Manipulation of co-pelletization for Chlorela vulgaris harvest by treatment of Aspergillus niger spore

doi:10.21203/rs.3.rs-3321113/v1

The co-pelletization of microalgae with filamentous fungi was a promising approach for microalgae harvest. However, the real conditions of microalgae growth limited the arbitrary optimization of co-pellets formation with filamentous fungi. Therefore, it is urgent to develop an approach to manipulate the co-pelletization through treatment of A. niger spores. In this study, Aspergillus niger and Chlorella vulgaris were used as the model species of filamentous fungi and microalgae to investigate co-pellets formation using A. niger spores after by different pH solutions treatment, swelling, snailase treatment. The importance of spore treatments on C. vulgaris harvest in sequence was claimed based on response surface methodology analysis. The pH solutions treatment, swelling, snailase treatment of A. niger spore contributed 21.0%, 10.5%, 40.7% of harvest ratio of C. vulgaris respectively, which guided the application of spore treatment into co-pelletization. Treatment of spore was showed as an efficient approach to manipulate co-pelletization for microalgae harvest in diverse microalgae condition. This results promoted the application of co-pelletization technology in microalgae harvest of various conditions.

Microalgae harvest

co-pellets

Aspergillus niger

Chlorella vulgaris

treatment of fungal spore

Microalgae harvest via filamentous fungal co-pellets technology was a hot topic because of it’s easy-handling, cost-saving, and good biodegradation since it was reported as a new concept in 2012 (Zhang and Hu 2012). These years, this technology was reviewed several times, and was considered as a break-through of microalgae harvest for biofuel production (Chu, Li et al. 2021, Leng, Li et al. 2021, Li, Liang et al. 2022, Satpati, Dikshit et al. 2023), other high-value products, such as proteins (Nyyssola, Suhonen et al. 2022), carbohydrates, carotenoids, pigments (Patel, Albarico et al. 2022), and other bioactive compounds (Chew, Yap et al. 2017). Therefore, the co-pelletization technology was dominant in this as easy as one-step (Wang, Yang et al. 2022) and eco-friendly process (Kumar Awasthi, Yuan et al. 2022) of diverse high value-added products production by microalgae, although co-culture of microalgae with other microbial species was also reported (Shu, Li et al. 2023). Traditionally, this successful co-pelletization process was committed by researchers to optimize co-culture conditions, such as carbon source concentration (Li, Liang et al. 2020), metal ion concentration (Zamalloa, Gultom et al. 2017), pH (Castrillo, Lucas-Salas et al. 2013), temperature, culture time, stir speed, light intensity and time for the highest harvest ratio of microalgae (Rosero-Chasoy, Rodríguez-Jasso et al. 2021). For example, Yang et al used 1% inocultion ratio of 1.5×10⁴/mL Aspergillus sp spores to microalgae to harvest 70% C. vulgaris in molasses wastewater (Yang, Li et al. 2019). Gultom et al optimized inocultion ratio of 2.5×10³/mL Aspergillus niger spores to microalgae as 3% to achieve 90% harvest ratio of C. vulgaris (Gultom, Zamalloa et al. 2014). Nasir optimized the pH value from 3.0 to 9.0 and rotate speed from 100 to 150 rpm to reach 97% harvest ratio of Chlorella sp (Nasir, Yunos et al. 2019). It was expected that the co-pelletization process was directed by a theoretical mechanism. Zhang et al proposed a mechanism of time spatial distribution of driving forces, such as electrostatic interaction, hydrophobic interaction, and salt bridge interaction for pelletization (Zhang and Zhang 2016). The electrostatic interaction came mainly from the carboxyl groups of melanin (Wargenau, Fleißner et al. 2011) although electrostatic interaction between fungi and microalgae surface was complex (Li, Wang et al. 2022). Hydrophobic interaction played a role through self-assemble property of hydrophobins at interfaces (Linder 2009). Salt bridge interaction was formed through groups of α-1, 3-glucan in polysaccharide chains and ions in cultivation broth (Fontaine, Beauvais et al. 2010). Based on this mechanism, researchers tried to verify the specific roles of cell components of fungal cell in microalgae harvest process. For example, Pei et al confirmed that tyrosine and tryptophan, precursors of melanin, played important roles in pelletization process using three-dimensional fluorescence spectrum (Pei, Ren et al. 2021). Li et al reported that a low molecular weight surface proteins of A. niger hsn26 and calcium played an important role in microalgae harvest process with the conclusion that co-pellets formation with microalgae was a surface proteins-mediated, calcium bridging-dependent and hydrophobic interaction-involved process (Li, Xu et al. 2017). Also, Bhattacharya et al reported that aromatic compounds, amines groups, phosphate groups, and carboxylic acids, played a vital role in the interaction between fungal hyphae and microalgae through Fourier transform infrared analysis (Bhattacharya, Mathur et al. 2017). Wang et al extracted secretions of A. fumigatus and Synechocystis sp. PCC6803 for infrared analysis to show their influence on microalgae harvest (Wang, Chen et al. 2021). Li et al also reported extracellular polymeric substances (EPS) containing proteins and polysaccharides of A. niger mycelium as the basis of symbiotic relationship between hyhpa and microalgae (Li, Xu et al. 2017). The role of polysaccharide of hypha on co-pellets formation with microalgae was also reported by Srinuanpan et al (Srinuanpan, Chawpraknoi et al. 2018). This mechanism was also confirmed by many researchers, such as Cairns et al (Cairns, Feurstein et al. 2019), Li et al (Li, Liu et al. 2020), and Miyazawa et al (Miyazawa, Yoshimi et al. 2020).

