# Enzyme-grafted nylon membranes for sequential filtration of Tetraselmis chui extract with enhanced protein recovery

Tim Schreiber, Martin Schmidt, Andrea Prager, Alexia Poidevin, Estelle Couallier, Agnes Schulze

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Tim Schreiber, Martin Schmidt, Andrea Prager, Alexia Poidevin, Estelle Couallier, et al. Enzyme-grafted nylon membranes for sequential filtration of Tetraselmis chui extract with enhanced protein recovery. Separation and Purification Technology, 2025, 369, pp.133131. 10.1016/j.seppur.2025.133131. hal-05341081

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Submitted on 31 Oct 2025

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# Enzyme-grafted nylon membranes for sequential filtration of *Tetraselmis chui* extract with enhanced protein recovery☆

Tim Schreiber<sup>a</sup>, Martin Schmidt<sup>a</sup>, Andrea Prager<sup>a</sup>, Alexia Poidevin<sup>b</sup>, Estelle Couallier<sup>b</sup>, Agnes Schulze<sup>a, *, ◎</sup>

## ARTICLE INFO

Editor name: Dr. B. Van der Bruggen

Keywords:

Polymer membrane

Nylon

Microalgae

Protein extraction

Trypsin

Lipase

*Tetraselmis chui*

## ABSTRACT

Microalgae are considered a promising and sustainable source for valuable biomolecules such as proteins. Extraction of proteins from microalgae suspensions by membrane technology is still limited by fouling and inefficient recovery due to the complex mixture of biomolecules. In this work an enzyme-modified nylon microfiltration membrane was prepared to efficiently recover proteins from an extract of the microalgae *Tetraselmis chui* using a simple sequential filtration setup. A mixture of hydrolytic enzymes, trypsin and a lipase, was successfully grafted onto flat-sheet membranes using an electron beam-based approach. It was shown that for generating high lipase activity, co-immobilization with trypsin or another protein is beneficial. A design of experiments approach was used to determine the effect size of selected parameters for the sequential filtration process. Derived empirical models were used for optimization and confirmed by experiments resulting in protein recovery within the filtrate of up to 1.4 g/L marking a significant increase compared to 0.8 g/L with the commercial reference membrane. Furthermore, up to 40 % of the total protein content from the microalgae dry matter could be recovered, compared to 24 % with the reference membrane. The sequential filtration was found to be advantageous compared to direct filtration allowing a combination of both enzymatic incubation and filtration. Various characterization methods were performed showing that the enzyme grafting layer reduces fouling.

## 1. Introduction

Microalgae are diverse, single-celled plant organisms that are found in a wide variety of aqueous environments such as oceans, lakes, ponds and rivers around the world [1]. While macroalgae such as seaweed have been used for thousands of years, particularly in Asian cuisine, and are associated with a balanced and healthy diet [2], microalgae are much less utilized so far. However, due to their nutrient-rich composition, rapid growth, and comparatively simple cultivation conditions, they have great potential as a sustainable source of valuable biomolecules such as proteins, fatty acids, and vitamins [3]. Thus, microalgae are currently the focus of research for a wide range of applications, such as the production of biofuels [4], as feed supplements in livestock farming and aquaculture [5], or as ingredients in innovative and sustainable food products [6,7].

Of particular interest is the extraction of proteins, which are

considered the basis of a balanced diet for both animals and humans [8]. Whole-cell proteins are protein-dense cellular structures that are most commonly added to microalgae formulated food products. The addition of whole cells has a beneficial effect due to the wide range of nutritious biomolecules in microalgae but is often limited by digestibility, strong color and a characteristic taste. With further extraction and purification steps, protein concentrates, isolates, hydrolysates, and bioactive peptides can be obtained that are used for various food products and supplements [9]. Several microalgae were already accepted as food additives by the European Union, such as *Tetraselmis chui*, which is a marine species of microalgae and has been added to the list of usable microalgae for food processes in 2014 [10]. Proteins can account for up to over 50 % of the dry mass, depending on the microalgae species, and contain a high content of essential amino acids [11], hence, industrial extraction of proteins is particularly interesting for both animal and human diet. To this end, the search for efficient and cost-effective

<footnote><sup>a</sup> Leibniz Institute of Surface Engineering (IOM), Permoserstraße 15, 04318 Leipzig, Germany</footnote>

<footnote><sup>b</sup> CNRS, Nantes Université, ONIRIS, Laboratoire de Génie des Procédés, Environnement et Agroalimentaire, GEPEA, F-44600 Saint Nazaire, France</footnote>

<footnote>* This article is part of a special issue entitled: 'EuroMembrane 2024 (invited only)' published in Separation and Purification Technology.</footnote>

<footnote>* Corresponding author.
E-mail address: agnes.schulze@iom-leipzig.de (A. Schulze).</footnote>
extraction methods is of great importance. Since most biomolecules in microalgae are present inside the cell, in the cytoplasm, or cell organelles, cell wall perforation is necessary. A simple thawing permeabilizes the cell wall and only 10% of the total proteins of *T. chui* are released in the aqueous phase [12], which means, 90% of the total proteins can still be used. Breaking up the cell structure is then an important step to release larger amount of valuable substances [13,14]. Among different processes, bead milling or sonication can be used for cell lysis. Once the cells are disrupted, clarification can be performed by centrifugation to separate cell fragments and the biomolecules dispersed in the aqueous phase as a dark green supernatant. The presence of chlorophyll can compromise the direct use of this extract [15]. Finally, an efficient separation of the ingredients is necessary in order to be able to utilize them in a targeted manner and exploit the full potential of microalgae in the extraction process within a biorefining context [16].

Common methods to separate desired biomolecules rely on precipitation [17,18], but precipitation of proteins from microalgae is not as efficient as with other crops (pea, soja) because of their variability and different isoelectric points. Another approach involves the implementation of a membrane filtration step as membrane technology is characterized by high selectivity, efficiency and mild filtration conditions and thus, especially polymer membranes are already used in processes such as biotechnology [19,20]. In addition, compared to other chemical-based extraction methods, additive-free separation can be performed which plays a particularly important role in recovery of potential food ingredients. Initial implementations in the process of extracting biomolecules from microalgae already showed the promising potential of membrane technology and demonstrated advantages compared to conventional methods [20]. However, in several cases the fractionation of biomolecules (lipids, polysaccharides, pigments, proteins) was restricted by a limited permeation of proteins through microfiltration membranes despite adapted membrane cut-off [20]. Two main hypotheses can be done to explain the high protein rejection [21]: the organization of biomolecules into aggregates larger than membrane pores, or as a result of membrane fouling that is still an essential challenge in membrane technology. Especially in the filtration of biomass, attractive interactions of solutes with the membrane material can occur leading to a reduced filtration performance over time [22]. A considerable proportion of operating costs in membrane-controlled processes can be attributed to irreversible fouling, which often requires the use of environmentally harmful or toxic chemicals [23,24]. Preventing or at least reducing fouling is therefore an important part of current research.

