1 Introduction

At a time when production and application of petroleum-based plastics and polymers are increasingly questioned, the demand for bio-based and sustainable alternatives is simultaneously rising. The requirements for these alternatives have increased again: they should be vegan, easy to functionalize and, depending on processing technique and application, soluble in aqueous solutions. One of these polymers facing all of these requirements is chitosan extracted from fungal sources.

Chitosan is a nitrogen-containing heteropolysaccharide consisting of (1→4)-β-D-glucosamines (GlcN) and a lower amount of N-acetyl-(1→4)-β-D-glucosamines (GlcNAc). It is the deacetylated form of chitin. The deacetylation degree (DD) states the molar fraction of GlcN in the polysaccharide. It determines the amount of free primary amine groups with a pKa of 6.3, which are responsible for the water solubility at pH values below 5–6 [1, 2]. Classification bases merely on the DD: If the DD is greater than 50%, the biopolymer is referred to as chitosan, otherwise as chitin. Due to its water solubility and free primary amine groups, the chitosan has, toward chitin, an extended application portfolio. There are already several commercial uses known, such as the application as flocculating agent, as wound dressing or as carrier of drugs [3]. Since the commercial application is often based on fishery-based chitosan, further commercial uses of fungal chitosan, beyond niche applications, needs to be identified. Fungal chitosan is characterized by a high DD and a low-molecular weight, which should enable the identification of further application fields in distinction to fishery-based chitosan [4,5,6].

Chitosan production from fungi, as is with the other sources benefits from the fact that larger amounts of the raw material are under-utilized. 60% of the biotechnology industry processes use ascomycota; moreover, they are also the largest producer of chitin in nature [7]. Fungal waste streams are produced during the biotechnological production of a wide range of products including organic acids like malic acid or citric acid but also industrial enzymes such as protease, cellulase or amylase [8].

The chitosan production from fungi includes at least one of the following three steps in variable order: 1. a deproteinization step for the removal of protein with low concentrated NaOH solution at elevated temperature [4, 9, 10], 2. an acid extraction of freely accessible chitosan with sulfuric or acetic acid, to obtain naturally present chitosan, and 3. a deacetylation (DA) process to convert the remaining purified chitin to chitosan as well [7]. The chitosan is afterward potentially purified in a solubilization-precipitation process [11, 12].

Many studies focusing structural investigations of the chitosan, rather than to yield the total chitin and chitosan content of the fungal cell wall omitting the DA step. For example, Tayel et al. [13] who used a deproteinization step with 1 M NaOH at 90 °C for 2 h followed by an acid extraction with 10% (v/v) acetic acid at 60 °C for 6 h. A different approach for chitosan extraction was suggested by Zamani et al. [14] and Cui et al. [15]. They took advantage of the chitosan’s solubility in hot diluted sulfuric acid which could be afterwards precipitated in the cold. A DA of the chitin remaining in the insoluble biomass after acid extraction can therefore increase the chitosan yield immensely, resulting in additional product with a high DD and a low-molecular weight [16].

Although all previous studies have provided new insights into the chitosan extraction from fungi, they primarily lack a comparable assessment of yield and purity. This is because the original chitin content of the mycelium was never determined in these studies. The definition of yield in relation to the biomass allows a literature comparison of the chitosan production process from fungi. With regard to chitosan purity, an evaluation is possible by measuring the solubility of chitosan in diluted acid as Tayel et al. [13] did. Unfortunately, this method is generally error-prone, since reaction by-products and contaminations such as salts also easily dissolve, thereby apparently increasing sample solubility [11, 13]. But in all other cases yields are only reported gravimetrically, so it is unclear whether the product consists mainly of chitosan or of other substances such as glucans, thus artificially increasing the yield. This study thus proposes to evaluate the individual steps based on their efficiency and recovery, not on an incomparable yield. To do so, the chitin content of the mycelium as well as the other compounds needs to be completely analyzed and determined before process development. Analysis of chitin and chitosan content can be challenging as many chemical methods include harsh conditions that can alter the polymer composition during analysis resulting in errors. Recently, new forms of enzyme assisted means to evaluate chitin and chitosan content have emerged solving those problems but have not yet been used for Aspergillus niger [17].

This study will on one hand provide a holistic and robust determination of the total chemical composition of A. niger mycelia with a specific focus on chitin content determination with two different methods. The goal was to establish a basis enabling a thorough evaluation of the purification of fungal chitin and the subsequent conversion to chitosan described in the second part of this study. Part of the process was the optimization of the deproteinization parameters, the variation of extraction solutions during acid extraction, the conducting of a DA as well as the evaluation of the need for a downstream purification. The individual products were then characterized by DD, purity and molecular weight distribution.

2 Materials and methods

2.1 Materials

The starting material for chitosan extraction is the air-dried mycelium of an A. niger production strain with a particle size distribution between 100 and 1,000 µm. For chitosan production, NaOH was purchased from Merck KGaA. Sulfuric acid (95–97% purity) and acetic acid (> 99% purity) were bought from Th. Geyer GmbH & Co.KG. Protein Assay Dye Reagent Concentrate was purchased from Bio-Rad, albumin protein standards were purchased from Thermo Scientific. The Glucan Assay Kit (Yeast & Mushroom) was acquired from Megazyme. To carry out NMR investigations, deuterium oxide (> 99.8% purity) was purchased from Scharlab S.L. and deuterium chloride (38%) from Carl Roth GmbH & Co. KG. All experiments were carried out with desalted water.