However, the real condition of microalgae cultivation was various and can’t be changed arbitrary to satisfy the requirement of co-pellets formation. Therefore, a simple and practical operation was needed urgently to achieve co-pelletization although theoretical mechanism of co-pelletization was successful. In this study, A. niger and C. vulgaris were used as the model species of filamentous fungi and microalgae to demonstrate an easy and practical approach for co-pelletization through treatment of A. niger spores. The treatments of A. niger spores showed significant influence on microalgae harvest through co-pelletization. Therefore, a simple and practical approach was developed for microalgae harvest through co-pelletization using treated A. niger spores.

2.1 Microbial species and chemicals

A. niger ATCC 1015 and C. vulgaris FACHB-8 were supplied by the American Type Culture Collection, (Manassas, Virginia, United States) and Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China), respectively. All chemicals were purchased from Sino-Pharm chemical reagent Co., Ltd (Shanghai, China). All bio-chemicals were purchased from Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China). Potato dextrose agar (PDA) medium, containing 37 g/L potato dextrose agar powder, was sterilized at 121 ℃ for 15 min. The blue green algae medium (BG-11) contained 0.04 g/L K₂HPO₄·3H₂O, 0.075 g/L MgSO₄·7H₂O, 0.036 g/L CaCl₂·2H₂O, 0.006 g/L citric acid, 0.006 g/L ammonium ferric citrate, 0.001 g/L EDTA, 1.5 g/L NaNO₃, 0.02 g/L Na₂CO₃, 1 mL trace element mixture A. Trace element mixture A contained 2.86 g/L H₃BO₃, 0.039 g/L Na₂Mo₄·2H₂O, 0.222 g/L ZnSO₄·7H₂O, 1.81 g/L MnCl₂·4H₂O, 0.074 g/L CuSO₄·5H₂O, 0.03 g/L CoCl₂. Autotrophic medium A contained 1.0 g/L KNO₃, 0.075 g/L KH₂PO₄, 0.1 g/L K₂HPO₄, 0.5 g/L MgSO₄·7H₂O, 0.0625 g/L CaCl₂·4H₂O, 0.01 g/L FeSO₄·7H₂O, 0.5 g/L yeast extract, 1 mL trace element mixture A, 115 ℃ sterilization for 20 min. Complex culture medium (CCM) contained 2.0 g/L glucose, 2.0 g/L NH₄Cl, 10 g/L KH₂PO₄, 0.5 g/L MgSO₄·7H₂O, 0.02 g/L FeSO₄·7H₂O, 1 mL trace element mixture A.

2.2 A. niger spore preparation

A. niger spores were transformed into PDA plate for 7 days cultivation at 28°C. Then the spores were collected by washing using sterile water, and stored at 4°C for further use. The absorbance of spore solution was measured by a UV spectrophotometer (720N, INESA instrument, Shanghai, China) at 620 nm. And the spores number was counted using a hemocytometer.

2.3 Melanin removal from A. niger spore by different pH solutions

Melanin of A. niger spores was extracted using different pH solutions according to the method of Smith et al (Smith, Chohan et al. 1998). The melanin from 1 mL 0.77×10⁸/mL spore was considered as removed completely after mixing with 1 mL 0.1 mol/L NaOH solution followed by 1 min ultrasonic treatment in a water bath and 150 rpm shaking for 20 min. Partial melanin removal from spores was carried out by mixing with HCl solution (pH 1, 2, 3.5), 200 mmol/L phosphate-citric acid buffer solution (pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), 0.125 mol/L tris-HCl buffer solution (pH 8.5) and sodium hydroxide solution (pH 10.5) respectively followed by 1 min ultrasonic treatment in a water bath and 150 rpm shaking for 20 min. The treated spores were separated from supernatant through a Ф0.22 µm filter for further use.

2.4 Hydrophobin removal from A. niger spores by swelling and chemical treatment

Swelling happened at the early stage of fungal spore germination with the changes of cell wall components (Ijadpanahsaravi, Punt et al. 2021), such as hydrophobins. Hydrophobin content of A. niger spores decreased with the extension of swelling time (Singh, Saikia et al. 2004). In this study, hydrophobin removal experiments were carried out through spores swelling and chemical treatment respectively.

For swelling approach, 1 mL of 0.77×10⁸/mL spores suspension was mixed with 4 mL CCM medium, and cultivated at 28 ℃, 150 rpm for 3 h. Swelled spores were sampled every 30 min, and separated from supernatant through a Ф0.22 µm filter for further use.

For chemical treatment approach, 1 mL of 0.77×10⁸/mL spore suspension was mixed with 4 mL sodium dodecyl sulfate (SDS) and formic acid solution with the final concentration of 0.01, 0.025, 0.05, 0.075 and 0.1%, respectively, followed by 3 h cultivation at 28℃, 150 rpm. Treated spores were separated from supernatant through a Ф0.22 µm filter for further use.

2.5 Treatment of A. niger spores by snailase treatment for polysaccharide removal

Polysaccharide removal by snailase digestion were carried out according to literature (Sadovskaya and Guérardel 2019). One milliliter of 0.77×10⁸/mL spore suspension was centrifuged at 12,000 rpm for 2 min to remove supernatant. Then, 50 µL snailase (A600870, Sangon Biotech) and 600 µL reaction buffer (B518257, Sangon Biotech) were added to suspend the spores for 6 h at 37℃. The treatment time and amount of snailase were optimized by 6 h, 12 h, 18 h, 24 h, and 25 µL, 50 µL, 75 µL, 100 µL respectively. Spores after treatment was centrifuged at 12,000 rpm for 2 min to remove supernatant.

