Potential role of N-acetyl glucosamine in Aspergillus fumigatus-assisted Chlorella pyrenoidosa harvesting
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Algal harvesting is a major cost which increases biofuel production cost. Algal biofuels are widely studied as third-generation biofuel. However, they are yet not viable because of its high production cost which is majorly contributed by energy-intensive biomass harvesting techniques. Biological harvesting method like fungal-assisted harvesting of microalgae is highly efficient but poses a challenge due to its slow kinetics and poorly understood mechanism.
In this study, we investigate Aspergillus fumigatus–Chlorella pyrenoidosa attachment resulting in a harvesting efficiency of 90% within 4 h. To pinpoint the role of extracellular metabolite, several experiments were performed by eliminating the C. pyrenoidosa or A. fumigatus spent medium from the C. pyrenoidosa–A. fumigatus mixture. In the absence of A. fumigatus spent medium, the harvesting efficiency dropped to 20% compared to > 90% in the control, which was regained after addition of A. fumigatus spent medium. Different treatments of A. fumigatus spent medium showed drop in harvesting efficiency after periodate treatment (≤ 20%) and methanol–chloroform extraction (≤ 20%), indicating the role of sugar-like moiety. HR-LC–MS (high-resolution liquid chromatography–mass spectrometry) results confirmed the presence of N-acetyl-d-glucosamine (GlcNAc) and glucose in the spent medium. When GlcNAc was used as a replacement of A. fumigatus spent medium for harvesting studies, the harvesting process was significantly faster (p < 0.05) till 4 h compared to that with glucose. Further experiments indicated that metabolically active A. fumigatus produced GlcNAc from glucose. Concanavalin A staining and FTIR (Fourier transform infrared spectroscopy) analysis of A. fumigatus spent medium- as well as GlcNAc-incubated C. pyrenoidosa cells suggested the presence of GlcNAc on its cell surface indicated by dark red dots and GlcNAc-specific peaks, while no such characteristic dots or peaks were observed in normal C. pyrenoidosa cells. HR-TEM (High-resolution Transmission electron microscopy) showed the formation of serrated edges on the C. pyrenoidosa cell surface after treatment with A. fumigatus spent medium or GlcNAc, while Atomic force microscopy (AFM) showed an increase in roughness of the C. pyrenoidosa cells surface upon incubation with A. fumigatus spent medium.
Results strongly suggest that GlcNAc present in A. fumigatus spent medium induces surface changes in C. pyrenoidosa cells that mediate the attachment to A. fumigatus hyphae. Thus, this study provides a better understanding of the A. fumigatus-assisted C. pyrenoidosa harvesting process.
KeywordsBioflocculation N-acetyl glucosamine Fungi Algae Atomic force microscopy
atomic force microscopy
Fourier transform infrared spectroscopy
high-performance liquid chromatography
high-resolution liquid chromatography–mass spectrometry
potato dextrose agar
potato dextrose broth
scanning electron microscopy
transmission electron microscopy
Microalgae showcase diverse applications for wastewater treatment and subsequent bioenergy generation. Large quantity of microalgal biomass is the foremost requirement in the biofuel route, for which several high-end/advanced photobioreactors have already been designed by several researchers [1, 2, 3]. However, the commercialization of algal biofuels still lags behind due to high cost of investment towards energy-intensive biomass harvesting techniques like centrifugation, membrane filtration and chemical based flocculation from photobioreactors [4, 5, 6]. Chemical processes are highly efficient, but the requirement of a lot of flocculant dosages renders the harvested biomass contaminated with undesirable chemicals [7, 8]. Biologically induced harvesting of algal cells is now being explored as a replacement for the conventional algal dewatering processes which contribute towards 3–15% of the algal biomass production cost [9, 10]. The harvested biomass can be processed for biofuel generation without any loss of quality [11, 12]. There have been quite a few studies on the algal–algal and algal–bacterial interactions for bio-harvesting of algal cells from bulk media [13, 14, 15, 16, 17, 18, 19, 20]. A very recent approach is the use of filamentous fungi for harvesting algal cells [21, 22, 23]. In spite of having the potential for becoming a cost-effective process for algal harvesting, the lack of knowledge regarding the causative factors for the algal attachment to fungal pellets limits its application.