2.2 Methods for characterization of mycelia composition

All analytical methods for the determination of the mycelia composition were conducted in triplicates. For analytical purposes, the mycelium was milled with a ZM1 ultracentrifugal mill (Retsch) and a ring sieve (200 µm pore size). This disintegrates the membrane and makes its components more accessible for the various analytical procedures.

Ash content, fat and moisture were measured according to Hahn et al. [18]. The content of α- and β-Glucans was determined using the glucan assay kit (Yeast and Mushroom) from Megazyme. The content of the carbon, hydrogen and nitrogen of mycelia and products were measured via elemental analysis with a Euro EA from HEKAtech.

To determine the different sugars (but chitosan) in the biomass, a total hydrolysis was conducted. For this, 0.3 g of the biomass was mixed with 3 mL of 72% (w/w) sulfuric acid and incubated for 1 h at 30 °C in a water bath. The solution was then transferred and filled up with 84 mL of water to reach a final concentration of 4% (w/w) sulfuric acid. This procedure was also carried out with a 1 g/L glucose solution as a control for degradation during the process and a mean for correction. The samples were autoclaved at 121 °C, 2 bar for 1 h. Solids were separated with a 5–13 µm filter. The supernatant was adjusted to pH 4–5 by adding calcium carbonate and sterile filtered with 0.22 µm nylon filters. For sugar analysis a Rezex ion-exchange column (ROA-Organic Acid H+ (8%) 300 x 7.8 mm) was used. As the mobile phase 5 mM H2SO4 solution was chosen and the HPLC was operated with a flow rate of 0.6 mL/min, an injection volume of 20 μL for 30 min.

Residual organic acids still present from the fermentation of A. niger were determined during washing of the mycelium with water.

2.2.1 Protein determination by standard addition procedure

For the determination of the protein content of the mycelia, the supernatant from the deproteinization method (see methods for chitosan production) was exploited. To minimize matrix influences on the measurement, a standard addition method was used for the microassay. First, the solution was filtered through a (0.22 µm pore size) nylon syringe filter to remove any particles. The filtrate was then neutralized with a HCl solution of the same molarity and an equal volume as the NaOH solution used for deproteinization. For the standard addition procedure, 10 µL of sample and 10 µL of BSA protein standards with concentrations of 0, 0.25, 0.375, 0.5, 0.675 and 0.75 mg/mL were added to a 96-well plate and 180 µL of 1:5 (v/v) diluted protein staining solution (Bio-Rad) was added. The plate was then orbitally shaken for 30 sec, incubated for 10 min and measured photometrically at 595 nm wavelength.

The absorbance of the samples with standard is plotted on the ordinate axis, the concentration of the added standards is plotted on the abscissa. The percentage protein content (cProt) of the mycelium was calculated from the absolute value of the intersection point of the function with the abscissa (SA), the dilution factor including neutralization (DF), the volume of NaOH solution used (V) and the amount of biomass used in the experiment (m) provided in Eq. (1):

$$c_{{{\text{Prot}}}} (\% ) = \frac{{\left| {{\text{SA}}} \right| \cdot {\text{DF}} \cdot V}}{m} \cdot 100$$
(1)

To evaluate the complete protein content, the same biomass was deproteinized twice and the supernatant was analyzed for proteins. The sum of the protein content was then assumed to be the total protein content.

2.2.2 Chitin and chitosan determination by DD, protein and nitrogen content

For the determination of chitin and chitosan content in the mycelia, a process was proposed adapting existing methods, which considers the protein content, the average DD of chitin and chitosan as well as the elemental composition of the mycelia.

The DD was determined according to Ottoy et al. [19] by 13C CP MAS NMR spectroscopy on a Avance III 400WB spectrometer at a resonance frequency of 100.6 MHz. Briefly, the samples were spun in 4 mm rotors at 8 kHz, contact pulses between 0.5 and 3 ms, and a delay of 5 sec was applied in between the scans. The acetylation degree was approximated from the ratio of methyl groups to the peaks clearly distinguishable from possible impurities (C2 peak). The determination of chitosan and chitin content within this method considers a nitrogen balance. For calculation purposes, it was assumed that the only nitrogen-containing biomolecules in the A. niger mycelium are proteins, chitin, and chitosan. In addition, an average protein conversion factor of 6.25 was assumed [20]. Based on the total nitrogen concentration of the mycelium (NCHN), the protein concentration (cProt), the nitrogen content of the proteins (NProt) and the calculated nitrogen content of a 36% deacetylated chitin (N36 % CHIT = 7.55%), an average chitin content in the mycelia (cChitin+Chitosan) can be calculated according to Eq. (2):

$$c_{{{\text{Chitin}} + {\text{Chitosan}}}} (\% ) = \frac{{N_{{{\text{CHN}}}} \cdot (100 - N_{{{\text{Prot}}}} ) \cdot c_{{{\text{Prot}}}} }}{{N_{{36 \% {\text{ CHIT}}}} }}$$
(2)