2.6 Co-cultivation of A. niger spores and C. vulgaris

The C. vulgaris was transferred from BG-11 medium plate containing 2% agar into in 100 mL sterile BG-11 medium in 250 mL flask with a cycling of 12 h light and 12 h dark for 7 days at 25°C, 100 rpm. Twenty milliliter of 1.9 ×10⁷ /mL C. vulgaris fermentation broth was centrifuged at 1700 g for 5 min to remove supernatant. Then, 20 mL CCM medium was added into C. vulgaris cells to suspend. One milliliter of 0.77×10⁸ /mL spore was added to the C. vulgaris medium for a 24 h cultivation at 28 ℃ and 130 rpm and another 24 h cultivation at 150 rpm in sequence. One milliliter of 200 g/L glucose solution was added to the cultivation system.

Effects of pH value of melanin removal (A), swelling time (B) and snailase treatment time (C) were carried out by one factor of one experiment firstly. And then, response surface methodology (RSM) analysis by Box-Behnken design experiments were carried out according to the factors and levels in Table 1.

Table 1

Factors and levels in RSM design
Factors	Level (-1)	Level (0)	Level (1)
A: pH solution	3.5	6.5	8.5
B: Swelling time (min)	60	120	180
C: Snailase treatment time (h)	6	15	24

2.7 Application of co-cultivation of A. niger and C. vulgaris in different media

To demonstrate microalgae harvest process, C. vulgaris, obtained from 20 mL CCM fermentation broth through centrifugation of 1700 g for 5 min at 4℃, was mixed with 1 mL of 0.77×10⁸/mL A. niger spore in 100 mL BG-11, medium A, and CCM respectively for cultivation at 28 ℃, 130 rpm for the first 24 h, and 150 rpm for another 24 h. One milliliter of 200 g/L glucose solution was added to CCM medium only at 24 h cultivation.

2.8 Analytic methods

The melanin content in supernatant was determined using absorbance at 430 nm (OD₄₃₀) (Wargenau, Fleißner et al. 2011). The residual melanin ratio was calculated using Eq. 1.

Residual melanin ratio (%)= (1-\(\frac{{\text{O}\text{D}}_{430} \text{o}\text{f} \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{s}\text{u}\text{p}\text{e}\text{r}\text{n}\text{a}\text{t}\text{a}\text{n}\text{t}}{{\text{O}\text{D}}_{430} \text{o}\text{f} \text{s}\text{u}\text{p}\text{e}\text{r}\text{n}\text{a}\text{t}\text{a}\text{n}\text{t} \text{a}\text{f}\text{t}\text{e}\text{r} 0.1 \text{m}\text{o}\text{l}/\text{L} \text{N}\text{a}\text{O}\text{H}})\times 100\) Eq. 1

The hydrophobicity determination was carried out according to literature (Smith, Chohan et al. 1998) with modification as follows: A. niger spores were washed by 5 mL phosphate urea magnesium sulphate (PUM) buffer (0.2 g/L MgSO₄·7H₂O, 17.0 g/L K₂HPO₄, 7.3 g/L KH₂PO₄, 1.8 g/L urea, pH7.0 adjusted by 1 mol/L phosphate saline buffer) firstly, and measured absorbance at 470 nm. The spore suspension was mixed with 500 µL hexadecane for 30 s shaking, and stored at 4 ℃ for 30 min. The absorbance of the spore suspension at 470 nm was measured again after hexadecane removal. Residual hydrophobicity was calculated according to Eq. 2.

\(\text{R}\text{e}\text{s}\text{i}\text{d}\text{u}\text{a}\text{l} \text{h}\text{y}\text{d}\text{r}\text{o}\text{p}\text{h}\text{o}\text{b}\text{i}\text{c}\text{i}\text{t}\text{y} \left(\text{%}\right)=\frac{{\text{O}\text{D}}_{470 \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}}}{{\text{O}\text{D}}_{470 \text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}}}\times 100\) Eq. 2

Polysaccharide content of supernatant was calculated by a colorimetric method for reduction sugar determination (Dubois, Gilles et al. 1951). Residual polysaccharide ratio was calculated according to Eq. 3.

\(\text{R}\text{e}\text{s}\text{i}\text{d}\text{u}\text{a}\text{l} \text{p}\text{o}\text{l}\text{y}\text{s}\text{a}\text{c}\text{c}\text{h}\text{a}\text{r}\text{i}\text{d}\text{e} \text{r}\text{a}\text{t}\text{i}\text{o} \left(\text{%}\right)=\frac{\text{R}\text{e}\text{d}\text{u}\text{c}\text{i}\text{n}\text{g} \text{s}\text{u}\text{g}\text{a}\text{r} \text{i}\text{n} \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{g}\text{r}\text{o}\text{u}\text{p}}{\text{R}\text{e}\text{d}\text{u}\text{c}\text{i}\text{n}\text{g} \text{s}\text{u}\text{g}\text{a}\text{r} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{g}\text{r}\text{o}\text{u}\text{p}}\times 100\) Eq. 3

C. Vulgaris cell density was measured using absorbance at 680 nm using a UV spectrophotometer (720N, INESA instrument, Shanghai, China). Harvest ratio was defined as total cell density of C. Vulgaris minus the C. Vulgaris cell density in supernatant, and divided by the total cell density in the initial fermentation broth (Eq. 4).