When fungus is grown under submerged conditions with agitation, it forms a dense, compact hyphal structure termed as pellets . When these pellet-forming filamentous fungi (PFF) were co-cultivated with algal cells, there is an orderly attachment of algal cells on the fungal pellets [22, 23, 25]. This phenomenon of algal–fungal attachment is of interest as it solves the problem of algal harvesting to a large extent. However, the studies with co-cultivation of fungi and algae have reported the interaction time to be between 24 and 72 h [22, 25, 26, 27, 28, 29, 30, 31]. In a more straight-forward approach, it has been observed that the attachment of algal cells (green algae or cyanobacteria) to fungal pellets could take place within 4–6 h using pre-cultivated fungal biomass [12, 32]. Such interaction of algae and PFF  is intriguing as it does not follow the simple kinetics of bio-harvesting [13, 33, 34]. Our recent study confirms that in addition to a specific set of physical conditions, the metabolically active fungus is mandatory for efficient attachment of the Chlorella pyrenoidosa cells to the Aspergillus fumigatus pellet . This fact raises further queries on the biological context of C. pyrenoidosa–A. fumigatus attachment, which although seems far simpler than the complex algal–fungal associations in nature.
A complex symbiotic association between fungus and cyanobacteria exists in the form of lichens in natural systems . In artificial systems, such as co-culturing of fungus (Aspergillus nidulans) with microalgae (Chlamydomonas), a mutualistic association for nutrient exchange has been reported as reflected by thinning of microalgal cell wall by enzymatic action of fungus. However, this mutualism may sometimes lead to antagonism, when the algal death occurs due to over secretions of these enzymes . Relatively simple phytoplankton–parasitic fungal (Chytrids) interactions controlled by cell-to-cell contact also exist in nature, which are driven by chemotaxis . In our recent studies, somewhat similar action of fungus was observed in algal–fungal pellets as fungus used algal cells as nutrient source by enzymatically degrading it . However, what drives the interaction between C. pyrenoidosa and A. fumigatus at the molecular level is not clear. There are also no specific molecules reported for PFFs so far, although enough evidence for extracellular polymeric substances (EPS)-mediated aggregation exists [38, 39, 40, 41].
The present study aims to find out the causative mechanism behind this interesting phenomenon. It also tries to answer questions like why a specific set of biological conditions is necessary for this process to occur. Experiments were designed to address several questions like is there any chemical signaling/mediating molecule which mediates the attachment of C. pyrenoidosa and A. fumigatus, what is the origin of this mediating molecule, i.e., C. pyrenoidosa or A. fumigatus, what is the type/nature of this mediating molecule, how does this molecule mediate the attachment, etc. However, this study raises further questions regarding the nature and type of receptors which may be responsible for the attachment process that needs to be investigated further.
Role of extracellular metabolites in C. pyrenoidosa–A. fumigatus harvesting
Nature and characterization of the mediating molecule
Category of compounds obtained after HR-LC–MS of A. fumigatus spent media
Category of compound
Name of compounds
Sugars (Monosaccharides, Polysaccharides)
Sugar derivatives (Sugar alcohols, ketones, aldehydes, esters)
N-Acetyl-d-glucosamine, 12a-hydroxy-5-deoxydehydromunduserone, Furo[3,4-b]pyridine-3-carboxylic acid, 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxo-, methyl ester, acetyl tyrosine ethyl ester
Organic acids and its derivatives
l-2-Aminoadipic acid, 11-amino-undecanoic acid, phthalic acid mono-2-ethylhexyl ester
Hydrocarbons (Long chain and short chain)
Lipid, Fatty acids and its methyl esters
11-Amino-undecanoic acid, 6’-Hydroxysiphonaxanthin, N-ethyl arachidonoyl amine
Nitrates, Nitrites, amines and other nitrogen derivatives
Protein metabolites (amino acid sequences)
Arg Arg Gln, Val Arg Gly, Ser Asn Gly, Leu Ala Arg
Vitamins and its derivatives