2.2.3 Chitin and chitosan characterization by acid detergent-fiber (ADF) and total hydrolysis

To verify the results of the new proposed method for chitin and chitosan content, a second method was conducted for comparison. First the chitin content was measured by the ADF and acid detergent lignin (ADL) procedure according to ISO 13906:2008 and Hahn et al. [21]. As confirmed by elemental analysis, the fiber applied for gravimetric determination did not only contain chitin but glucans as well. Therefore, an additional totally hydrolysis (see above) with succeeding chromatographic analysis of the ADF regarding its glucan content was conducted. The adjusted chitin content was then determined by subtracting the glucan amount (mGlucan) from the ADF (mADF) divided by the original amount of mycelia (mMycelia) according to Eq. (3):

$${\text{c}}_{\text{Chitin}} \, (\%) = \frac{{\it{m}}_{\text{ADF}}-{\it{m}}_{\text{Glucan}}}{{\it{m}}_{\text{Mycelia}}}$$
(3)

As the ADF-method only measures chitin, and not the native chitosan occurring in the mycelia, the content of the native chitosan in the biomass was determined according to Zamani et al. [14] and Cui et al. [15] (see chitosan analytics).

2.3 Methods for chitosan production

Experiments regarding chitosan production were done in triplicates, except for DA whereby the experiments were conducted in duplicates.

2.3.1 Deproteinization of fungal mycelia

The Tornado reaction system IS6 from R.B. Radley Co. Ltd was used for deproteinization. To investigate the optimum process conditions for deproteinization, biomass to solution ratio and the NaOH molarity were varied. 100 mL of NaOH solution of varying molarity was added to the reaction vessels and heated until the liquid reached a temperature of 90 ± 4 °C. Then, a defined amount of biomass of A. niger mycelium was added and stirred for 2.5 h at approximately 250 rpm. Next, the biomass was separated with a paper filter (pore size 5–13 μm) under vacuum and washed with water until pH neutrality (0.8–1.0 L). The neutralized biomass was subsequently washed with 200 mL acetone and dried at 105 °C. The material was weighed to determine the biomass loss and yield. Deproteinization efficiency was calculated as the ratio of protein amount in the supernatant compared to the total protein content which was determined before.

2.3.2 Acid extraction by acetic acid and sulfuric acid

The dried, deproteinized biomass contains unmodified and not-bound chitosan that was extracted with either acetic acid or sulfuric acid.

The first method was adapted from Tayel et al. [13]. For the extraction, 16 g of deproteinized biomass was suspended in 640 mL of 2% (v/v) acetic acid (S/L 1:40 (w/v)) in a round-bottomed flask and heated to 60 °C with an oil bath. The incubation is carried out under reflux to avoid liquid loss. After 6 h of incubation, the alkali- and acid-insoluble material (AAIM, solid) is separated by centrifugation at room temperature (RT) and 4,872 x g for 15 min from the acid-soluble material (AASM, liquid). The supernatant contains dissolved chitosan, while the pellet contains, besides other natural compounds, the insoluble chitin. To wash the pellet, it was resuspended in 100 mL water, centrifuged again (RT, 4,872 x g, 15 min) and the supernatant discarded. This step was repeated until the supernatant was pH-neutral. Subsequently, the pellet was washed two times with 50 mL acetone and dried. The chitosan-containing supernatant was titrated with 40% (w/w) NaOH solution to pH 8–9. The solution was then incubated at 4 °C for at least 48 h until the chitosan had completely precipitated. The flocs were then separated using a glass filter frit (POR 3, 16–40 μm, 125 mL). And the retentate was washed with water (400 mL) and acetone (200 mL). The product was dried and weighed.

A sulfuric acid extraction of chitosan from fungi was first described by Zamani et al. [14] and Cui et al. [15]. According to the authors, chitosan does not dissolve in cold diluted sulfuric acid, but does readily dissolve in the acid at a temperature exceeding 70 °C. For this study, the procedure was slightly adapted. The aim was to achieve partial hydrolysis of the glucans by the use of sulfuric acid and thus to increase the accessibility for subsequent DA and consequently the purity of the product. First, 6.25 g of dried, deproteinized mycelia was weighed and transferred to 500 mL shake flasks with bottom baffles. Subsequently, 250 mL of 72 mM sulfuric acid was added (S/L 1:40 (w/v)) and the mixtures were autoclaved (121 °C, 20 min). The biomass was then rapidly filtrated while being hot using a hot glass filter frit (POR 2, 40–100 μm) to prevent precipitation of chitosan prior to filtration. The chitosan-containing filtrate was immediately titrated to pH 8–10 with 8 M NaOH solution before cooling at 4 °C overnight whereby the chitosan precipitated in form of white flocs. The flocs were then separated using a glass filter frit (POR 3, 16–40 μm, 125 mL). The retentate was then washed with water (400 mL) and acetone (200 mL). The product was dried and weighed. To wash the chitin-containing retentate, it was resuspended in 100 mL water, centrifuged (RT, 4,872 x g, 15 min), and the supernatant was discarded until the washing water was pH-neutral (5–10 times). The pellet was then washed with acetone (200 mL), dried and weighed.