Enzymes have already been used with microalgae, mostly for cell disruption compared to conventional methods for microalgae processing such as bead milling or ultrasonication [12], and beneficial effects of using digestive enzymes have already been reported [16,25,26]. It was shown that enzyme mixtures containing lipase and protease can be used to degrade aggregates and release the proteins into the permeate. However, a disadvantage of this method is that fresh enzyme must be applied for each extraction process, which can lead to high operating costs in a continuous process. In this study, an integrated method of hydrolytic enzymes immobilized onto nylon flat-sheet microfiltration membranes was developed and used for recovery of proteins from an extract of microalgae *Tetraselmis chui*. For this purpose, a mixture of lipase and protease was immobilized by electron beam-induced grafting. Electron beam irradiation is considered a promising technology to covalently bind proteins and enzymes onto polymer membranes in a reagent-free approach [27,28,29]. The aim was to combine enzymes as "green" biocatalysts for digestion of bio-aggregates to facilitate the release of proteins, and the separation potential of a polymer membrane, in order to develop an innovative biocatalytic membrane reactor that efficiently extracts proteins from a microalgae suspension. The influence of the enzymes on protein recovery and membrane fouling mitigation was systematically investigated.

## 2. Experimental

### 2.1. Enzyme immobilization

A commercial polyamide (nylon) microfiltration flat-sheet membrane (Nytran® N, GE Healthcare Life Sciences Whatman, Germany; 0.45 µm) was employed. Immobilization was performed according to prior studies [22,23]. Briefly, nylon membranes (diameter: 44 mm) were impregnated with an aqueous solution of enzymes in phosphate buffered saline (PBS, pH 7.4) for 5 min. As enzymes, trypsin (Try, from bovine pancreas), and lipase (Lip, from *Candida rugosa*) were purchased from Sigma Aldrich (St. Louis, USA). The samples were then irradiated by transporting them on a conveyor belt through an electron beam. In this way, the membrane and the impregnated enzymes are irradiated simultaneously [30]. The applied dose was adjusted between 20 and 200 kGy by changing the speed of the conveyor belt (2.1–3.0 m/min). Acceleration voltage was set to 160 kV in order to penetrate the whole membrane cross-section. Subsequently, the samples were washed three times with PBS buffer for 20 min at 450 rpm.

### 2.2. Preparation of microalgae extract

A *Tetraselmis chui* extract was produced and provided by GEPEA (Saint-Nazaire, France). The frozen biomass was thawed one night at 4 °C at a dry matter concentration of 100 g/L. Then the initial biomass was sonicated by a Sonics® Vibra cell ultrasound probe (model 75185). The lysis parameters were: 5 min with an amplitude of 100 % corresponding to an energy of 8000 J, that is 130 W for 35 mL in an ice bath. Destruction rate is up to >90 %. After that, the lysate was diluted by two and macerate for 30 min to 1 h at 4 °C. Finally, the diluted lysate was centrifuged at 5000 g for 4 min at 4 °C to obtain the supernatant, also named microalgae extract. Supernatant of *Tetraselmis chui* was then lyophilized to facilitate the storage and transportation. The amount of dry matter present in the supernatant was measured by drying at 105 °C for 48 h of at least 4 g of supernatant. The protein content present in the supernatant was measured by bicinchoninic acid (BCA) assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific, USA). Thus, the protein composition of the lyophilizate was quantified at 59 % of the dry matter.

### 2.3. Microalgae extract filtration

For filtration, the lyophilized microalgae extract was resuspended in deionized water in Millipore® quality and treated with ultrasonic for 20 min to ensure an even dispersion (pH of suspension: 9.0). The modified membranes were freshly prepared the day of the filtration and then put into a stirred cell (Amicon model 8050, Merck, Germany). Filtrations were performed in a dead-end setup. First, 25 mL water was filtered at 0.1 bar. Then, microalgae suspension was added, and sequential filtration was performed at 0.1 bar under continuous stirring (450 rpm) at 23 °C. In total, 4 filtration steps were performed filtering each 5 mL of the algae suspension (total 20 mL). Before every filtration step the whole Amicon cell was incubated at 37 °C under continuous stirring (450 rpm) in order to increase enzyme activity. Permeate samples were used to determine the protein concentration, PC, by BCA assay. Bovine serum albumin (BSA) was used for calibration of the BCA assay. All measurements were performed at least in quadruplicate. Control experiments with protein-grafted membranes were conducted by immobilization of BSA (Sigma Aldrich, USA). Statistical comparison of means was performed using the method of Tukey and a significance level of 0.05 within OriginPro software (2024b, OriginLab, USA). The membrane samples were rinsed with water to remove reversible fouling and dried overnight at 37 °C for further characterization.

### 2.4. Immobilized enzyme activity

For the assessment of the immobilized enzyme activity, 10 mm
<table><caption>**Table 1**

Investigated process factors with lower and upper factor level limits. All factors were of numeric type.</caption><thead><tr><td>Factor</td><td>Name</td><td>Unit</td><td>Lower limit</td><td>Upper limit</td></tr></thead><tbody><tr><td>A</td><td>Lipase concentration</td><td>g/L</td><td>0</td><td>5</td></tr><tr><td>B</td><td>Trypsin concentration</td><td>g/L</td><td>0</td><td>15</td></tr><tr><td>C</td><td>Electron beam dose</td><td>kGy</td><td>20</td><td>200</td></tr><tr><td>D</td><td>Dry matter concentration</td><td>g/L</td><td>1</td><td>10</td></tr><tr><td>E</td><td>Incubation time</td><td>min</td><td>10</td><td>90</td></tr></tbody></table>

samples of the modified membranes with a central hole (diameter: 4.5 mm) were used. Photometric substrate conversion assays with continuous monitoring using a microplate reader (Infinite M200 Pro, Tecan, Switzerland) were performed according to previous work [29,31,32]. Briefly, membrane samples were placed in a 48-well plate and 300 µL of assay solution was added: (1) protease assay (0.5 mM BAPNA (N-benzoyl-D,L-arginin-p-nitroanilide) in HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 8.0) with 5 % dimethyl sulfoxide, DMSO); and (2) lipase assay (0.2 mM pNP-C12 (p-nitrophenyl laurate) in PBS buffer (pH 8.0) with 5 % DMSO).

Immediately after the addition of the assay solution, the change in absorbance was monitored for 12 h at 400 nm to record the enzyme catalyzed progress curve. Enzyme activity was observed via the release of a colored product from the reaction of the enzyme with the chromogenic substrate. Observed absorbance signals were converted into a concentration signal by employing a calibration curve. Reaction rates were determined by calculating the slope of the initial (linear) part of the progress curve (trypsin: 20–30 min, lipase: 1–5 min). One enzyme unit (U) corresponds to the release of 1 µmol of colored product per minute under the conditions of the test (pH 8.0, 25 °C). All measurements were performed at least in quadruplicate.

## 2.5. Design of experiments (DoE)

To systematically investigate various parameters influencing the filtration process, a design of experiments approach was carried out using the response surface method within the Design-Expert software (v23.1.1, Stat-Ease, USA). Five relevant process parameters were investigated (Table 1): (A) lipase concentration (0–5 g/L), (B) trypsin concentration (0–15 g/L), (C) electron beam dose during immobilization (20–200 kGy), (D) concentration of algae extract dry matter in the suspension (1–10 g/L), and (E) incubation time at 37 °C between the filtration steps (10–90 min). Filtrate protein concentration in g/L as well as protein recovery yield were selected as responses for the filtration process. An I-optimal design with quadratic model was applied resulting in 32 experiments. For this purpose, according to Equation (1), the protein yield, PY, was calculated using the averaged protein concentration in the filtrate, PC, and the dry matter concentration in the feed, D, containing about 59 % protein content. Models were manually reduced to only include significant factors (p-value ≤ 0.05) via analysis of variance (ANOVA). Subsequently, an optimization approach was carried out yielding optimized process settings that were used to

## 2.6. Membrane characterization

The chemical surface composition of untreated and modified membranes was investigated by X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra, Kratos Analytical Ltd., UK). A monochromatic Al Kα source operated at 150 W (15 kV and 10 mA) as well as charge compensation and correction were applied to record survey spectra (160 eV pass energy, 1.0 eV resolution), and high-resolution spectra (40 eV pass energy, 0.1 eV resolution), respectively.

The morphology of the membrane surfaces with and without fouling was investigated by scanning electron microscopy (SEM, Ultra 55, Carl Zeiss Ltd., Germany). Magnifications ranged from 300- to 10,000-fold. The samples were cut manually and then coated with a thin (30 nm) chromium layer using the Z400 sputtering system (Leybold, Germany). Multiple spots were analyzed to provide a representative overview.

Surface wettability of membranes with water was determined using a static contact angle measuring system (DSA, 30E, Krüss, Germany) employing the sessile drop method and the Young–Laplace model. Dynamic behavior of contact angle change was investigated by measuring in time intervals of Δt = 0.1 s.

# 3. Results

## 3.1. Sequential filtration to recover protein from Tetraselmis chui extract

A design of experiments (DoE) approach was carried out, as described in Section 2.5, to systematically investigate the effect of five selected process parameters on the sequential filtration of Tetraselmis chui extract using enzyme-modified nylon membranes. The experimental design, i.e., combinations of factor levels and measured responses, is given in appendix (Table A1). Two responses (targets) were chosen leading to different empirical models: 1) filtrate protein concentration, PC, in g/L; and 2) filtrate protein yield, PY, in %.

### 3.1.1. Model R1: Filtrate protein concentration

Investigated factors were the concentration of enzyme in the impregnation solution (A, lipase: 0–5 g/L; B, trypsin: 0–15 g/L), the dose of the electron beam (C, 20–200 kGy), the algae extract dry matter concentration in the suspension (D, 1–10 g/L), and the incubation time at 37 °C between the 4 filtration steps (E, 10–90 min), Thus, a total incubation time of 40–360 min was achieved. The averaged protein concentration in the filtrate (PC) was selected as the target value and determined by BCA assay using BSA protein as calibration substance. Evaluation of the analysis of variance is shown in the appendix (Table A2). The derived empirical model consists of 13 terms, including all main factors, as well as 2-factor interactions and quadratic terms (Eq. (2)), where A-E corresponds to the investigated factors in Table 1. Please note that a square root transformation had to be applied). Evaluation showed a high quality of the model according to the very high adjusted correlation coefficient of $R_{adj}^2 = 0.9701$, and furthermore allowing reliable predictions with a prediction coefficient of $R_{pred}^2 = 0.9554$.

$$ \sqrt{PC} = -0.03170A + 0.06689B + 0.00310C + 0.60219D + 0.01809E + 0.00799AB + 0.00051BE - 0.00006CE + 0.00085DE - 0.00469B^2 \\ - 0.03210D^2 - 0.00012E^2 + 0.47537 \quad (2) $$

perform model confirmation by conducting four replicate experiments.

$$ PY (\%) = \frac{PC}{D \cdot 0.59} \cdot 100 \quad (1) $$

As expected, the concentration of dry matter in the microalgae extract suspension (D) has by far the largest influence on protein recovery. Consequently, a higher initial concentration results in a higher protein content in the filtrate. It can also be seen that both the trypsin concentration (B) and the incubation time (E) have a strong influence on
<figure><img src="image_4.png" /><figcaption>Fig. 1. Response surface plots for the filtrate protein concentration depending on (A) the dry matter concentration in the microalgae extract suspension and trypsin concentration, (B) the incubation time and trypsin concentration, (C) the lipase and trypsin concentration, and (D) the dry matter and lipase concentration. The following factor levels (if not varied) were used to produce the graphs: lipase 5 g/L, trypsin 15 g/L, electron beam dose 140 kGy, dry matter 10 g/L, and incubation time of 60 min.</figcaption></figure>

the target value. The response surfaces in Fig. 1 show the correlation between the selected parameters and the target, revealing a positive effect of trypsin on protein recovery. High concentrations of trypsin, and thus high tryptic membrane activity led to higher protein concentration in the filtrate (Fig. 1A, B). The model shows that without trypsin, a protein content of up to 0.9 g/L can be expected, while immobilization of 15 g/L trypsin increases the recovery up to 1.5 g/L. The influence of

### 3.1.2. Model R2: Filtrate protein yield

In addition to the protein concentration in the filtrate, an empirical model for the protein yield can also be obtained based on the experimental data. Evaluation of the analysis of variance is shown in the appendix (Table A3). The model consists of 8 terms, including all main factors, as well as 2-factor interactions (Eq. (3)), where A-E corresponds to the investigated factors in Table 1).

$$ PY = 1.45A + 5.02B + 0.7731C - 7.18D + 5.04E - 2.13BD - 1.66CE + 25.11 \quad (3) $$

lipase is rather small, but nevertheless not negligible leading to a difference of up to 0.2 g/L when 15 g/L trypsin and 5 g/L lipase is grafted. It is also evident that protein extraction benefits from longer incubation times, which indicates that the enzymes were able to release further proteins from the complex dry matter suspension, and thus, make a significant contribution to protein recovery, which has already been shown with free enzymes [26].

The evaluation showed that this model also has a high degree of accuracy and predictability with a correlation coefficient of $R^2_{adj} = 0.9417$ and a prediction coefficient of $R^2_{pred} = 0.9247$. Compared to the first model, it is noticeable that the influence of the parameters has changed considerably. Although the dry matter concentration (D) still has the largest influence on the target variable, it is no longer as dominant as with the protein concentration model. Instead, trypsin concentration (B) and incubation time (E) are factors that strongly
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Response surface plots for the protein yield in filtrate depending on (A) the trypsin and the dry matter concentration in the microalgae extract suspension, and (B) dry matter concentration and incubation time. The following factor levels (if not varied) were used to produce the graphs: lipase 5 g/L, trypsin 15 g/L, electron beam dose 140 kGy, and incubation time of 60 min.</figcaption></figure>

influence the recovered protein yield. Algae dry matter concentration has an inverse effect on protein yield, i.e., the yield increases with decreasing dry matter concentration (Fig. 2). A possible explanation could be that active sites of the enzymes become saturated at high algae content, ultimately leading to the formation of a blocking fouling layer. Additionally, mass transfer limitations due to a higher solid-to-solvent ratio could also lower the recovered protein yield. However, the trend of the influence of the remaining parameters can be confirmed. Higher enzyme concentrations and a longer incubation time led to an increased yield of up to 40 %.

### 3.1.3. Optimization of protein recovery

The derived models were used to perform an optimization approach within the Desing-Expert software. Optimization to achieve a maximum recovered protein concentration and a maximum recovered protein yield were done, resulting in following settings: lipase concentration of 5 g/L; trypsin concentration of 15 g/L; EB dose of 140 kGy; dry matter concentration in the suspension of 10 g/L for maximum protein concentration, or 1 g/L for maximum protein yield, respectively; and the incubation time fixed at 60 min for practical reasons. Since the model showed a rather wide optimal dose range between 80–170 kGy, the dose for all further experiments was set at 140 kGy, based on previous studies on trypsin immobilization [27]. Confirmation of both models was successful by carrying out four replicates with the specified process parameters (Table 2). Thus, with a dry matter suspension of 10 g/L up to 1.40 ± 0.04 g/L protein could be extracted using the developed

<caption>**Table 2**</caption>

Confirmation of models for response R1, recovered protein concentration, and response R2, recovered protein yield. The experimental data mean (n = 4) was compared to the predicted mean and the 95 % prediction interval (PI).

<table><thead><tr><th>Model confirmation</th><th>Predicted mean</th><th>95 % PI low</th><th>Data mean</th><th>95 % PI high</th></tr></thead><tbody><tr><td>R1: Protein conc. (sqrt) (g/L)<sup>a</sup></td><td>1.38</td><td>1.21</td><td>1.40</td><td>1.57</td></tr><tr><td>R2: Protein yield (%)<sup>b</sup></td><td>42.2</td><td>38.3</td><td>39.5</td><td>46.1</td></tr></tbody></table>

(a) Optimized settings for confirmation: lipase 5 g/L, trypsin 15 g/L, EB dose 140 kGy, algae dry matter 10 g/L, incubation time 60 min.

(b) Optimized settings for confirmation: lipase 5 g/L, trypsin 15 g/L, EB dose 140 kGy, algae dry matter 1 g/L, incubation time 60 min.

<caption>**Table 3**</caption>

Enzymatic activity of investigated membrane samples prepared by grafting using the specified enzyme/protein concentration at 140 kGy electron beam dose.

<table><thead><tr><th rowspan="2">Sample</th><th colspan="3">Used concentration (g/L)</th><th colspan="2">Membrane activity (U/m²)</th></tr><tr><th>Trypsin</th><th>Lipase</th><th>BSA</th><th>Trypsin</th><th>Lipase</th></tr></thead><tbody><tr><td>Nylon-Ref</td><td>0</td><td>0</td><td>0</td><td>0.3 ± 0.6</td><td>0.0</td></tr><tr><td>Nylon-g-Lip</td><td>0</td><td>5</td><td>0</td><td>0.0</td><td>25.7 ± 7.4</td></tr><tr><td>Nylon-g-Try</td><td>15</td><td>0</td><td>0</td><td>3.5 ± 0.2</td><td>0.3 ± 0.1</td></tr><tr><td>Nylon-g-Try/Lip</td><td>15</td><td>5</td><td>0</td><td>5.2 ± 0.8</td><td>95.1 ± 5.9</td></tr><tr><td>Nylon-g-BSA</td><td>0</td><td>0</td><td>20</td><td>0.0</td><td>9.0 ± 5.3</td></tr><tr><td>Nylon-g-BSA/Lip</td><td>0</td><td>5</td><td>15</td><td>0.0</td><td>98.2 ± 7.6</td></tr><tr><td>Nylon-g-BSA/Try</td><td>15</td><td>0</td><td>5</td><td>3.3 ± 1.6</td><td>3.2 ± 0.8</td></tr></tbody></table>

biocatalytic polymer membrane. Based on a protein content of 59 % of the dry mass of *Tetraselmis chui* extract, this results in a protein yield of around 24 %. When aiming for a maximized protein yield a dry matter suspension of 1 g/L is required, resulting in up to 40 % protein recovery yield.

Since the empirical models revealed a strong effect of trypsin, optimized membranes grafted with the mixture of trypsin and lipase (Nylon-g-Try/Lip) were compared with membranes grafted with solely trypsin (Nylon-g-Try, for preparation see Table 3). For this purpose, filtrations were carried out with 10 g/L dry matter of microalgae in the suspension at 60 min incubation time (Fig. 3). The pristine nylon reference membranes led to a recovered protein content of 0.8 g/L, which was much lower in contrast to Nylon-g-Try (1.27 g/L) and Nylon-g-Try/Lip (1.40 g/L). Thus, trypsin has a significant effect on protein recovery compared to the reference (+57 %), while addition of lipase (Nylon-g-Try/Lip) led to a further but smaller increase in recovered protein content (+73 %). Similar trends were observed when calculating the protein yield, however, the effect of trypsin was greater (Nylon-g-Try: +65 %, Nylon-g-Try/Lip: +66 %).

### 3.2. Enzyme immobilization

In order to investigate the effect of enzyme co-immobilization, activity measurements of nylon membranes grafted with trypsin, lipase, BSA, and mixtures of those were performed (Table 3, Fig. 4). Membrane
<figure><img src="image_6.png" /><figcaption>Fig. 3. Effect of grafted trypsin or trypsin/lipase mixture on recovered protein concentration and protein yield, respectively. The optimized settings according to model R1 and R2 were used, which effectively differ mainly in the employed algae dry matter concentration of the feed suspension.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 4. Immobilized enzyme activity of investigated modified nylon membranes compared to the reference membrane (for sample composition see Table 3).</figcaption></figure>

lipolytic activity revealed that co-immobilization of lipase with trypsin (Nylon-g-Try/Lip: 95.1 ± 5.9 U/m²), or BSA (Nylon-g-BSA/Lip: 98.2 ± 7.6 U/m²), respectively, resulted in much higher activity than immobilization of solely lipase (Nylon-g-Lip: 25.7 ± 7.4 U/m²). Samples without lipase expectedly showed no activity. In contrast, membrane tryptic activity was just slightly higher in case of co-immobilization with lipase (Nylon-g-Try/Lip: 5.2 ± 0.8 U/m²), but similar for the BSA-trypsin mixture (Nylon-g-BSA/Try: 3.3 ± 1.6 U/m²) and solely trypsin (Nylon-g-Try: 3.5 ± 0.2 U/m²). Samples without trypsin expectedly showed no activity. Data implies that co-immobilization of lipase with

another protein/enzyme is beneficial for achieving high lipolytic activity.

In recent work, a mechanism for electron beam radiation-induced graft immobilization (RIGI) of non-vinylic substances such as enzymes onto polymer membranes was proposed [33]. Briefly, high energy electrons lead to simultaneous activation of membrane and dissolved enzymes facilitated by water radiolysis. Activation refers mainly to the formation of radical species that are able to recombine with each other. During the grafting process, in order to induce the formation of covalent bonds between the nylon membrane and the enzyme, it is important that
<figure><img src="image_8.png" /><figcaption>Fig. 5. Dynamic water contact angle for reference membrane and enzyme-grafted membrane.</figcaption></figure>

the enzyme is in proximity to the membrane surface including the inner
surface of the membrane pores. The utilized nylon membrane is hy-
drophilic as confirmed by water contact angle measurements (Nylon-Ref
after 1.0 s: ca. 20°, Fig. 5). Lipase enzymes have a higher level of hy-
drophobicity than conventional proteins such as BSA or trypsin [34].
Therefore, it is assumed that trypsin and BSA lead to an increase in lipase
activity either by facilitating the attachment of lipase, e.g., by cross-
linking acting as sort of linkers, or by promoting a favorable orientation
of lipase molecules. So far, it has not been possible to answer the

hypothesis since BCA assay is not suitable to differ between enzymes
within an immobilized mixture. However, water contact angle mea-
surements of Nylon-Ref and Nylon-g-Try/Lip (Fig. 5) showed that the
water contact angle of the enzyme-grafted membrane is initially similar
to the reference but decreases more slowly in the first few seconds. This
indicates a difference in the surface wettability of the membrane, which
is likely caused by the immobilization of the enzymes and their hydro-
phobic functionalities. It is not expected that this decrease in wettability
has a major influence on the filtration performance of the modified
membrane, as after a few seconds immersed in water the membrane
becomes fully wetted. Permeability measurements with water revealed
that the enzyme grafted membrane has a similar permeance with 11,735
± 64 L/(m² h bar) compared to the reference membrane with 11,319 ±
293 L/(m² h bar).

3.3. Influence of enzymes

3.3.1. Influence on fouling behavior

The enzyme-modified optimized sample (Nylon-g-Try/Lip) was investigated in order to determine the influence of the enzymes on the fouling behavior after filtration with the algae suspension and compared with a reference membrane without enzymes. An indication of a difference in the fouling behavior gave scanning electron microscopy (Fig. 6). While both samples showed hardly any visible differences before the filtration process, a top layer covering a large part of the pores can be seen on the reference membrane after filtration. However, this cannot be seen on the enzyme-grafted membrane, which indicates that the immobilized enzymes either inhibit the formation of a fouling layer or degrade parts of it. In both samples, however, larger agglomerates can be seen, which are deposited on the surface and on which the enzymes appear to have no influence. These could be remnants of the cell debris of the microalgae or other aggregates that could not be degraded during

<figure><img src="image_9.png" /><figcaption>Fig. 6. Scanning electron microscopy images of the reference membrane (Nylon-Ref) and the optimized enzyme-grafted membrane (Nylon-g-Try/Lip) before and after filtration of the microalgae extract suspension (10 g/L dry matter, incubation time 60 min).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** XPS elemental ratio of the reference membrane (Nylon-Ref) and the optimized enzyme-grafted membrane (Nylon-g-Try/Lip) before and after filtration with 10 g/L algae dry matter.</figcaption></figure>

<caption>**Table 4**</caption>

XPS data (selected elemental compositions) of membrane samples before and after filtration with 10 g/L microalgae extract.

<table><caption>**Table 5**</caption><thead><tr><th rowspan="2">Sample</th><th colspan="6">Elemental composition [at.%]</th></tr><tr><th>C</th><th>O</th><th>N</th><th>P</th><th>Na</th><th>Ca</th></tr></thead><tbody><tr><td>Nylon-Ref</td><td>75.0 ± 0.1</td><td>18.4 ± 0.8</td><td>4.3 ± 0.8</td><td>-</td><td>0.4 ± 0.1</td><td>-</td></tr><tr><td>Nylon-Ref, fouled</td><td>49.5 ± 1.5</td><td>31.1 ± 1.0</td><td>7.0 ± 0.0</td><td>4.6 ± 0.0</td><td>3.2 ± 0.3</td><td>3.3 ± 0.1</td></tr><tr><td>Nylon-g-Try/Lip</td><td>69.1 ± 2.8</td><td>19.1 ± 1.1</td><td>8.2 ± 3.3</td><td>0.5 ± 0.3</td><td>1.8 ± 1.1</td><td>-</td></tr><tr><td>Nylon-g-Try/Lip, fouled</td><td>72.1 ± 0.2</td><td>17.1 ± 0.9</td><td>10.2 ± 1.3</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Nylon-g-Try, fouled</td><td>66.0 ± 0.6</td><td>21.1 ± 0.4</td><td>11.7 ± 0.0</td><td>-</td><td>-</td><td>0.5 ± 0.1</td></tr></tbody></table>

XPS data (selected elemental ratios) of membrane samples before and after filtration with 10 g/L microalgae extract.

<table><thead><tr><th rowspan="2">Sample</th><th colspan="5">Elemental ratio [%]</th></tr><tr><th>O/C</th><th>N/C</th><th>P/C</th><th>Na/C</th><th>Ca/C</th></tr></thead><tbody><tr><td>Nylon-Ref</td><td>24.6 ± 1.1</td><td>5.7 ± 1.1</td><td>-</td><td>0.5 ± 0.1</td><td>-</td></tr><tr><td>Nylon-Ref, fouled</td><td>62.9 ± 3.9</td><td>14.2 ± 0.5</td><td>9.2 ± 0.3</td><td>6.4 ± 0.8</td><td>6.7 ± 0.4</td></tr><tr><td>Nylon-g-Try/Lip</td><td>27.6 ± 0.4</td><td>12.0 ± 5.2</td><td>0.8 ± 0.5</td><td>2.6 ± 1.7</td><td>-</td></tr><tr><td>Nylon-g-Try/Lip, fouled</td><td>23.7 ± 1.1</td><td>14.1 ± 1.8</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Nylon-g-Try, fouled</td><td>32.0 ± 0.9</td><td>17.8 ± 0.2</td><td>-</td><td>-</td><td>0.7 ± 0.1</td></tr></tbody></table>

the incubation and filtration time.

XPS measurements were carried out to study the composition of the membrane surface (Fig. 7, Table 4, Table 5). The general composition of the reference membrane's top site is dominated by C (75.0 ± 0.1 %), O (18.4 ± 0.8 %), and N (4.3 ± 0.8 %) in accordance with the molecular structure of polyamide. However, a clear change in the membrane surface chemical composition can be observed after carrying out the filtration process. In addition, P and Ca appeared. These elements are often found in components of the cell such as the cell walls and can lead to fouling. Phosphorus is found in phospholipids, which are considered

important building blocks for cell membranes and can therefore serve as a fouling indicator. In addition, the relative elemental ratio for oxygen (O/C, from 24.6 ± 1.1 % to 65.9 ± 3.9 %) and nitrogen (N/C, from 5.7 ± 1.1 % to 14.2 ± 0.5 %) has increased, respectively. This also suggests the accumulation of organic biomass on the membrane surface, as these elements occur in cellular components or ingredients. Proteins in particular, with their high oxygen content, are a reasonable and well-studied cause of fouling.

Compared to Nylon-Ref, however, the Nylon-g-Try/Lip shows significantly less drastic differences in the elemental composition after filtration. The relative oxygen content (O/C) even decreased slightly from 27.6 ± 0.4 % to 23.7 ± 1.1 %, while the relative nitrogen content (N/C) showed no significant change with 12.0 ± 5.2 % before and 14.1 ± 1.8 % after filtration, respectively. The optimized membrane also shows no residues of phosphorus and calcium after filtration on its surface.

The comparison of the fouled Nylon-g-Try/Lip with the fouled Nylon-g-Try shows that not only the trypsin but also the lipase is beneficial to the prevention of fouling. With 32.0 ± 0.9 % (O/C) and 17.8 ± 0.2 % (N/C), the fouling indicating substances seem to be more abundant on Nylon-g-Try than on Nylon-g-Try/Lip after filtration. This difference must therefore be caused by the lipase. Lipases, which belong to the group of esterases, catalytically cleave ester bonds such as those found in lipids and fats via hydrolysis. This could lead to the degradation of hydrophobic cell components reducing the adsorption and agglomeration on the membrane surface.

Trypsin, which is classified as a protease, primarily cleaves peptide bonds via hydrolysis, which results in larger protein structures and agglomerates being converted into smaller products. Proteins consist of a concatenation of several amino acids, which have a high oxygen content. The comparison of Nylon-g-Try to Nylon-Ref shows a clear difference in O/C content, which is about twice as high in the reference after filtration as in the trypsin-immobilized membrane. This indicates that a large part of the fouling is caused by the formation of a top layer through adsorption of biomolecules, e.g., protein structures, which is supported by XPS measurements and can also explain the increased protein content in the filtrates of the optimized membrane.

### 3.3.2. Influence on protein recovery

To investigate the effect of the immobilized enzymes on the protein recovery performance, studies comparing Nylon-g-Try/Lip (enzymes) with Nylon-g-BSA (protein) and unmodified nylon were performed (Fig. 8). First, the protein concentration in filtrate for each step of sequential filtration (incubation time: each 1 h) was determined

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Influence of time and filtration mode (sequential filtration: 4 × 1 h, batch filtration: 1 × 4 h) on protein concentration in filtrate for enzyme-, protein-, and non-grafted nylon membrane.</figcaption></figure>
revealing a significant time dependence for the enzyme-grafted membrane. Protein concentration increased with increasing total incubation time (1 h: 1.16 g/L; 2 h: 1.33 g/L; 3 h: 1.47 g/L; 4 h: 1.65 g/L). In contrast, control experiments using untreated or BSA-grafted nylon showed no time dependence ranging between 0.75–0.97 g/L. Since enzymatic action is a biocatalytic reaction, longer incubation times would result in higher product yield supporting the beneficial effect of immobilized enzymes for protein recovery from microalgae extracts. Furthermore, sequential filtration after 4 h (i.e., 4 × 1 h incubation) led to a higher protein concentration of 1.65 g/L within this permeate fraction compared to only 1.36 g/L in batch filtration (1 × 4 h incubation). It is assumed that sequential filtration might facilitate the transport of biomolecules within the feed into the membrane pore structure and thus, in proximity to immobilized enzymes within the membrane's inner surface improving the extraction process. Moreover, repeated filtration steps could remove degradation products from the inside of the membrane and at the same time flush new substrate towards the enzymes. While the beneficial effect of the enzymes on the protein recovery has been demonstrated, the exact mechanisms of the enzymatical reactions need to be investigated in a follow-up study.

## 4. Summary and outlook

As part of this work, an enzymatically active nylon membrane was successfully developed which is suitable for protein extraction by sequential filtration of microalgae *Tetraselmis chui* extract. Immobilization of enzyme mixtures consisting of the hydrolases trypsin and lipase from *Candida rugosa* was conducted in a one-step electron beam-based procedure. Predictive empirical models were derived by employing the response surface methodology within a design of experiments approach. A nylon membrane modified with an impregnation solution of 15 g/L trypsin and 5 g/L lipase exposed to an electron beam dose of 140 kGy, resulted in an optimized protein recovery of 1.40 ± 0.04 g/L using a dry matter concentration of 10 g/L in the microalgae extract suspension and an incubation time of 60 min per filtration step, which

corresponds to a yield of a 24 %. It was shown that the immobilization of lipase and trypsin led to an increase in extraction capacity of +0.6 g/L (+75 % compared to pristine reference membrane). By filtrating a 1 g/L algae dry matter suspension, an optimized protein recovery yield of 40 % was achieved. Studies on the immobilized enzyme activity showed that high immobilized lipase activity is only achieved by co-immobilization in a mixture with another protein such as trypsin or BSA. Various characterization methods confirmed that immobilization of the enzymes significantly improves the anti-fouling behavior of the membrane during filtration of microalgae extract suspension. SEM as well as XPS data suggest that a fouling layer is either partly inhibited or degraded by the enzymatically active membrane surface. Future work should aim at investigating the enzymatic reactions in detail and address scalability, long term stability of immobilized enzymes, as well as the possibility of using immobilized enzymes in different filtration setups or membrane systems such as crossflow filtration and hollow-fiber membranes.

## Funding

This work was funded by the German Academic Exchange Service (DAAD), Campus France (PHC PROCOPE) and supported by the Federal State of Germany and the Free State of Saxony.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

Pascale Gillon (GEPEA, France) is acknowledged for her participation in the enzyme selection.

# Appendix

<caption>**Table A1**</caption>

I-optimal design of experiments for investigating the protein recovery from microalgae extract using enzyme-grafted nylon membranes. Given are the investigated five factors (A-E) and measured responses (R).

<table><thead><tr><th>#</th><th>A<br>Lipase conc. (g/L)</th><th>B<br>Trypsin conc. (g/L)</th><th>C<br>EB dose (kGy)</th><th>D<br>Dry matter conc. (g/L)</th><th>E<br>Incubation time (min)</th><th>R1<br>Protein conc. (g/L)</th><th>R2<br>Protein yield (%)</th></tr></thead><tbody><tr><td>1</td><td>1.7</td><td>0</td><td>20</td><td>5.8</td><td>68</td><td>13.4</td><td>20</td></tr><tr><td>2</td><td>0</td><td>8</td><td>200</td><td>4</td><td>52</td><td>13.5</td><td>29</td></tr><tr><td>3</td><td>1.1</td><td>15</td><td>200</td><td>1</td><td>90</td><td>5.3</td><td>45</td></tr><tr><td>4</td><td>0</td><td>0</td><td>200</td><td>10</td><td>90</td><td>17.9</td><td>15</td></tr><tr><td>5</td><td>2</td><td>15</td><td>140</td><td>7.6</td><td>90</td><td>23.9</td><td>27</td></tr><tr><td>6</td><td>3.3</td><td>13.5</td><td>80</td><td>2</td><td>90</td><td>9.8</td><td>42</td></tr><tr><td>7</td><td>1.8</td><td>11</td><td>20</td><td>5.7</td><td>10</td><td>11.0</td><td>16</td></tr><tr><td>8</td><td>5</td><td>15</td><td>200</td><td>1</td><td>90</td><td>5.4</td><td>46</td></tr><tr><td>9</td><td>4.5</td><td>0</td><td>200</td><td>1</td><td>10</td><td>2.6</td><td>22</td></tr><tr><td>10</td><td>5</td><td>6</td><td>25</td><td>10</td><td>90</td><td>28.4</td><td>24</td></tr><tr><td>11</td><td>1.7</td><td>0</td><td>25</td><td>5.8</td><td>68</td><td>14.4</td><td>21</td></tr><tr><td>12</td><td>2</td><td>3.9</td><td>140</td><td>1</td><td>90</td><td>4.2</td><td>36</td></tr><tr><td>13</td><td>5</td><td>10.7</td><td>140</td><td>5.8</td><td>10</td><td>16.6</td><td>24</td></tr><tr><td>14</td><td>3.4</td><td>7</td><td>200</td><td>10</td><td>47</td><td>19.1</td><td>16</td></tr><tr><td>15</td><td>0</td><td>15</td><td>200</td><td>10</td><td>10</td><td>16.4</td><td>14</td></tr><tr><td>16</td><td>0</td><td>8</td><td>80</td><td>10</td><td>52</td><td>25.9</td><td>22</td></tr><tr><td>17</td><td>0.4</td><td>0</td><td>200</td><td>1</td><td>10</td><td>2.6</td><td>22</td></tr><tr><td>18</td><td>0</td><td>15</td><td>25</td><td>1</td><td>90</td><td>4.9</td><td>42</td></tr><tr><td>19</td><td>5</td><td>0</td><td>140</td><td>5.7</td><td>68</td><td>13.0</td><td>19</td></tr><tr><td>20</td><td>3.4</td><td>7</td><td>200</td><td>10</td><td>47</td><td>25.5</td><td>22</td></tr><tr><td>21</td><td>0</td><td>0</td><td>20</td><td>1</td><td>10</td><td>1.6</td><td>14</td></tr><tr><td>22</td><td>4.9</td><td>7.9</td><td>25</td><td>1</td><td>52</td><td>4.3</td><td>36</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table A1 (continued)**</caption><thead><tr><th>#</th><th>A<br>Lipase conc. (g/L)</th><th>B<br>Trypsin conc. (g/L)</th><th>C<br>EB dose (kGy)</th><th>D<br>Dry matter conc. (g/L)</th><th>E<br>Incubation time (min)</th><th>R1<br>Protein conc. (g/L)</th><th>R2<br>Protein yield (%)</th></tr></thead><tbody><tr><td>23</td><td>0</td><td>8</td><td>200</td><td>4</td><td>52</td><td>12.8</td><td>27</td></tr><tr><td>24</td><td>0</td><td>8</td><td>80</td><td>10</td><td>52</td><td>22.8</td><td>19</td></tr><tr><td>25</td><td>2</td><td>15</td><td>140</td><td>1</td><td>31</td><td>4.2</td><td>36</td></tr><tr><td>26</td><td>5</td><td>0</td><td>20</td><td>10</td><td>10</td><td>10.9</td><td>9</td></tr><tr><td>27</td><td>1.8</td><td>11</td><td>20</td><td>5.7</td><td>10</td><td>11.8</td><td>18</td></tr><tr><td>28</td><td>3.3</td><td>0.8</td><td>80</td><td>2.9</td><td>26</td><td>8.3</td><td>24</td></tr><tr><td>29</td><td>1.2</td><td>0</td><td>140</td><td>7.8</td><td>10</td><td>14.9</td><td>16</td></tr><tr><td>30</td><td>5</td><td>15</td><td>25</td><td>10</td><td>42</td><td>24.4</td><td>21</td></tr><tr><td>31</td><td>2</td><td>15</td><td>140</td><td>1</td><td>31</td><td>4.4</td><td>37</td></tr><tr><td>32</td><td>5</td><td>15</td><td>25</td><td>10</td><td>42</td><td>25.9</td><td>22</td></tr></tbody></table>

<caption>**Table A2**</caption>

ANOVA results for response R1, recovered protein concentration in filtrate. Factors with a p-value above 0.05 are included only due to hierarchical reasons, e.g., C is included due to significant CE.

<table><caption>**Table A3**</caption><thead><tr><th>Source</th><th>Sum of squares</th><th>df</th><th>mean squares</th><th>F-value</th><th>p-value</th></tr></thead><tbody><tr><td>Model</td><td>43.07</td><td>12</td><td>3.59</td><td>84.79</td><td>&lt; 0.0001</td></tr><tr><td>A-Lipase concentration</td><td>0.0876</td><td>1</td><td>0.0876</td><td>2.07</td><td>0.1665</td></tr><tr><td>B-Trypsin concentration</td><td>1.56</td><td>1</td><td>1.56</td><td>36.81</td><td>&lt; 0.0001</td></tr><tr><td>C-EB dose</td><td>0.0163</td><td>1</td><td>0.0163</td><td>0.39</td><td>0.542</td></tr><tr><td>D-Dry matter concentration</td><td>29.91</td><td>1</td><td>29.91</td><td>706.47</td><td>&lt; 0.0001</td></tr><tr><td>E-Incubation time</td><td>1.83</td><td>1</td><td>1.83</td><td>43.15</td><td>&lt; 0.0001</td></tr><tr><td>AB</td><td>0.2198</td><td>1</td><td>0.2198</td><td>5.19</td><td>0.0344</td></tr><tr><td>BE</td><td>0.2268</td><td>1</td><td>0.2268</td><td>5.36</td><td>0.032</td></tr><tr><td>CE</td><td>0.4508</td><td>1</td><td>0.4508</td><td>10.65</td><td>0.0041</td></tr><tr><td>DE</td><td>0.2121</td><td>1</td><td>0.2121</td><td>5.01</td><td>0.0374</td></tr><tr><td>B<sup>2</sup></td><td>0.363</td><td>1</td><td>0.363</td><td>8.57</td><td>0.0086</td></tr><tr><td>D<sup>2</sup></td><td>2.19</td><td>1</td><td>2.19</td><td>51.82</td><td>&lt; 0.0001</td></tr><tr><td>E<sup>2</sup></td><td>0.1997</td><td>1</td><td>0.1997</td><td>4.72</td><td>0.0427</td></tr><tr><td>Residual</td><td>0.8044</td><td>19</td><td>0.0423</td><td></td><td></td></tr><tr><td>Lack of Fit</td><td>0.5002</td><td>13</td><td>0.0385</td><td>0.76</td><td>0.6835</td></tr><tr><td>Pure Error</td><td>0.3042</td><td>6</td><td>0.0507</td><td></td><td></td></tr><tr><td>Cor Total</td><td>43.88</td><td>31</td><td></td><td></td><td></td></tr></tbody></table>

ANOVA results for response R2, recovered protein yield in filtrate. Factors with a p-value above 0.05 are included only due to hierarchical reasons, e.g., C is included due to significant CE.

<table><thead><tr><th>Source</th><th>Sum of squares</th><th>df</th><th>Mean squares</th><th>F-value</th><th>p-value</th></tr></thead><tbody><tr><td>Model</td><td>2840.85</td><td>7</td><td>405.84</td><td>55.38</td><td>&lt; 0.0001</td></tr><tr><td>A-Lipase concentration</td><td>37.51</td><td>1</td><td>37.51</td><td>5.12</td><td>0.033</td></tr><tr><td>B-Trypsin concentration</td><td>486.41</td><td>1</td><td>486.41</td><td>66.38</td><td>&lt; 0.0001</td></tr><tr><td>C-EB dose</td><td>12.06</td><td>1</td><td>12.06</td><td>1.65</td><td>0.2118</td></tr><tr><td>D-Dry matter concentration</td><td>1070.4</td><td>1</td><td>1070.4</td><td>146.07</td><td>&lt; 0.0001</td></tr><tr><td>E-Incubation time</td><td>390.51</td><td>1</td><td>390.51</td><td>53.29</td><td>&lt; 0.0001</td></tr><tr><td>BD</td><td>50.68</td><td>1</td><td>50.68</td><td>6.92</td><td>0.0147</td></tr><tr><td>CE</td><td>32.89</td><td>1</td><td>32.89</td><td>4.49</td><td>0.0447</td></tr><tr><td>Residual</td><td>175.87</td><td>24</td><td>7.33</td><td></td><td></td></tr><tr><td>Lack of Fit</td><td>148.37</td><td>18</td><td>8.24</td><td>1.8</td><td>0.2407</td></tr><tr><td>Pure Error</td><td>27.5</td><td>6</td><td>4.58</td><td></td><td></td></tr><tr><td>Cor Total</td><td>3016.72</td><td>31</td><td></td><td></td><td></td></tr></tbody></table>

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