26,26,26,27,27,27-hexafluoro-1alpha, 24-dihydroxyvitamin D3, 1alpha, 25-dihydroxy-26,27- dimethyl-20,21,22,22,23,23-hexadehydro-24ahomovitamin D3, 2alpha-Fluoro-19-nor-22-oxa—1alpha, 25-dihydroxyvitamin D3, Phylloquinone (Vitamin K1)
Nucleic acids and its derivatives
Propylthiouracil glucuronide, Pseudouridine, 7,8-didehydroastaxanthin
Other metabolites and compounds
Zolpidem Metabolite I, dihydrodeoxystreptomycin
Confirmation of the mediating molecule in C. pyrenoidosa–A. fumigatus interaction
Various treatments of A. fumigatus spent medium indicated sugar-like molecule to be responsible for harvesting. Hence, experiments were conducted to study the role of glucose and GlcNAc as a replacement of A. fumigatus spent medium during C. pyrenoidosa–A. fumigatus harvesting. Glucose, being the simplest form of sugar, was tested first. C. pyrenoidosa cells were incubated with 100-mM glucose and then harvesting experiments were carried out using washed A. fumigatus pellets devoid of any A. fumigatus spent medium. Results showed > 75% harvesting after 5 h, suggesting that glucose assisted in the harvesting process. Negative control of C. pyrenoidosa incubated without glucose did not show any harvesting with washed A. fumigatus pellets (Additional file 1: Figure S1).
Microscopic analysis and surface characterization of A. fumigatus spent medium-/GlcNAc-incubated C. pyrenoidosa cells during harvesting
Chemical harvesting, although quick, contaminates the biomass with unnecessary chemical contaminants . Previous reports have suggested that biological harvesting of micro-algae improves the quality of the biomass for biofuel purpose. Wrede et al.  reported that there is a significant increase in lipid yield when microalgal cells were harvested with A. fumigatus. Increase in lipid content for algal–fungal pellets was also reported by Bhattacharya et al. . In another study, Botryococcus braunii was harvested using A. fumigatus and the resultant biomass did not show any significant variation in the biomass composition. Apart from biodiesel aspect, harvesting of microalgae using filamentous fungi also increases its biogas potential. Prajapati et al.  reported enhanced bio-methane production from algal–fungal pellets due to the enzymatic degradation of microalgal cell wall by the fungi. All these studies show that harvesting algae with filamentous fungi is a favorable process for biofuel production. However, as the underlying mechanism of the process is not clearly understood, it is difficult to replicate the process at large scale. This study gives an insight into the communication mechanism between A. fumigatus and C. pyrenoidosa which could be exploited for fungal-assisted microalgal harvesting processes at large.
Chemical communication between microbial cells is an inherent property which dictates the attachment and aggregation of cells [41, 43]. Such communications mediated by secretion of extracellular chemical signaling molecules could impose inter-beneficial partnerships for both the species . In natural systems, the algae and fungus exhibit a host–parasite relationship as seen in chytrid–phytoplankton association . In artificial systems, the co-culturing of Chlamydomonas reinhardtii and A. nidulans strains has been reported to show obligate mutualism in terms of nitrogen and carbon exchange within the species. The fungus converts glucose into CO2, which is being used by algae for its growth. On the other hand, the nitrogen fixing Chlamydomonas sp. reduces nitrite to ammonia, which fungus can use as a nitrogen source [36, 45]. During this nutrient exchange, the cell wall of Chlamydomonas sp. attached to fungus was reported to show thinning due to the action of fungal remodeling enzymes .
The present study can be closely related to the above-mentioned cases as it also reports extracellular secretions from A. fumigatus, mediating cell wall changes in the C. pyrenoidosa cells. These secretions might help A. fumigatus to provide signal to the C. pyrenoidosa cell and bring it into close vicinity for nutritional benefits or enzymatic degradation . Similar phenomenon of microalgal–fungal attachment has been reported by our group with wide range of microalgal species encompassing green algae (Chlorella sp.), blue–green algae (Chroococcus sp.) as well as mixed consortia [12, 32].
