal cells. These changes in outer charges and the presence of EPS could play a critical role in the flocculation harvesting of microalgal cells using fungal bioflocculants [5, 41].

However, it should be considered that the pH of microalgal cultures usually increased up to 9 and sometimes 10 at the late stationary phase which led to the precipitation of microalgal cells (autoflocculation) [4].

3.4.4 Effect of microalgal cultural density

The microalgal cultures differ in their cell densities due to different cultural and environmental factors. The influence of cultural density of microalga on FE using the fungal bioflocculant has been investigated at three levels of cultural optical densities, i.e., OD680 nm at 1, 2, and 3 values. In this experiment, the optical density of the 2-week-old Chlorella culture was adjusted at the specific values either by centrifugation or by dilution by cell-free cultural filtrate of the same microalga. According to data illustrated in Fig. 8, the FE of Chlorella culture with OD680 nm of 1 was 81.3%. This percentage was significantly increased to 86.4 and 91.2% by increasing the cultural cell density up to OD680 nm of 2 and 3, respectively. These results showed that the same amount of bioflocculant is capable of causing more microalgae biomass flocculation in denser cultures. This conclusion is consistent with the conclusion of [46], who reported that the higher the density of microalgae biomass concentration, the lower the amount of flocculant required per unit amount of biomass. Similarly, [47] reported a linear correlation between the concentration of initial microalgae and the increase in harvest efficiency with the same amount of flocculant.

Fig. 8
figure 8

Effect of Chlorella cultural density on FE using fungi/rice straw bioflocculant

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3.4.5 Effect of fungal algal ratio

The ration between flocculant substance and the targeted particles (microalgae for instance) is a critical factor that could affect the whole performance of the flocculation process [1, 4, 5, 11, 48]. Hence, the ration of A. terreus/rice straw bioflocculant to the Chlorella culture was investigated to optimize its harvesting efficiency for biofuel production. The bioflocculant was added to the targeted microalgal culture at 10, 20, and 30% v/v ratios for 2 h and left half an hour for sedimentation. According to the results of HE presented in Fig. 9, the maximum HE, i.e., 93.2%, was obtained due to application of 30% bioflocculant. However, the percentage of sedimentation was significantly lower as 89 and 83.1% for 20 and 10% bioflocculant:microalga (v/v), respectively. In a similar study [14], obtained around 99% HE due to five h application of A. oryzae fungal pellets to at 1:1 to C. vulgaris culture. In another study, around 97% HE was obtained for C. vulgaris by A. nomius pellets when fungal mycelium to microalgae biomass ratio 1:2 (w/w) was established after 3-h agitation [8].

Fig. 9
figure 9

Effect of A. terreus–based bioflocculant/algal ratio on FE of Chlorella sp

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3.5 Production of biodiesel as renewable fuel source

The biomass of oleaginous microalgae is considered a renewable feed stock for sustainable biodiesel production [21, 49]. The biomass of Chlorella sp. (as oleaginous microalga) was harvested under optimized conditions by the lipid-rich cellulolytic fungus A. terreus grown on rice straw and applied for biodiesel production (Fig. 1S, 2S). The total lipids of microalga/bioflocculant biomass mixture have been extracted (30% of dry weight) and subjected to transesterification reaction with methanol to produce biodiesel fuel. The produced fatty acid methyl ester (biodiesel) was subjected to characterization using GC–MS to determine its suitability as biofuel. The characterization of biodiesel was performed based on the fatty acid profile resulted by GC analysis. According to obtained results (Fig. 3S, Table 1S), the percentage of unsaturated fatty acids was around 67.2% of the total identified fatty acids. Linoleic acid (18:2) and oleic acid (C18:1) fatty acids represent 29.2 and 38% of total lipids, which indicates the suitability of produced biodiesel as fuel. The structural characteristics of fatty acids, such as their chain length, degree of unsaturation, and chain branching, have an impact on the physiochemical properties of biofuel. Accordingly, the best fraction for the production of biodiesel is C16-C18 fatty acids [50]. Generally, bioflocculation using fungi is applicable and an ecofriendly approach to harvest microalgal biomass as feedstock for biofuel production [5].

4 Conclusion

Many fungi have the ability to cause flocculation harvesting of microalgal cells, with the possibility of using the obtained biomass in the production of biodiesel, if its fat content and composition are suitable. Fungi capable of degrading cellulose present an advantage in microalgae harvesting, as their production cost can be reduced by growing them on cheap agricultural cellulose waste such as rice straw. Obtained results of this study revealed the applicability of the lipid rich cellulolytic fungus A. terreus grown on rice straw as bioflocculating agent for microalgae harvesting. The lipid profile of the obtained fungus/microalga biomass represents a suitable fraction for biodiesel properties. Microalgal pH, age, and cell density as well as bioflocculant/culture ratio and contact time are important factors affecting the efficiency of the bio-harvesting process.