\(\text{H}\text{a}\text{r}\text{v}\text{e}\text{s}\text{t} \text{r}\text{a}\text{t}\text{i}\text{o} \text{o}\text{f} C. Vulgaris \left(\text{%}\right)=1-\frac{\text{R}\text{e}\text{s}\text{i}\text{d}\text{u}\text{a}\text{l} \text{c}\text{e}\text{l}\text{l} \text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}}{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{c}\text{e}\text{l}\text{l} \text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}}\times 100\) Eq. 4

2.9 Statistical analysis

All experiments in this study were carried out in triple. The standard deviation was calculated using Microsoft 2016 (Seattle, WA, USA).

3.1. Effects of pH treatment on A. niger spore and co-pellets with C. vulgaris

Considering the complex structure of fungal cell wall containing melanin, hydrophobin, and polysaccharide simultaneously, Fig. 1 showed the influences of pH treatment of spore on residual melanin ratio, residual hydrophobicity, residual polysaccharide ratio of A. niger spores, harvest ratio of C. vulagris and co-pellets morphology with C. vulgaris after 48 h cultivation. With the increasing of pH value from 1.0 to 10.5, the residual melanin ratio increased from 28.3–62.8% sharply, and kept at about 65% (Fig. 1A). The residual hydrophobicity of A. niger spores showed a Gauss curve with the maximum value of 45.8% at pH 6.0 and 6.5 after melanin extraction (Fig. 1B). The residual polysaccharide ratio increased from about 94.6% to about 98.5% minorly (Fig. 1C). Therefore, extremely low pH value led to relative low ratios of residual melanin, hydrophobicity, and polysaccharide. The harvest ratio of C. vulgaris decreased from 40% to about 12% when spores were treated by solutions of pH value from 1.0 to 10.5 (Fig. 1D). However, the sizes of co-pellets did not show any significant difference in Fig. 1E.

3.2. Effects of swelling on A. niger spore and co-pellets with C. vulgaris

A. niger hydrophobin formed an amphipathic rodlet structure film of nanometer thickness at the surface of spores, which provided hydrophobicity as an important driving force for spore aggregation. During swelling, the ordered rodlet structure of hydrophobin disappeared by proteases digestion gradually (Valsecchi, Lai et al. 2019), and accompanied by changing of melanin and polysaccharide content simultaneously (Fig. 2). The residual melanin ratio showed an inversed bell-shape curve in Fig. 2A, although the residual melanin ratio fluctuated in a relatively narrow range of 76.4%-83.6%. And the residual hydrophobicity decreased sharply from 42.2–13.9%, and then fluctuated to 8.8% gradually during 180 min swelling (Fig. 2B). The residual polysaccharide ratio showed a half Gauss curve of decreasing from 92.2–68.6% (Fig. 2C). The harvest ratio of C. vulgaris showed as a bell-shaped curve with the maximum value of 56.3% using spores of 120 min swelling (Fig. 2D). The co-pellets size using spores of 150 min swelling was significantly larger than that of others although all pellets were formed well (Fig. 2E).

3.3. Removal of two classes hydrophobin by chemical treatment

Fungal hydrophobins were divided into two classes according to their solubility (Smith, Chohan et al. 1998). The hydrophobin class I only dissolved in trifluoroacetic acid and formic acid. The hydrophobin class II dissolved in ethanol or sodium dodecyl sulfate (SDS). To clarify the contributions of two classes hydrophobins, formic acid and SDS were used to remove hydrophobin class I and class II respectively with the results in Fig. 3. All the residual ratios of melanin, hydrophobicity, and polysaccharide decreased with the increasing of formic acid concentration and SDS concentration. The residual melanin ratios only decreased from 94.5–86.1% (Fig. 3A), and 94.7–90.8% (Fig. 3F) at formic acid and SDS treatment respectively. And, residual polysaccharide ratios decreased from 89.5–56.6%, and 82.7–62.2% at formic acid and SDS treatment respectively. Therefore, hydrophobin removal experiments influenced melanin contents insignificantly, and influenced polysaccharide content significantly. The harvest ratio of C. vulgaris also decreased with different curves simultaneously at formic acid and SDS treatment respectively. The harvest ratio of C. vulgaris at the condition of formic acid treatment decreased sharply from 80.2–5.8% with the increasing of formic acid concentration from 0.01 to 0.05%, and kept stable till 0.1% formic acid used. While harvest ratio of C. vulgaris at the condition of SDS treatment showed a linear decreasing from 45.7–16.7% with the increasing of SDS concentration from 0.01 to 0.1%. Based on significant changes of hydrophobicity and polysaccharide, the harvest ratio of C. vulgaris by co-pellets dived significantly. The larger range of harvest ratio of C. vulgaris at the condition of hydrophobin class I removal than that of hydrophobin class II removal indicated a more important role of hydrophobin class I than hydrophobin class II during this process.