Fungi produce various exopolysaccharides during its growth , which have been known to mediate cell–cell adhesion. The present study has identified an extracellular causative factor produced by the fungus A. fumigatus which is responsible for the C. pyrenoidosa–A. fumigatus attachment. Experiments done with washed and unwashed A. fumigatus pellets have shown the presence of a mediating molecule in the A. fumigatus spent medium that is crucial for the attachment process. Washed A. fumigatus pellets could not induce attachment since they were devoid of any A. fumigatus spent medium. Fresh PDB also did not have the mediating molecule, and its harvesting efficiency harvesting was similar to the washed A. fumigatus pellets. This observation suggested that the causative factor is the extracellular metabolite produced by actively growing fungi. The result was also in agreement with our recent observations that relatively old (72-h grown) or autoclaved A. fumigatus pellets failed to harvest the C. pyrenoidosa cells due to their low metabolic activity and damaged hyphae . A. fumigatus spent medium is a cocktail of extracellular metabolites including EPS which is a complex mixture of polysaccharides, proteins, nucleic acids and amyloids . EPS production by Aspergillus sp. is reported for bio-flocculation . However, to pin-point the causative factor, it is necessary to know the nature of the mediating molecule.
The autoclaving of the A. fumigatus spent medium did not affect the harvesting efficiency of the fungus. This indicated that the mediating factor was not proteinaceous in nature as autoclaving would denature the protein. Chloroform and methanol mixture was able to extract the polar organic components from the A. fumigatus spent medium. Loss of activity after this treatment indicated that the mediating factor was a polar organic compound. This was further confirmed using hexane as a solvent for extraction. Treatment of the A. fumigatus spent medium with hexane did not show any loss in harvesting activity. As hexane is able to extract only non-polar organic compounds, it further confirmed that the molecule is a polar organic compound. Further insights into the nature of the molecule were obtained by treating the A. fumigatus spent medium with sodium per-iodate. Per-iodate targets any saccharide/polysaccharide leading to its oxidation. Loss of harvesting activity after per-iodate treatment of the A. fumigatus spent medium strongly suggested that the mediating factor was a sugar-like molecule. The HR-LC–MS analysis of the A. fumigatus spent medium also showed the presence of two sugars, glucose and GlcNAc which might be the triggering factors for the attachment process. Previously, the bioflocculant produced by Achromobacter xylosoxidans was found to be composed mainly of carbohydrate hetero polymer . In another study, characterization of a bioflocculant produced by Aspergillus flavus showed that it contained 69.7% sugar of which 1.8% was amino sugar-like GlcNAc . In a recent study, it has been found that an amino sugar galactosaminogalactan mediates attachment of A. fumigatus to epithelial cells .
In the present study, we were able to pinpoint the particular molecule that could replicate the C. pyrenoidosa–A. fumigatus attachment process in the absence of A. fumigatus spent medium. Results of several spent treatments corroborated with HR-LC–MS results suggested the target molecule to be glucose or GlcNAc. The presence of either GlcNAc or A. fumigatus spent medium is mandatory for the attachment process. The t test between harvesting kinetics of control and GlcNAc-incubated C. pyrenoidosa cells did not show any significant difference. When GlcNAc was used for harvesting studies, it was clearly seen that the harvesting process was significantly faster as compared to glucose. HR-LC–MS and HPLC results confirmed that glucose is converted into GlcNAc by A. fumigatus pellets. Conversions of sugar-like molecules into GlcNAc have been reported by saprophytic fungus using degrading/hydrolytic enzymes [49, 50]. Kinetics of glucosamine formation using glucose and other sugars as substrate by fungus like Aspergillus sp. has also been reported during submerged fermentation . Similar harvesting experiments with glucose anomers (Galactose and Mannose) showed only 30% harvesting which further suggests the role of GlcNAc in the harvesting process since these glucose anomers cannot be converted into GlcNAc (Additional file 4: Figure S3). The present result explains the previously observed pre-requisites for C. pyrenoidosa–A. fumigatus attachment (the presence of metabolically active fungi, temperature of 38 °C and neutral pH) , which seem to favor GlcNAc production. Earlier report showed that GlcNAc production by Aspergillus sp. was dependent on the pH of the system, where higher pH inhibited its formation . Comparative study of GlcNAc production among three wild-type fungi viz. A. fumigatus, Rhizopus oligosporus and Monascus pilosus showed that A. fumigatus had the highest production capacity among the three fungi.