2.3.3 DA of chitin

For the DA of chitin to chitosan, 400 mL 40% w/w NaOH was filled into a 500 mL PFA round bottom flask and heated to 120 °C. Once the temperature was reached, 20 g of biomass (from mycelium, AIM or AAIM) was added to the flask (1:20 (w/v)). Then, DA was performed for 2.5 h at 120 °C and 200 rpm with a magnetic stir bar under reflux. The treated biomass was separated from the DA solution using a 40 μm paper filter and the retentate was removed. This retentate was then washed by resuspension in 200 mL water and the liquid was separated by centrifugation (RT, 4,872 x g, 5 min) till a neutral pH was reached (10–15 times). Subsequently, 200 mL of acetone was added to the biomass, centrifuged again (RT, 4,872 x g, 5 min) and the supernatant was discarded. After the acetone had evaporated under the fume hood, the chitosan was dried in an oven at 105 °C and chitosan as well as the filter were analyzed gravimetrically.

2.3.4 Solubilization and purification for chitosan purification

To purify the deacetylated chitosan, 1 g of chitosan was dissolved in 100 mL of 1% (v/v) acetic acid while stirring overnight at RT. Subsequently, residual non-solubilized particles were removed through a 40 µm paper filter, which was previously dried at 105 °C and weighed. Next, the filtrate was titrated to a pH of 8–10 by an 8 M NaOH solution, which precipitated the dissolved chitosan. After at least 48 h incubation at 4 °C, the chitosan was fully precipitated and was separated using a paper filter (pore size: 2–4 µm). It was washed with 300 mL water and 100 mL acetone. The chitosan was then dried at 105 °C overnight, weighed and characterized.

2.4 Chitosan analytics

2.4.1 Determination of the DD by 1 H NMR

1H NMR spectroscopy was performed according to Hahn et al. [18]. DD (%) was calculated by the area ratio of the acetyl peak \({\text{I}}_{{\text{CH}}_{3}}\) and the mean area of the additional hydrogen molecules \({\text{I}}_{\left({\text{H}}_{2}-{\text{H}}_{6}\right)}\) according to Fernandez-Megia et al. [22] by Eq. (4):

$${\text{DD}} \left( \% \right) = \left( {1 - \frac{{\frac{1}{3} \cdot I_{{{\text{CH}}_{3} }} }}{{ \frac{1}{6} \cdot I_{{\left( {{\text{H}}_{2} - {\text{H}}_{6} } \right)}} }}} \right) \cdot 100$$
(4)

2.4.2 Purity of chitosan by elemental analysis and solubility

To determine purity by elemental analysis, the elemental composition of pure chitosan or chitin needs to be considered. The aim was to compare the nitrogen content of the sample to the theoretical nitrogen content of a completely pure chitosan at a given DD. The theoretical sample nitrogen content Ncalculated was calculated according to Eq. (5) with a nitrogen content of chitin at 0% DD (NChitin = 6.86%) and chitosan at 100% DD (NChitosan = 8.64%) [23]:

$$N_{{{\text{calculated}}}} (\% ) = (N_{{{\text{Chitin}}}} + \left( {N_{{{\text{Chitosan}}}} - N_{{{\text{Chitin}}}} } \right) \cdot {\text{DD}})$$
(5)

The calculation of the theoretical nitrogen content of chitin and chitosan was done under the assumption that the nitrogen is completely attributable to the biopolymer and that no nitrogen-containing impurities such as proteins are present in the purified chitosan product. Furthermore, the DD had to be known by another method, in this case 1H NMR spectroscopy. In addition, it was theoretically assumed that the biopolymers are an infinitely long chain, since the length of the individual polymers is not known which can affect the hydrogen content.

The determined nitrogen content was further corrected by the moisture content (Ms) of the sample. To determine the purity, the ratio of the nitrogen content of the chitosan sample determined by elemental analysis (Nsample) and the nitrogen content of the theoretically pure product (Ncalculated) was calculated Eq. (6):

$${\text{Purity}}\,(\% ) = \frac{{N_{{{{\rm sample}}}} }}{{N_{{{{\rm calculated}}}} \cdot (100 - M_{S})/100}}$$
(6)

Solubility was used as a second measure to evaluate chitosan purity. For evaluation, 100 mg of dried chitosan was dissolved in 10 mL of sodium acetate buffer pH 4.4 for 12 h at RT while stirring. Insoluble chitosan was separated using a filter crucible (POR 2, 40–100 µm) and weighed.

Molecular weight analysis was conducted as stated in previous work [18].

2.5 Statistical analyses

Deviations between the mean values were first analyzed using an F-test. Afterward, a t-test was carried out for equal variance and Welch's t-test for different variance with a significance level of α = 0.05. To compare several mean values, a one-way analysis of variance was carried out, also with a significance level of α = 0.05.

3 Results and discussion

The first step in the production of chitosan is the characterization of the mycelium, which is the only way to properly characterize the subsequent process. The results of the subsequent chitosan production in this publication are intended as an example of the potential that can be found in the processing of fermentation residues containing fungi to produce a sustainable, vegan chitosan.

3.1 Biomass composition of A. niger mycelia

3.1.1 Elemental content

The A. niger mycelia exhibited an elemental composition of 44.8 ± 0.1% carbon, 7.0 ± 0.1% hydrogen and 2.6 ± 0.1% nitrogen. Since chitin, chitosan and also proteins are nitrogen-containing molecules, the determined nitrogen content cannot be used to directly conclude to the mycelium components. Nevertheless, it is relevant for the subsequent calculation of the content of chitin and chitosan.