3.4. Effects of snailase treatment on A. niger spore and co-pellets with C. vulgaris

The polysaccharide of fungal spore cell wall was mainly composed of β- (1,3) -glucan, mannose and chitin, which could be degraded into monosaccharides due to enzymatic degradation (Latge and Beauvais 2014). Figure 4 showed influences of snailase amount and its treatment time on A. niger spores and co-pellets with C. vulgaris. With the increasing of snailase amount and treatment time, all the residual melanin ratio, hydrophobicity, and residual polysaccharide ratio decreased, although with different curves. Residual melanin ratios at different amounts of snailase loaded and treatment time showed similar curves of decreasing from 100–22.0% when snailase increased from 0 uL to 100 uL (Fig. 4A), and 100–11.0% when treatment time was extended to 24 h (Fig. 4F). Residual hydrophobicity at just stated two experiments also decreased from 48.3–37.5% and 15.9% respectively with different curves (Fig. 4B and 4G). The residual polysaccharide ratio decreased significantly from 94.4–8.1% (Fig. 4C), and 91.9–29.7% (Fig. 4H) respectively. The harvest ratio of C. vulgaris kept about 65% at the condition of 0 uL -75 uL snailase loaded, and decreased sharply to 6.8% when 100 uL snailase was loaded (Fig. 4D). However, the harvest ratio of C. vulgaris decreased linearly when the treatment time of 50 uL snailase extended to 24 h (Fig. 4I). although the properties of spores changed significantly by snailase treatment, spores and C. vulgaris formed co-pellets successfully after 48 h cultivation in Fig. 4E and 4J.

Based on the results of Fig. 1 - Fig. 4, it was showed that all the residual melanin ratio, hydrophobicity, residual polysaccharide ratio of spores changed connectively at all the conditions of pH treatment, swelling and chemical treatment, and snailase treatment. This interference among these three parameters came from the complex structure of spore wall although melanin, hydrophobin, and polysaccharide located in different layers of spore wall (Beauvais, Fontaine et al. 2014). Currently, gene knockout was an effective approach to regulate single one component. For example, alb1 gene and RodA gene knockout blocked melanin synthesis (Priegnitz, Wargenau et al. 2012) and a hydrophobin production (Ball, Kwan et al. 2020) respectively. Furtherly, the clustered regularly interspaced short palindromic repeats (CRISPR) technology also demonstrated its capability of gene editing in Aspergillus niger (Zhang, Ouyang et al. 2019, Zheng, Zheng et al. 2019). However, gene editing technology changed gene sequence forever, which was not flexible for properties of A. niger spore, and provided an issue for the safety of A. niger.

Table 2 summarized the variation tendencies of spore properties and corresponding harvest ratios of C. Vulgaris. It was difficult to identify a general relationship between spore properties and harvest ratio. Therefore, to identify the order of importance of each parameters of spores, RSM was used to analyze the effects of treatments of spores on harvest ratio of C. vulgaris through normalization.

Table 2 Correlation of spore properties and harvest ratios of microalgae

(Red: increasing; Purple: decreasing)

Treatment	Melanin	Hydrophobin	Polysaccharide	Harvest ratio
pH	●	●→●	●	●
Swelling	●→●	●	●	●→●
Formic acid	●	●	●	●
SDS	●	●	●	●
Snailase time	●	●	●	●
Snailase loaded	●	●	●	●

3.5. Analysis of spore treatments for harvest ratio through RSM

The pH solution (A), swelling time (B) and snailase treatment time (C) were used as three independent variables with their response of C. Vulgaris harvest ratio in Table 3. RSM analysis using fitting quadratic polynomial model was showed in Table 4. And the ternary quadratic multinomial regression equations of harvest ratio on pH solution, swelling time, and snailase treatment time was obtained as Eq. 4.

Y = 44.0-2.6A-1.8B-8.4C + 1.1AB + 2.4AC + 1.9BC-10.4A²-12.3B²-9.5C² Eq. 4

It was significant for this model because the P value was lower than 0.05. The P value of lack of fit was higher than 0.05, indicating that the model had a good fitting degree. Therefore, this model accurately reflected the effects of pH solution, swelling time and snailase treatment time on the harvest ratio. The absolute value of each coefficient in Eq. 4 directly reflected the influence degree of each factor on the harvest ratio. The importance order of factors affecting the harvest ratio was snailase treatment time, pH solution, swelling time in sequence, indicating the contribution strength for harvest ratio was salt bridge interaction, electrostatic interaction, hydrophobic interaction in sequence. The contribution ratios of pH solution, swelling time, and snailase treatment time were 21.0%, 10.5%, and 40.7% respectively according to the RSM analysis.

Table 3

Factor levels and results of RSM experiments
No.	A	B	C	Harvest ratio (%)
1	1	1	0	19.6
2	-1	0	-1	37.6
3	0	-1	-1	35.8
4	0	0	0	45.9
5	0	-1	1	14.5
6	0	1	-1	26.4
7		0	1	16.7
8	-1	-1	0	25.4
9	0	0	0	44.5
10	-1	1	0	21.9
11	0	0	0	46.0
12	0	0	0	45.5
13	1	0	-1	26.9
14	0	1	1	12.6
15	0	0	0	38.2
16	1	0	1	15.6
17	1	-1	0	18.8

Table 4

Variance analysis of fitting quadratic polynomial model
Source	SS	df	MS	F value	P value
model	2314.80	9	257.20	32.73	< 0.0001
pH (A)	53.98	1	53.98	6.87	0.0344
Swelling time (B)	24.43	1	24.43	3.11	0.1212
Snailase treatment time (C)	566.50	1	566.50	72.09	< 0.0001
AB	4.56	1	4.56	0.58	0.4712
AC	22.42	1	22.42	2.85	0.1350
BC	13.80	1	13.80	1.76	0.2267
A²	451.11	1	451.11	57.41	0.0001
B²	631.40	1	631.40	80.35	< 0.0001
C²	377.66	1	377.66	48.06	0.0002
Residual	55.01	7	7.86
Lack of fit	11.45	3	3.82	0.35	0.7923
Pure error	43.56	4	10.89
Cor total	2369.81	16