We could demonstrate the presence of GlcNAc on the spent medium-incubated and GlcNAc-incubated C. pyrenoidosa using the lectin Concanavalin A (Con A) stain, and FTIR. Although, Con A gives signal for glucose as well as glucosamine, C. pyrenoidosa suspended in BG11 only do not show any signal as seen in the Fig. 4a. When C. pyrenoidosa is incubated with A. fumigatus spent media or glucosamine, Con A shows signals on the algal cell surface (Fig. 4b, c). When C. pyrenoidosa cells were incubated in the presence of glucosamine alone, the concentration of glucosamine decreases over time. The FTIR pattern of glucosamine-incubated cells show peaks similar to glucosamine. The Con A staining results complement the findings of the FTIR data. The mechanism of interaction could be further elaborated by examining the effect of A. fumigatus spent medium on C. pyrenoidosa cell’s morphology, ultrastructure and cell surface roughness. Roughness analysis is one of the parameters which indicates the changes in the cell surface due to changes in the growing environment. The root mean square (rms) value corresponds to the roughness of the sample. AFM analysis showed that the roughness of the A. fumigatus spent medium-incubated C. pyrenoidosa cells was increased compared to the normal C. pyrenoidosa cells. Increase in roughness is an essential step for the cellular attachment process . It has also been reported that in case of a green fouling alga Enteromorpha, increased roughness of cells caused more fouling compared to smooth cells with low roughness . SEM micrographs (Fig. 5c) of spent medium-incubated cells depicted elongated C. pyrenoidosa cells embedded in a sticky matrix. Extracellular release of mucilage for entrapment is well documented for parasitic type of A. fumigatus species .
The fact that both A. fumigatus spent medium-incubated C. pyrenoidosa and GlcNAc-incubated algae showed the presence of GlcNAc-specific peaks in the FTIR spectra suggests that similar cell surface changes may be induced by A. fumigatus spent medium and GlcNAc. In this connection, SEM micrographs of GlcNAc-incubated C. pyrenoidosa cells also showed elongation and wrinkles on the cell surface. TEM images clearly show that A. fumigatus spent medium induces the formation of villi-like structures on C. pyrenoidosa surface. It was interesting to note that similar villi-like structures were induced by incubating C. pyrenoidosa cells with GlcNAc. On the other hand, washed A. fumigatus pellets (without A. fumigatus spent medium) failed to induce such changes. The above results strongly suggest that GlcNAc present in A. fumigatus spent medium is responsible for inducing surface changes in C. pyrenoidosa cells that mediates the attachment to A. fumigatus hyphae .
Organism and culture conditions
Microalgal species Chlorella pyrenoidosa was obtained from National Collection of Industrial Microorganisms (NCIM), NCL Pune (India). C. pyrenoidosa was maintained in 2% algae culture agar (HiMedia M343-500G) slants supplemented with BG11 (HiMedia, M1541-500G) in a plant growth chamber (Daihan Labtech, LGC-5101). Liquid cultures were maintained in BG11 broth at 120 rpm without CO2 supplementation. For experimental purposes, C. pyrenoidosa culture was grown in 2.5-L flasks under continuous light in a greenhouse maintained at 25 ± 1 °C and a light intensity of ≈ 3500 Lx . The fungal strain Aspergillus fumigatus (Accession no. KY241789), previously isolated from wastewater , was used as the harvesting organism. A. fumigatus was maintained on sterile potato dextrose agar (PDA) slants (Himedia M096-500G) at 28 ± 1 °C.
C. pyrenoidosa and A. fumigatus harvesting experiments
To check the viability of normal C. pyrenoidosa cells and C. pyrenoidosa cells incubated with A. fumigatus spent medium during harvesting (2.5 h), the cells were stained with a nucleic acid stain, i.e., SYTOX green (Cat. No. S7020, Invitrogen), which specifically stains the dead or damaged cells. Following this, dual fluorescence was used to show red autofluorescence for live C. pyrenoidosa cells and green fluorescence of SYTOX green (Excitation/Emission: 504/523 nm) for dead cells .