3.1.2 Protein content of mycelia by consecutive deproteinization

The protein content was determined by two subsequent deproteinizations followed by an analysis of the supernatant regarding the protein content via spectrophotometric assay. A previous neutralization of the supernatant and the use of a standard addition procedure to decrease negative influences of the sample matrix on the protein assay was crucial for a reliable protein detection. In the frame of the first deproteinization, 19.7 ± 0.3% of protein content was calculated by measuring the protein amount in the supernatant. In the second deproteinization, 2.5 ± 0.6% protein in the supernatant was determined (Fig. S1). Thus, the A. niger mycelium has an approximate total protein content of 22.2 ± 0.7% assuming all protein was extracted. Comparable data were found by Cai et al. [12] who determined a protein content of 23.1% by Kjeldahl method. The found protein content was also in the range of the general protein concentration of fungi, which is between 20 and 50% [24]. These results laid the foundation for the following calculation of the chitin and chitosan content by a nitrogen mass balance.

3.1.3 Chitin and chitosan content

For determination of the chitin and chitosan content of the mycelia, two different methods were applied and compared: The first one used a nitrogen mass balance calculation. The second one combined a gravimetric method and a chromatographic measurement after total hydrolysis to determine the content via an ADF and total hydrolysis procedure.

3.1.4 Chitin and chitosan determination method 1: nitrogen mass balance

The chitin and chitosan content was determined by a combination of different analytical tools resulting in a nitrogen mass balance. Additional to the determination of the elemental composition and the protein content, the average DD of chitin/chitosan in the unprocessed mycelium has to be measured.

A tool used to determine DD in this study was 13C CP MAS NMR spectroscopy applying cross polarization (CP) with a contact time of 1 ms, according to literature (Fig. 1) [19]. The respective spectrum of the mycelia, as shown in Fig. 1, was characterized by eight distinctive peaks. C1–C6 peaks between δ13C = 50–120 ppm were assigned the respective carbon atoms of the glucosamine ring. In addition, a carbonyl group giving rise to a peak at δ13C = 175 ppm and an acetyl group resulting in a peak at δ13C = 23 ppm [25].

Fig. 1
figure 1

Gaussian fit of the 13C CP MAS NMR spectra of unprocessed Aspergillus niger mycelia regarding the C2 and CH3 peak of the acetyl group indicated.

Full size image

Only the peak assigned to the C2 atom is, with δ13C ≈ 55 ppm, out of the shift range δ13C where peaks are potentially affected by impurities [26]. To crosscheck the validity of the literature method, the applied contact time was screened between 0.5 and 3.0 ms (Fig. S2) to make sure that the peak intensity ratio between C2 peak and CH3 group remain constant [19]. Impurities that could lead to statistical errors would have different polarization transfer characteristics and thus cause changing peak intensities. However, the peak of the C2 atom and the ratio remain constant after a contact time of 1 ms and above (Fig. S2). This supports further that the evaluation method of Ottoy et al. [19] to approximate the DD at 1 ms contact time is also applicable, if exclusively the intensity of the C2 peak instead of the mean C1–C6 peak intensity in Eq. 3 is considered (Fig. 1). This resulted in a mean DD of 40%, which is consistent with a DD of 37–42% that Heux et al. [25] determined with their method on similar material.

Subsequently, a total chitin and chitosan content of 16.0 ± 0.8% was calculated by Eq. 3. Regarding the 13C CP MAS NMR, it has to be mentioned that the influence of the mean DD is quite low on the final chitin and chitosan content. It must be noted that with this form of content determination of chitin and chitosan no individual differentiation between the chitin and chitosan was possible, since the mean DD was used in the calculation and a quantity distribution of the DD over the polymers was not possible.

3.1.5 Chitin determination method 2: the ADF and total hydrolysis procedure

Following the ADF-ADL method of Hahn et al. [21], different compounds of the fungal mycelium were removed, such as proteins, fats, free sugars and soluble minerals, to purify the chitin and gravimetrically determine its content [21, 27]. The method was originally developed and used to determine the fiber content in feedstuff, but was already successfully adapted to determine chitin content in insects. With this method, an ADF content of 52.5 ± 3.7% was measured. Since this value clearly exceeds the results of the other measuring method, it was determined by means of elemental analysis that a high level of organic impurities was still present because of a low nitrogen content. In particular due to glucans, which increased the carbon to nitrogen ratio (C/N = 14.7), while pure chitin would be around 6.9 [23]. Therefore, total hydrolysis was selected as the method of choice to determine the glucan content. Hereby, the fiber was hydrolyzed and the remaining number of glucans in the fiber could be determined by calculating the sum of glucose which could be detected chromatographically. Thus, a glucan content of 63.8% was measured in the fiber, resulting in a chitin content of 36.2% in the fiber and, respectively, 19.7 ± 3.5% considering the chitin content of the total mycelium. Since the glucan content in the ADF was lower than in the mycelia, it was obvious that glucans were only partially hydrolyzed under these conditions. The ADL content was below the determination limit of the ISO norm and was therefore negligible. Since fungal biomass, unlike other chitin sources, also contains chitosan, it was mandatory to determine whether the chitosan was also present in the ADF or whether it was removed during the process. As Zamani et al. [14] and Cui et al. [15] already confirmed, chitosan is only soluble in hot sulfuric acid and not in cold one. Chitosan was therefore treated according to the ADF protocol. Chitosan was found to be soluble in the hot ADF solution, so it can be assumed that the chitosan embedded in the mycelium is removed during the ADF process. Therefore, chitin was determined by the ADF procedure and chitosan from acid extraction (see acid extraction results below) resulting in a total chitin and chitosan content of 19.9 ± 3.7%.