The interactions of two factors were showed as contour diagram and corresponding surface diagram in Fig. 5. At the condition of 15 h snailase treatment (Fig. 5A and 5B), the harvest ratio had the optimum value when pH value or swelling time was 6.4–6.6 and 90–120 min respectively. This optimum value of harvest ratio was also observed in Fig. 5C-5F when swelling time and pH value was fixed at 120 min and 6.5 respectively. Therefore, the optimal treatment conditions of A. niger spore was snailase treatment time 15 h, pH 6.5, 120 min swelling. Contribution ratios of pH solution, swelling time, and snailase treatment time for C. Vulgaris harvest were 21.0%, 10.5%, and 40.7% respectively using multivariate regression analysis (Table 5). Therefore, the approaches of spore treatment were ranked and quantified to guide the harvest ratio of microalgae using co-pelletization.

Table 5

Contribution ratios of treatments
Treatment	Contribution ratio (%)
pH solution	21.0
Swelling time	10.5
Snailase treatment time	40.7

3.6. Application of co-pellets in different media

Three general media were used to demonstrate the harvest ratio of C. Vulgaris via co-pellets with results in Fig. 6. The harvest ratios were obtained as 2.0%, 32.8%, and 90.0% in medium BG-11, medium A, and medium CCM respectively (Fig. 6A). Low harvest ratio of C. vulgaris in BG-11 was attributed into no organic carbon source in BG-11 for A. niger spore germination and growth, which was constant to the minimum nutrients requirement for A. niger spore germination (Ijadpanahsaravi, Punt et al. 2021). While, Chen et al obtained Penicillium sp. pellets in BG-11 supplemented with 10 g/L glucose (Chen, Leng et al. 2018). In medium A, 0.5 g/L yeast extract served as carbon source for A. niger pellets formation which led to 32.8% harvest of C. vulgaris. At the condition of medium CCM containing 10 g/L glucose, 90.0% harvest ratio was obtained accompanying with A. niger pellets formation perfectly (Fig. 6B).

Facing the diverse conditions of C. Vulgaris, this study actively changed the physiological condition of A. niger spore to achieve the C. Vulgaris harvest efficiently via pellets. This approach expended the operation strategies as some factors of microalgae cultivation could not be changed arbitrarily. For example, it was not reasonable to add anymore nutrients to wastewater for microalgae cultivation (Chen, Leng et al. 2018). And factors, such as temperature, rotate speed of large scale cultivation of microalgae, also were not easy to be changed and matched the requirement. This approach of spore treatment broadened the conditions of microalgae harvest via pellets formation.

Furtherly, considering the complex mechanism of fungal pellets formation and co-pelletization with microalgae, this study claimed that order of factors affecting the harvest ratio was snailase treatment time, pH solution, swelling time in sequence, without excluding any others. For example, calcium ion was a controversial factor as Li et al (Li, Nie et al. 2022) and Vandamme et al (Vandamme, Foubert et al. 2012) draw totally different conclusions based on their results. The even-changing components of A. niger cell wall during pellets formation added more difficult on theoretical direction of microalgae harvest via fungal pellets (Lin, Chen et al. 2022). Li et al reported another three factors, polysaccharide, specific surface of pellets, and zeta potential, of microalgae harvest of 84.22%, 5.17%, and 10.61% respectively (Li, Liu et al. 2020). Li et al also listed polysaccharide as the major driving force source of microalgae harvest, although with a different, new and difficultly regulated factor of specific surface.

To manipulate C. Vulgaris harvest via A. niger pellets, spores were treated by immerse in different pH solution, swelling, and snailase treatment to regulate the harvest ratio of C. vulgaris although the interaction of these three treatments. After optimization of one factor by one experiment and RSM, the importance order of affecting the harvest ratio was snailase treatment time, pH solution, swelling time in sequence, with the contribution ratios of 40.7%, 21.0%, and 10.5% respectively. These results help the process of microalgae harvest via fungal pellets in practical place efficiently.

Data Availability Statement

Data available on request from the authors

Author Contributions

XZ and ZJ conceived the study. XZ performed all the experiments and drafted the original manuscript. WC and MW monitored methodology validation part, supervised the experiments. SG, HZ and JG prepared, reviewed, and edited the manuscript.

Declaration of Competing Interests

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the International Science and Technology Cooperation Foundation of Shanghai (Grant No. 19230742900).