Role of extracellular factors
To assess the role of an extracellular substance for the attachment process, A. fumigatus biomass was washed twice with distilled water removing all the spent medium from it. The A. fumigatus medium present after the growth of the fungus has been termed as A. fumigatus spent medium throughout the text. The A. fumigatus biomass was subjected to 5 harvesting experimental sets viz: Set I—Control (unwashed C. pyrenoidosa and A. fumigatus pellets), Set II—Unwashed C. pyrenoidosa and washed A. fumigatus pellets, Set III—Unwashed C. pyrenoidosa and washed A. fumigatus pellets resuspended in its spent medium, Set IV—Unwashed C. pyrenoidosa and washed A. fumigatus pellets resuspended in fresh PDB, Set V—Washed C. pyrenoidosa and unwashed A. fumigatus pellets. Harvesting of all the above sets was performed similarly as described above for 4 h. Experiments were done in triplicates for each and every experimental set. C. pyrenoidosa culture without any A. fumigatus biomass was run as a negative control to check the sedimentation rate of C. pyrenoidosa culture. The experimental setup is graphically shown in Fig. 1a.
To study the effect of an extracellular factor (present in A. fumigatus spent medium) on the C. pyrenoidosa cell surface, scanning electron microscopy (SEM) and High-Resolution Transmission electron microscopy (HR-TEM) were performed before harvesting (normal C. pyrenoidosa cells) and after designated time during harvesting (2.5 h). For comparison, the SEM and HR-TEM of C. pyrenoidosa cells incubated with washed A. fumigatus pellets (without A. fumigatus spent medium) from Set II were also performed. The atomic force microscopy (AFM) of normal C. pyrenoidosa cells and cells during harvesting was performed to evaluate the change in cell height and roughness.
Treatment and characterization of A. fumigatus spent medium
By the above preliminary experiments, the role of some extracellular factors presents in the A. fumigatus spent medium was highlighted. Hence, various treatments of A. fumigatus spent medium like autoclaving, sodium periodate treatment, Methanol: Chloroform extraction and hexane extraction were performed followed by testing the harvesting efficiency of these pre-treated A. fumigatus spent medium when added to washed A. fumigatus pellets and C. pyrenoidosa cells. For every experiment, A. fumigatus pellets were first washed to remove any spent medium present. Harvesting experiments were done with normal C. pyrenoidosa cells and A. fumigatus pellets resuspended in fungal spent medium after treatments (autoclaving, sodium periodate treatment, Methanol: Chloroform extraction and hexane extraction). A mixture of C. pyrenoidosa cells and A. fumigatus pellets with untreated spent was used as a positive control. A negative control comprising C. pyrenoidosa mixed with washed A. fumigatus pellets was run to ensure that the pellets of A. fumigatus did not have the fungal spent media.
Aspergillus fumigatus spent medium was autoclaved for 15 min at 121 °C and 15 psi pressure before using for harvesting experiments. As autoclaving would denature any protein component in the spent A. fumigatus medium, the role of protein molecule (if any) could be found out. Sodium periodate targets any saccharide/polysaccharide leading to the oxidation of these residues. The spent A. fumigatus media (100 ml) were pre-treated with 20-mM sodium periodate (Cat No. S1147, Sigma) solution prepared in an oxidation buffer (50-mM sodium acetate and 50-mM acetic acid, pH-4.5). Since sodium periodate is light sensitive, the preparation of the solution and pre-treatment of A. fumigatus spent medium were performed in the dark at 28 °C and 150 rpm. To stop the activity of sodium periodate, the sample was exposed to light for 15 min to remove the remaining periodate in the system.
The methanol: chloroform extraction was performed by mixing an equal volume of methanol: chloroform mixture (1:1) with the A. fumigatus spent medium for 1 h at 28 °C and 150 rpm to identify any polar molecules responsible for the harvesting process. The aqueous fraction was separated from the mixture by a separating funnel and the organic fraction was discarded. Washed A. fumigatus pellets suspended in this aqueous fraction were then subjected to harvesting of C. pyrenoidosa cells. A similar experiment was done using the hexane-extracted aqueous fraction (extracted following the similar protocol used for Methanol: Chloroform aqueous extract) to identify the presence of any non-polar molecule which aided in the harvesting process. The A. fumigatus spent medium was analyzed using high-resolution liquid chromatography–mass spectrometry (HR-LC–MS).
Determination of mediating molecule
Based on the HR-LC–MS analysis and preceding A. fumigatus spent medium treatments, glucose and N-acetyl glucosamine (GlcNAc) were suspected to play the most important role in harvesting process. C. pyrenoidosa biomass was exposed to 100-mM glucose and GlcNAc, respectively, by incubation for 2 h at 38 °C and 100 rpm, followed by removal of residual glucose and GlcNAc by centrifugation (4000g for 10 min) and resuspension of biomass in fresh BG11. C. pyrenoidosa after glucose/GlcNAc incubation was then subjected to harvesting with washed A. fumigatus pellets (without spent medium) for 5 h as described in “C. pyrenoidosa and A. fumigatus harvesting experiments” section. Another set of C. pyrenoidosa suspension was also incubated under the same conditions without supplementation of glucose/GlcNAc followed by its harvesting with washed A. fumigatus pellets. To analyze the concentration of glucose and GlcNAc at different stages of experiment, high-performance liquid chromatography (HPLC) was performed at three levels: (i) supernatant of C. pyrenoidosa suspension immediately after glucose or GlcNAc addition (initial); (ii) supernatant of C. pyrenoidosa suspension after 2 h of incubation with glucose or GlcNAc and (iii) supernatant of respective sets at the end of harvesting for 5 h. The results were compared with HPLC of glucose and GlcNAc standard as a reference. HR-LC–MS of the supernatants from all the set after harvesting was also done to confirm the result of HPLC.
GlcNAc-incubated C. pyrenoidosa biomass was also subjected to Fourier Transform Infra-Red Spectroscopy (FTIR) and was compared to FTIR spectra of C. pyrenoidosa biomass during harvesting (after 2 h) as well as normal C. pyrenoidosa. The FTIR of GlcNAc powder was also performed to correlate the presence of similar peaks on the incubated C. pyrenoidosa biomass. To further confirm the role of GlcNAc and A. fumigatus spent medium in the harvesting process, C. pyrenoidosa cells incubated with GlcNAc (100 mM) and A. fumigatus spent medium was observed under a fluorescent microscope and high-resolution transmission electron microscope (HR-TEM, Sect. 2.7.5) and was compared with normal C. pyrenoidosa cells. For fluorescent microscopy, the cells were stained with Concanavalin A (Con A) conjugated with Alexa Fluor® 594 (Cat No. C11253, ThermoFisher Scientific) to detect the presence of GlcNAc on the C. pyrenoidosa cell surface , followed by viewing of cells under a fluorescent microscope (Nikon Eclipse Ti-U). Con A is a lectin which binds d-glucose, d-fructose, d-mannose, N-acetyl-d-glucosamine and related monosaccharides . Since Con A does not have fluorescence, it is tagged with a fluorescent dye Alexa Fluor 594 (Excitation/Emission: 590/617 nm). To 100 µl of C. pyrenoidosa cells, 0.1 µl of the dye was added and kept in the dark for 5 min at room temperature. The stained cells were then observed under the microscope.
Sample preparation for high-performance liquid chromatography (HPLC) and high-resolution liquid chromatography–mass spectrometry (HR-LC–MS)
Samples were made salt free by passing through polymeric cartridge Strata-X-CW (Cat No. 8B-S035-JEG, Phenomenex). The cartridge was first conditioned by adding 10-ml methanol followed by 10-ml distilled water. The flow-through was discarded, and then 10 ml of the sample was added to the cartridge. The flow-through from the cartridge was collected, tested for harvesting activity and then analyzed using HPLC and HR-LC–MS.
Standards for glucose (Cat No. 47829) and GlcNAc (Cat No. PHR1432-1G) were purchased from Sigma. Standards were dissolved in HPLC grade water (Cat No. AS077-1L, Hi-Media). HPLC was performed according to manufacturer’s protocol using Agilent 1260 series machine with Agilent Hi-Plex H column and RI detector. The mobile phase was 0.005-M H2SO4 (Cat No. 5438270100, Sigma) at a flow rate of 0.6 ml min−1. The column temperature was kept at 60 °C and the run time was 20 min. All samples were degassed prior to analysis.
HR-LC–MS was done to identify the nature of the compounds present in the A. fumigatus spent medium. The A. fumigatus spent medium is a mixture of various types of compounds like proteins, carbohydrates, organic acids, sugars and A. fumigatus metabolites. Hence, HR-LC–MS was done for these compounds. The aliquot was subjected to HR-LC–MS analysis (ACCUCORE RP-MS), and was outsourced to Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology (IIT), Mumbai. The column used was ZORBAX ECLIPSE C-18 (Agilent Technologies). The sample was run isocratically for 30 min using acetonitrile (95%) as a solvent. The compounds were analyzed using Quadrupole—Time of Flight mass spectrometer (Q-TOF MS; Agilent iFunnel G6550A) giving the probable hits from the database library provided by the manufacturer.
Scanning electron microscopy (SEM)
SEM analysis of (i) normal C. pyrenoidosa cells, (ii) C. pyrenoidosa cells incubated with A. fumigatus spent medium, (iii) GlcNAc-incubated C. pyrenoidosa and (iv) washed A. fumigatus pellets were done using a previously described protocol . The samples for SEM analysis were first washed with PBS and then fixed with 1% glutaraldehyde for 4 h at room temperature. The samples were centrifuged and the fixative was discarded. Samples were then lyophilized (Allied Frost FD3) for SEM analysis using a ZEISS EVO 50 instrument under the following analytical condition: EHT = 20.00 kV, WD = 9.5 mm, Signal A = SE1.
High-resolution Transmission electron microscopy (HR-TEM)
The C. pyrenoidosa cells ((i) normal C. pyrenoidosa cells, (ii) C. pyrenoidosa cells incubated with A. fumigatus spent medium, (iii) GlcNAc-incubated C. pyrenoidosa and (iv) washed A. fumigatus pellets) were centrifuged at 4000g for 10 min and prepared for viewing as outlined by Gola et al. . The samples were examined by high-resolution transmission electron microscope (Tecnai G2 20) operated at 200 kV.
Atomic force microscopy (AFM)
To confirm the observations of TEM analysis, AFM studies of the C. pyrenoidosa cells (normal cells and A. fumigatus spent medium-incubated C. pyrenoidosa cells) were done. C. pyrenoidosa cells were centrifuged and suspended in 50-mM citrate–phosphate buffer (pH 3) for conditioning. The buffer was removed by centrifugation, and the pellets were kept on glass cover slip for 30 min followed by washing with de-ionized water and then air drying. AFM micrographs were obtained using Bruker INNOVAA2 Sys instrument in tapping mode.
Fourier transform infrared spectroscopy (FTIR)
For FTIR measurements, the samples were first washed with phosphate buffer saline (PBS) and then lyophilized. The lyophilized powder was then used for FTIR analysis using a Nicolet Is50 (Thermo Scientific) instrument.
All experiments were performed in triplicates and results were represented as mean ± S.D wherever applicable. Graphs were drawn using Microsoft Excel® (Part of Microsoft Office 2013 package). Significance test has been done using one-way ANOVA (p < 0.05). t Test (two-tailed) was also performed to check the significance between two data sets.
The authors are grateful to Nano Research Facility (NRF) and Central Research Facility (CRF), IIT Delhi, for help with HPLC, FTIR, AFM,SEM and HR-TEM analysis. Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, for HR-LCMS analysis and SAIF AIIMS, New Delhi for TEM section cutting.
AB and AM designed the experiments. AB, MM and PK conducted the experiments. AB and MM have written the manuscript. AM has thoroughly scrutinized the manuscript. All authors read and approved the final manuscript.
The present work was carried out with financial support from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (SB/S3/CEE/0002/2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethics approval and consent to participate
Not applicable for this study.
Consent for publication
The authors declare that they have no competing interests.
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