3.1.6 Comparison of the methods for the determination of chitin and chitosan content

The difference between both methods was not significant (α = 0.05, p = 0.26, p > α) whereby both methods confirm each other. While method 2 caused a higher standard deviation due to the number of manual steps, method 1 was the more precise method, but required more sophisticated equipment such as solid state 13C NMR and elemental analysis. In general, the calculation according to method 1 showed that fluctuations in the degree of DA have only a very minimal influence on the chitin content, while the protein content has a greater one.

The results were in the same order of magnitude with Wu et al. [28] who found 13% of chitin in the mycelia of A. niger. This group monitored the chitin content in relation to the time of fermentation. They measured an increasing chitin content during the fermentation period with a maximum content of 13% at the end of the exponential phase. The biomass used in this study was isolated at the end of a fermentation, thus also after completion of growth. Zhao and Wang [29] found a higher chitin amount of 20–22% which is in line compared to the results of this study. It needs to be stated that the chitin content can be heavily affected by cultivation conditions and the strain used [30, 31].

The previous findings with regard to the chitin and chitosan content formed the basis for the evaluation of the chitosan production process and the determination of the chitosan yield obtained.

3.1.7 Glucan and total sugar content

For the A. niger mycelia a β-glucan content of 48.0 ± 1.4% and an α-glucan of 0.6 ± 0.1% was determined in quintuplicates resulting in a total glucan content of 48.6 ± 1.4%. These results were consistent with data published by McCleary and Draga [32] who also measured the glucan content of A. niger and determined a content of 50.9% β-glucan and 1.0% α-glucan. That means that a certain amount (9.9%) was not identified yet. This is why the total hydrolysis method was also performed as an additional analysis method to evaluate the results. These experiments resulted in a total sugar content of the mycelia of 58.0 ± 1.3%. The deviation between the results of the glucan assay and the total sugar analysis were significant (α = 0.05, p = 0.0003, p ≦ α). This indicates that there were other carbohydrates contained in the mycelia additional to glucans. While the glucan assay selectively targeted glucans via specific enzymes, the total acid hydrolysis releases all monosaccharides in the mycelia that could be detected by the chromatography. The measured monosaccharides consisted exclusively of glucose. Possible explanation could be the accumulation of starch in the mycelia as previously reported for other fungi [33, 34]. In addition to that, the mycelia was simply dried after fermentation making it plausible that carbohydrates from the fermentation process simply adhered to the surface of the biomass increasing the total sugar content of the mycelia. Mannan or mannose, which are often part of glycoproteins of fungi, were not identified [30].

3.1.8 Summary of mycelia composition

The share of minerals (1.0%), fat (1.4%) and organic acids (0.9%) in the mycelia composition were low and could be neglected for further calculations. Based on these findings, for the first time methods to determine the total composition of the A. niger mycelia were established. The results are summarized and presented in Fig. 2.

Fig. 2
figure 2

Percentage composition of the dry content of Aspergillus niger mycelia as determined in this publication, for chitin and chitosan content results of 13C CP MAS NMR method were taken.

Full size image

In addition to the target substances chitin and chitosan, it was noticeable in the composition that proteins and glucans also make up the two largest proportions of the fungal mycelium. Therefore, the purity of the chitosan products after the process is mostly affected by the efficiency of protein and glucan removal. For prospective zero waste concepts, it would also be crucial to isolate the components in a cascade approach.

3.2 Chitosan production

Chitosan production from A. niger mycelium was performed according to three different process sequences (Fig. 3). Individual parameters were varied, methods were compared and the necessity of the process steps for chitosan production was investigated.

Fig. 3
figure 3

Different chitosan production pathways that were proposed and researched in this study comparing the necessity of each process step including an optional (dotted lines) purification step after direct deacetylation. As the yields after sulfuric acid extraction were higher, only these were considered further and subsequently deacetylated. DA: deacetylation, DPDA: deproteinization and deacetylation, DPAEDA: deproteinization, acid extraction and deacetylation.

Full size image

3.2.1 DA

After direct DA without purification, 23.5% of the biomass could be recovered. The yield exceeded the chitin content in the mycelia indicating that the product is impure and still contains large amounts of glucans, which was also confirmed through the high C/N ratio of 14.5. Taking into account that the mean C/N ratio of protein is around 3.2, the removal of proteins had thus additionally increased the C/N ratio of the DA product [35]. Pure chitin has a ratio of 6.85, chitosan of 5.15 [36].

3.2.2 Deproteinization and deacetylation (DPDA)

To increase the purity of the chitosan, deproteinization was performed as former step of DA. In this step, a low concentrated base, preferably NaOH at an increased temperature was used to extract the proteins from the mycelia. Under these conditions, a breakdown of covalent bonds, like disulfide and peptide bonds, occurs [37,38,39]. The largely unbound proteins could then be easily extracted, as solubility was increased in the basic environment due to the ionization of the proteins and amino acids [40]. The aim was to use the lowest possible concentration of NaOH and a high amount of biomass per volume of NaOH solution without compromising protein removal to achieve an efficient deproteinization process. As a measure, the protein concentration in the supernatant was determined via standard addition procedures, calculated as deproteinization efficiency and shown in Fig. 4.

Fig. 4
figure 4

Influence of variations in the dry biomass to NaOH ratio at 2 M NaOH (A) and the molar concentration of NaOH (B) on biomass loss, deproteinization efficiency and removed protein. Deproteinization efficiency was calculated based on the total protein content determined above.

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No significant differences in biomass loss or deproteinization efficiency were observed when varying the biomass to NaOH ratio. The C/N ratio of the residual biomass ranged between 16.0 and 16.4 with no clear trend toward a specific ratio. Thus, the amount of biomass does not have a negative effect on deproteinization. Therefore, a ratio of 1:15 (w/v) was chosen for chitosan production. While the variation of ratios was performed with 2 M NaOH, the NaOH concentration was evaluated as well at a solid-to-liquid ratio of 1:15.

The loss of biomass is in line with expectations. Higher concentrations of NaOH had a higher impact on the mycelium and thus resulted in an increasing biomass removal. The purity of the intermediate product from our study only increased slightly from a C/N ratio of 17.1 in the unprocessed mycelia to 15.6 (1 M), 16.1 (2 M) and 16.4 (4 M) in the deproteinized biomass. On the other hand, it is striking that the deproteinization efficiency with 1 M (88.8%) was approximately twice as high as under higher base concentrations (48.6% resp. 40.0%). Possible explanation of the phenomenon could be that the protein dye used in the standard addition procedure only binds to peptides larger than 3 kDa according to the manufacturer. Harsher conditions result in common in small peptides and even amino acids. Cai et al. [12] achieved a lower deproteinization efficiency of just 62.3% through measurement with the Kjeldahl method of the deproteinization supernatant by using an untreated mycelia and a low process time (20 min). When we used a not ground mycelia for deproteinization the deproteinization rate lowered to 74.0% (1 M NaOH, 1:15 (w/v)). This shows that pretreatment can increase the deproteinization rate, but due to the low reduction in comparison, milling will not be used in future work.

Using the deproteinized and not ground biomass (1 M NaOH, 1:15 (w/v)) for a subsequent DA 14.9% of chitosan-containing product was obtained.

3.2.3 Deproteinization, acid extraction and deacetylation (DPAEDA)

An additional acid extraction was performed after deproteinization to extract the naturally occurring chitosan prior to DA. Two different methods for chitosan extraction were carried out following Tayel et al. [13] using acetic acid and Zamani et al. [41] using sulfuric acid. The idea is that deproteinization had already partially broken down the cell wall of the mycelium by removing glycoproteins that were covalent bound to it, making the chitosan more accessible and allowing direct extraction of chitosan with acid [42]. The extractions were evaluated by residual biomass (AAIM), biomass loss and yield (Fig. 5).

Fig. 5
figure 5

Comparison of the two extraction methods for chitosan based on acetic acid or, respectively, sulfuric acid. Evaluated were the gravimetric content of alkali- and acid-insoluble material (AAIM) after extraction, the loss of biomass as well as the extracted chitosan without taking purity in account.

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The chitosan resulting from both protocols had a lightly brown and orange color. The biomass loss using diluted acetic acid as solvent was 12.6% lower than with sulfuric acid. The high temperature combined with a low concentrated sulfuric acid (72 mM) initiated a partial hydrolysis of the glucans in the mycelia increasing the biomass loss. The chitosan yields were 0.7 ± 0.1% with acetic acid and 1.5 ± 0.3% with sulfuric acid in relation to the untreated biomass. The chitosans are distinguished by a low purity with a C/N ratio of 23.6, respectively, 39.1 and a mediocre solubility in acids. In contrast to purity, the DD measured by 1H NMR of the chitosans were high, ranging from 88.2 to 91.1% without DA. The yields found in this study are contradictory to similar studies of chitosan extraction, which achieved much higher yields of 3.5–11.6% during similar conditions using acetic acid [13, 43]. Dhillon et al. [44] used the same sulfuric acid-based method with A. niger mycelia yielding between 3.9 and 6.4%. In the literature of both extraction methods, disintegration methods such as milling or freeze-drying are used to disintegrate the mycelium beforehand. Apparently, the chitosan is still too integrated into the membrane even after deproteinization treatment and cannot be completely extracted in this study. Using prior biomass milling, as shown in the analytical methods, would probably have resulted in a more accessible chitosan and an increased yield.

However, the extraction of natural occurring chitosan without further pretreatment was not efficient in this case. The procedure had again a positive effect on chitin purification before DA, lowering the C/N ratio of the AAIM from 15.6 after deproteinization to 13.6 (acetic acid) and 11.5 (sulfuric acid). For the DA, the AAIM of the sulfuric acid extraction was used, which resulted in a yield of chitosan of 6.3% in this three-step process.

3.2.4 Summary of the three chitosan production pathways

This section summarizes and characterizes the chitosan products after the three different processes to produce chitosan (Table 1). The standard deviations are shown in Table S1.

Table 1 Summary of the chitosan production process by direct deacetylation (DA), with additional deproteinization (DPDA) and additional acid extraction (DPAEDA) without further purification steps
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The major difference that was observed concerning the yield was also noticeable regarding the purity. The number of the purification steps apparently increased the purity while lowering the recovery of the product. The recovery was calculated in the relation to the amount of chitin determined in the unprocessed mycelium (16.0%), while taking into account the purity. But it was also visible that despite having a low chitosan yield itself the acid extraction had a major influence on the purity after DA, increasing it by 32.7%. When conducting a single factor analysis of variance there was no significant difference regarding DD between the DA at different stages (α = 0,05; F = 1,22; FKrit = 4,26; F < FKrit). Thus, the direct DA was the most efficient procedure to obtain a chitosan with a higher DD at the cost of a low purity. Compared to other studies that conducted a direct DA like Muñoz et al. [6] having a chitosan with a DD of only 72.9%. This is probably because of the high solid-liquid ratio (1:5 w/v) and lower temperature (110 °C, 4 h) compared to the study presented here.

3.2.5 Solubilization and precipitation to increase chitosan purity (DA + purification)

Since direct DA, as well as DA with prior deproteinization, gave insufficient results in terms of purity, it was decided to carry out an additional purification step. The chitosan after direct DA was subjected to succeeding purification investigation. Since it has only a slightly lower purity, but a significantly higher chitosan content, respectively, recovery compared to the other samples. After purification, 5.5 ± 2.2% of an orange-colored chitosan was obtained.

3.2.6 Overview and characterization of the two proposed methods to produce chitosan

An overview of the chitosans from the two most promising processes in this study compared to commercial chitosans is shown in Table 2. Standard deviations can be obtained from Table S2.

Table 2 Overview of the characteristics of the chitosan from different production processes
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The data include the chitosan from acid extraction (DPAEDA), respectively, including the chitosan from purification after direct DA. Since the chitosan from the DPDA process were of insufficient purity, this process sequence was not considered further below. In general, both chitosans exhibited a high DD (~90%) combined with a very low-molecular weight (~20 kDa). Despite the harsh conditions of using sulfuric acid and autoclaving, no noticeable difference between a direct DA and a prior acid extraction regarding Mw and DD of chitosan was found. Therefore, we could conclude that the conditions during DA seemed to be the main factor for degradation impacting molecular weight. It was worth noting that only the chitosan resulting from direct DA and following purification is completely soluble in sodium acetate buffer pH 4.4. Commercial chitosan from Pleurotus ostreatus dissolved only partly indicating that there were still glucans cross-linked yet. This trend was also recognizable in the purity of the different chitosans. Besides glucans, the largest impurity was found to be acetic acid and sodium acetate which could be detected in the 1H NMR spectra of the process with the purification step (DA + purification) at 1.85 ppm, respectively, and 1.96 ppm indicating that the purity can be increased by an adapted wash or evaporation process. While having the lowest yield, the direct DA followed by purification had the highest recovery. Interestingly, in the three-step process, the recovery is only slightly lower, suggesting that the main chitin losses occurred during DA and not in the deproteinization or acid extraction. This is probably due to depolymerization and degradation during the harsh conditions of the DA. In comparison with other studies, the yield is comparable or slightly lower (Table 3).

Table 3 Literature comparison of yield, molecular weight and DD of different chitosans obtained from Aspergillus niger
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As assumed, the chitosan from A. niger mycelia were also characterized in the literature by a low-molecular weight and a moderate DD. The molecular weight seemed to be generally low and consistent with the results here, while the DD was often lower than in this study implying that the parameters used in the DA should be adapted. Only Cai et al. [12] and Muñoz et al. [6] achieved an above-average higher molecular weight due to a low NaOH concentration (4%) or a high solid/liquid ratio (1:5 w/v), whereby the chitosan was spared by mild conditions, but only a low DD was achieved. A comparison regarding the yield was difficult, since the literature studies currently did not provide information with regard to purity or recovery. Recovery in particular would be a useful measure for evaluating the processes, since the yield must always be considered in relation to the original chitin content of the mycelium enabling to express a reasonable conclusion about the process efficiency.

4 Conclusion

As it is shown in the study, a direct DA and purification process is suitable to produce chitosan with high DD (90.5%), low-molecular weight (18 kDa) and increased purity (89.6%). The succeeding application was not in the focus of the study. It needs thus to be confirmed in further application-specific investigations that the fungal chitosan is eligible for applications where a low-molecular weight and vegan chitosan could be suitable rather than a arthropods-based one.

To assess chitosan production process, we developed and compared for the first time an analysis of the total biomass composition. In this sense, we observed that a direct DA would degrade the major part of the biomass. This means that the other compounds such as proteins and glucans (in total 70.8%) as main components could not be valorized any more although these represent valuable compounds itself. It could be also assumed that an economic process for chitosan production is not viable without the isolation of the other compounds. A chemical or better a biotechnological process to obtain the compounds in a cascade approach would thus be favorable in the future.

With the adaption of the known analytical methods, we achieved valuable tools to initiate and foster this research. This has also contributed to provide our readers with a thorough evaluation of the chitosan production by using defined product or process characteristics such as purity and recovery. This can also be seen as plea for other chitin purification and chitosan production investigations to characterize the whole process enhancing a comprehensive understanding of the process. Although all three process strategies resulted in a non-purified chitosan, the investigations revealed that a direct DA with preliminary steps followed by purification led to a high-purified chitosan. In prospective investigations, it would be worthy to subject also the “contaminated” chitosan to application-specific tests in order to avoid the purification step.