Ball, S. R., A. H. Kwan and M. Sunde (2020). Hydrophobin rodlets on the fungal cell wall. The fungal cell wall : An armour and a weapon for human fungal pathogens. J.-P. Latgé. Cham, Springer International Publishing: 29-51.
Beauvais, A., T. Fontaine, V. Aimanianda and J.-P. Latgé (2014). Aspergillus cell wall and biofilm. Mycopathologia 178(5): 371-377.
Bhattacharya, A., M. Mathur, P. Kumar, S. K. Prajapati and A. Malik (2017). A rapid method for fungal assisted algal flocculation: Critical parameters & mechanism insights. Algal Res 21: 42-51.
Cairns, T. C., C. Feurstein, X. Zheng, P. Zheng, J. Sun and V. Meyer (2019). A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger. Biotechnol Biofuels 12: 149.
Castrillo, M., L. M. Lucas-Salas, C. Rodriguez-Gil and D. Martinez (2013). High pH-induced flocculation-sedimentation and effect of supernatant reuse on growth rate and lipid productivity of Scenedesmus obliquus and Chlorella vulgaris. Bioresour Technol 128: 324-329.
Chen, J., L. J. Leng, C. S. Ye, Q. Lu, M. Addy, J. H. Wang, J. Liu, P. Chen, R. Ruan and W. G. Zhou (2018). A comparative study between fungal pellet- and spore-assisted microalgae harvesting methods for algae bioflocculation. Bioresour Technol 259: 181-190.
Chew, K. W., J. Y. Yap, P. L. Show, N. H. Suan, J. C. Juan, T. C. Ling, D.-J. Lee and J.-S. Chang (2017). Microalgae biorefinery: High value products perspectives. Bioresour Technol 229: 53-62.
Chu, R. Y., S. X. Li, L. D. Zhu, Z. H. Yin, D. Hu, C. C. Liu and F. Mo (2021). A review on co-cultivation of microalgae with filamentous fungi: Efficient harvesting, wastewater treatment and biofuel production. Renew Sust Energ Rev 139: 110689.
Dubois, M., K. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith (1951). A colorimetric method for the determination of sugars. Nature 168(4265): 167.
Fontaine, T., A. Beauvais, C. Loussert, B. Thevenard, C. C. Fulgsang, N. Ohno, C. Clavaud, M.-C. Prevost and J.-P. Latge (2010). Cell wall α1-3 glucans induce the aggregation of germinating conidia of Aspergillus fumigatus. Fungal Genet Biol 47(8): 707-712.
Gultom, S. O., C. Zamalloa and B. Hu (2014). Microalgae harvest through fungal pelletization-co-culture of Chlorella vulgaris and Aspergillus niger. Energies 7(7): 4417-4429.
Ijadpanahsaravi, M., M. Punt, H. A. B. Wösten and W. R. Teertstra (2021). Minimal nutrient requirements for induction of germination of Aspergillus niger conidia. Fungal Biol 125(3): 231-238.
Kumar Awasthi, A., Z. Yuan, M. Kumar Awasthi, M. Li, S. Mishra and A. Kumar Pandey (2022). Bioprocess potential of Eco-friendly fungal isolates converting organic waste to bioresource. Bioresour Technol 346: 126586.
Latge, J.-P. and A. Beauvais (2014). Functional duality of the cell wall. Curr Opin Microbiol 20: 111-117.
Leng, L., W. Li, J. Chen, S. Leng, J. Chen, L. Wei, H. Peng, J. Li, W. Zhou and H. Huang (2021). Co-culture of fungi-microalgae consortium for wastewater treatment: A review. Bioresour Technol 330: 125008.
Li, L., T. Liang, W. Liu, Y. Liu and F. Ma (2020). A comprehensive review of the mycelial pellet: Research status, applications, and future prospects. Indu Eng Chem Res 59(39): 16911-16922.
Li, L., T. Liang, M. Zhao, Y. Lv, Z. Song, T. Sheng and F. Ma (2022). A review on mycelial pellets as biological carriers: Wastewater treatment and recovery for resource and energy. Bioresour technol 355: 127200-127200.
Li, L., W. Liu, T. Liang and F. Ma (2020). The adsorption mechanisms of algae-bacteria symbiotic system and its fast formation process. Bioresour Technol 315: 123854.
Li, N., P. Wang, S. Wang, C. Wang, H. Zhou, S. Kapur, J. Zhang and Y. Song (2022). Electrostatic charges on microalgae surface: Mechanism and applications. J Environ Chem Eng 10(3): 107516.
Li, Y., H. Nie, H. Zhang, W. Niu, S. Li and H. Wang (2022). Promotion effect of bacteria in phycosphere on flocculation activity of Aspergillus niger on Synechocystis biomass. Aquaculture 550: 737833.
Li, Y., Y. Xu, L. Liu, P. Li, Y. Yan, T. Chen, T. Zheng and H. Wang (2017). Flocculation mechanism of Aspergillus niger on harvesting of Chlorella vulgaris biomass. Algal Res 25: 402-412.
Lin, W., L. Chen, Z. Tan, Z. Deng and H. Liu (2022). Application of filamentous fungi in microalgae-based wastewater remediation for biomass harvesting and utilization: From mechanisms to practical application. Algal Res 62: 102614.
Linder, M. B. (2009). Hydrophobins: Proteins that self assemble at interfaces. Curr Opin Colloid Interface Sci 14(5): 356-363.
Miyazawa, K., A. Yoshimi and K. Abe (2020). The mechanisms of hyphal pellet formation mediated by polysaccharides, α-1,3-glucan and galactosaminogalactan, in Aspergillus species. Fungal Biol Biotechnol 7: 10.
Nasir, N. M., F. H. M. Yunos, H. H. W. Jusoh, A. Mohammad, S. S. Lam and A. Jusoh (2019). Subtopic: Advances in water and wastewater treatment harvesting of Chlorella sp. microalgae using Aspergillus niger as bio-flocculant for aquaculture wastewater treatment. J Environ Manage 249: 109373.
Nyyssola, A., A. Suhonen, A. Ritala and K.-M. Oksman-Caldentey (2022). The role of single cell protein in cellular agriculture. Curr Opin Biotechnol 75: 102686.
Patel, A. K., F. P. J. B. Albarico, P. K. Perumal, A. P. Vadrale, C. T. Nian, H. T. B. Chau, C. Anwar, H. M. U. D. Wani, A. Pal, R. Saini, L. H. Ha, B. Senthilkumar, Y.-S. Tsang, C.-W. Chen, C.-D. Dong and R. R. Singhania (2022). Algae as an emerging source of bioactive pigments. Bioresour Technol 351: 126910.
Pei, X.-Y., H.-Y. Ren and B.-F. Liu (2021). Flocculation performance and mechanism of fungal pellets on harvesting of microalgal biomass. Bioresour Technol 321: 124463.
Priegnitz, B. E., A. Wargenau, U. Brandt, M. Rohde, S. Dietrich, A. Kwade, R. Krull and A. Fleissner (2012). The role of initial spore adhesion in pellet and biofilm formation in Aspergillus niger. Fungal Genet Biol 49(1): 30-38.
Rosero-Chasoy, G., R. M. Rodríguez-Jasso, C. N. Aguilar, G. Buitrón, I. Chairez and H. A. Ruiz (2021). Microbial co-culturing strategies for the production high value compounds, a reliable framework towards sustainable biorefinery implementation – an overview. Bioresour Technol 321: 124458.
Sadovskaya, I. and Y. Guérardel (2019). Simple protocol to purify cell wall polysaccharide from gram-positive bacteria and assess its structural integrity. Methods Molr Biol 1954: 37-45.
Satpati, G. G., P. K. Dikshit, N. Mal, R. Pal, K. C. Sherpa, R. C. Rajak, S.-u. Rather, S. Raghunathan and M. Davoodbasha (2023). A state of the art review on the co-cultivation of microalgae-fungi in wastewater for biofuel production. Sci Total Environ 870: 161828.
Shu, L., J. Li, J. Xu and Z. Zheng (2023). Nutrient removal and biogas upgrade using co-cultivation of Chlorella vulgaris and three different bacteria under various GR24 concentrations by induction with 5-deoxystrigol. World J Microbiol Biotechnol 39(9): 245.
Singh, T., R. Saikia, T. Jana and D. K. Arora (2004). Hydrophobicity and surface electrostatic charge of conidia of the mycoparasitic Trichoderma species. Mycol Prog 3(3): 219-228.
Smith, S. N., R. Chohan, R. A. Armstrong and J. M. Whipps (1998). Hydrophobicity and surface electrostatic charge of conidia of the mycoparasite Coniothyrium minitans. Mycol Res 102: 243-249.
Srinuanpan, S., A. Chawpraknoi, S. Chantarit, B. Cheirsilp and P. Prasertsan (2018). A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi and the application in phytoremediation of secondary effluent. Int J Phytoremediation 20(10): 1017-1024.
Valsecchi, I., J. I. Lai, E. Stephen-Victor, A. Pillé, A. Beaussart, V. Lo, C. L. L. Pham, V. Aimanianda, A. H. Kwan, M. Duchateau, Q. G. Gianetto, M. Matondo, M. Lehoux, D. C. Sheppard, Y. F. Dufrene, J. Bayry, J. I. Guijarro, M. Sunde and J. P. Latgé (2019). Assembly and disassembly of Aspergillus fumigatus conidial rodlets. Cell Surf 5: 100023.
Vandamme, D., I. Foubert, I. Fraeye, B. Meesschaert and K. Muylaert (2012). Flocculation of Chlorella vulgaris induced by high pH: Role of magnesium and calcium and practical implications. Bioresour Technol 105: 114-119.
Wang, J., R. Chen, L. Fan, L. Cui, Y. Zhang, J. Cheng, X. Wu, W. Zeng, Q. Tian and L. Shen (2021). Construction of fungi-microalgae symbiotic system and adsorption study of heavy metal ions. Sep Purif Technol 268: 118689.
Wang, S.-K., K.-X. Yang, Y.-R. Zhu, X.-Y. Zhu, D.-F. Nie, N. Jiao and I. Angelidaki (2022). One-step co-cultivation and flocculation of microalgae with filamentous fungi to valorize starch wastewater into high-value biomass. Bioresour Technol 361: 127625.
Wargenau, A., A. Fleißner, C. J. Bolten, M. Rohde, I. Kampen and A. Kwade (2011). On the origin of the electrostatic surface potential of Aspergillus niger spores in acidic environments. Res Microbiol 162(10): 1011-1017.
Yang, L., H. Li and Q. Wang (2019). A novel one-step method for oil-rich biomass production and harvesting by co-cultivating microalgae with filamentous fungi in molasses wastewater. Bioresour Technol 275: 35-43.
Zamalloa, C., S. O. Gultom, A. Rajendran and B. Hu (2017). Ionic effects on microalgae harvest via microalgae-fungi co-pelletization. Biocatal Agric Biotechnol 9: 145-155.
Zhang, J. and B. Hu (2012). A novel method to harvest microalgae via co-culture of filamentous fungi to form cell pellets. Bioresour Technol 114: 529-535.
Zhang, J. and J. Zhang (2016). The filamentous fungal pellet and forces driving its formation. Crit Rev Biotechnol 36(6): 1066-1077.
Zhang, Y., L. Ouyang, Y. Nan and J. Chu (2019). Efficient gene deletion and replacement in Aspergillus niger by modified in vivo CRISPR/Cas9 systems. Bioresour Bioproc 6(1): 4.
Zheng, X., P. Zheng, K. Zhang, T. C. Cairns, V. Meyer, J. Sun and Y. Ma (2019). 5S rRNA promoter for guide RNA expression enabled highly efficient CRISPR/Cas9 genome editing in Aspergillus niger. ACS Synth Biol 8(7): 1568-1574.

No competing interests reported.

GraphicalAbstract.jpg

Manipulation of co-pelletization for Chlorela vulgaris harvest by treatment of Aspergillus niger spore

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods