The aquaculture waste as the carbon source for nanocellulose production by Komagataeibacter europaeus

Abstract

Nanocellulose produced by bacteria has attracted worldwide attention owing to its excellent mechanical properties, water retention capacity, non-toxicity, antibacterial ability, and high plasticity, making it applicable in various fields. In this study, the collected aquaculture waste biomass such as Chaetomorpha crassa and C. linum were hydrolyzed with cellulose and viscozyme to be employed as the carbon sources in HS medium for Komagataeibacter europaeus 14,148 to produce bacterial cellulose (BC). The sugar obtained after enzymatic hydrolysis of biomass of C. crassa by cellulase (CCH) and by viscozyme (CVH) was 1.40 ± 0.02 g/100 mL, 1.17 ± 0.03 g/100 mL, respectively, and similarly, C. linum by cellulase (LCH) and by viscozyme (LVH) 0.78 ± 0.01 g/100 mL, and 0.82 ± 0.04 g/100 mL, respectively. The glucose saccharification of enzymatic hydrolysate in CCH and CVH was 34.9% and 17.3% which were both higher than LCH and LVH. The yield of BC by K. europaeus 14,148 from different enzymatic hydrolysate of green seaweed was 0.26 ± 0.02, 0.51 ± 0.07, 0.48 ± 0.06, 1.77 ± 0.12, and 1.69 ± 0.03 g/L in HS medium, CCH, CVH, LCH, and LVH, respectively. Macroalgal hydrolysate by carbohydrase was a better carbon source for producing high-quality BC. The high concentration of total phenol compound in the enzymatic hydrolysate obtained from green seaweed enhanced the bacterial activity of K. europaeus 14,148, conversely contributing to enhancing BC production and improving radical scavenging activity of BC. This study provided value addition to aquaculture waste to be utilized as carbon source to produce high-value products supporting sustainable development goals no. 12 “Responsible consumption and production.”

Keywords

Bacterial cellulose enzymatic saccharification sustainable development goals no. 12

Introduction

The increased practice of aquaculture in the world caused attention to the impact of its exported by-products on the environment¹ and drove the research trend on harnessing the potential of by-products to decrease its negative effect.² As the global environmental sustainability issue arose by the over usage of antibiotics³ and chemicals⁴ in the aquaculture practice; research on eco-friendly technology by introducing green seaweed Caulerpa lentillifera,⁵ Ulva lactuca,⁶ brown seaweed Saccharina latissima,⁷ microalgae,⁸ bacteria,⁹ probiotics,¹⁰ and biofloc¹¹ contributed to improved water quality, and disease control. However, invasive species that are not intended for culturing, such as macroalgae¹² and green seaweeds,¹³ may be used as feedstock for bioprocess due to their higher carbohydrate content.

Aquaculture farming waste includes microalgae and seaweed, which contain re-utilizable ingredients suitable for consumption by humans as food source, food packaging, cosmetics, medicines,¹⁴ fertilizer,¹⁵ nutrient recycling,¹⁶ food sources for holothurians,¹⁷ and bioremediation.¹⁸ Mostly, the efficient extraction of nutrients such as carbohydrates and proteins is attributed to chemical solvent extraction; the detoxification of toxic waste and pollutants is required before releasing them into the environment. Hence, enzymatic hydrolysis of above said biomass provides a non-toxic, environmentally friendly technology to extract nutrients.

Seaweed is macroalgal biomass that contains bioactive ingredients like carrageenan, agar (red seaweed), alginate, fucoidan (brown seaweed), ulvan (green seaweed).¹⁹ The traditional methods to extract these compounds were by ultrasound, hydrothermal, and chemical solvent treatment, which accompany additional treatment on toxic chemical residues. However, enzymatic treatment was considered a more sustainable method to obtain bioactive, antioxidant, antibacterial, and phenolic compounds from seaweed.²⁰ Enzymatic saccharification was improved by pre-autoclave treatment in Chaetomorpha linum, Ulva fasciata, and Caulerpa taxifolia.²¹ The cocktail of carbohydrase and protease employed for enzymatic hydrolysis of seaweed enhanced the solubilization of carbohydrates, protein, and antioxidants of total phenolic compound in green seaweed, red seaweed, and brown seaweed.²²

Cellulose is the most abundant and renewable polysaccharide available worldwide, mainly derived from wood and natural fibers, however, it can also be secreted and synthesized by algae and bacteria.²³ Komagataeibacter is an aerobic bacteria found in traditional fermented vinegar and was well-studied for the production of bacterial cellulose (BC). This bacterium was able to utilize the enzymatic hydrolysate from agro-waste²⁴ as a low-cost carbon source to produce natural nanoscale cellulose²⁵ with excellent water retention capabilities, high crystallinity, strong tensile strength, and high thermal stability.²⁶

Recently the carbon source from agricultural waste, which is rich in cellulose and other sugars, like pineapples,²⁷ fruit processing waste,²⁸ fruit and vegetable peels²³ have been used to replace the glucose in the HS medium to improve BC production efficiency, quality and reduce the cost of media.

This work is based on utilization of seaweeds which is an aquaculture waste as it was collected from aquaculture pond where it grew by itself getting nutrition from fish excretion and fish feed nutrients. These seaweeds need to be removed for space for pisciculture. Green seaweeds enzymatic hydrolysate was used as carbon source for BC production by K. europaeus. Enzymatic hydrolysis of green seaweed enhanced the yield of phenolic compounds. Phenol compounds of seaweed's enzymatic hydrolysate did not inhibit BC production. Enzymatic hydrolysate of green seaweed improved BC radical scavenging activity. There are no reports on hydrothermal pretreatment and enzymatic hydrolysis of green seaweeds to be utilized as carbon source for K. europaeus for BC production.

The BC production efficiency was related to the concentration of reducing sugar and total phenolic compounds of green seaweed were emphasized. The characteristic analysis of BC was shown by water-holding capacity, scanning electron microscope (SEM), and Fourier-transform infrared (FTIR). This green enzymatic process creates a circular bio-economic process for the sustainable utilization of aquaculture waste.

Material and method

Seaweed and its collection

Two green seaweed species, Chaetomorpha crassa and C. linum, were collected from an aquaculture farm in Dapeng Bay, Pingtung County, Taiwan. Samples were packed in plastic bags and transported to the laboratory. After washing the samples with clean water, it was dried at 60 °C for 24 hours. The dried biomass was homogenized RT-04 (Rong Tsong, Taiwan) at 25,000 rpm for 30 seconds and filtrated through 30 mesh to obtain a particle size of less than 0.590 mm, and then kept in a desiccator for subsequent experiments.

Enzymatic hydrolysis of seaweed

Two commercial enzymes, cellulase (Trichoderma reesei, ≥700 units/g) and viscozyme (Aspergillus sp., 100 FBG/g), were obtained from NAVIGATION (Taiwan). The method of enzymatic hydrolysis was a modified version of Mittal and Raghavarao²⁹ and Zu et al.³⁰ The dried powders of C. crassa and C. linum were mixed with water at a 1:10 ratio (wt. basis) and kept at 121 °C for 20 minutes. The enzymes were added at the concentration of 10 units per gram of biomass on dry wt. basis during hydrolysis with no addition of water or buffer. The hydrolysis condition with cellulose and viscozyme was set at 55 °C and pH 4.8 for 24 hours at 120 rpm in a water bath. The pH was estimated by inoLab^® pH7110 (Xylem, Germany). The hydrolyzed sample was centrifuged at 8000 rpm for 10 minutes by Himac CF15R (Hitachi, Japan), and the supernatant was used for chemical analysis.

Production of bacterial cellulose by Komagataeibacter europaeus

The K. europaeus BCRC 14,148 used in this experiment was purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute, Taiwan. Strains were inoculated into standard HS liquid medium and cultured at 30 °C for 48 hours.

HS medium³¹ was the control medium, and per liter contained 5 g peptone, 5 g yeast extract, 3.75 g Na₂PO₄⋅12H₂O, and 1.1 g citric acid. The 20 g/L glucose was added to the HS medium, which was used as a control, and the enzymatic hydrolysate of seaweed was added into the HS medium to attend to the reducing sugar contents of 0.5, 1.0, 1.5, and 2.0%, respectively. All prepared media were sterilized at 121 °C for 20 min before inoculating the bacteria. The cultivation protocol was modified by Tseng et al.²³ 5% K. europaeus BCRC 14,148 was used as inoculum to 5 mL of individual medium and cultured at 30 °C for 7 days. Each experimental trial was repeated with three samples, and the results were expressed as mean values with standard deviation. The harvested BC was immersed in 5 mL 1 M sodium hydroxide solution at 60 °C for 12 hours to remove residual bacteria. After treatment, BC was washed with water until the pH value was neutral then freeze-dried at −25 °C for 24 hours by Kingmech FD4.5-8P-D (RBS, Taiwan). The weight of freeze-dried BC was used to calculate the yield of BC as follows:

Yield (g / L) = \frac{A}{B}

(1)

A: weight of BC after freeze-drying (g)

B: the volume of cultivated medium (L)

Crude composition and reducing sugar analysis

In accordance with the testing methods of the National Standard of the Republic of China (CNS) the moisture (CNS5033, N6114), crude ash (CNS5034, N6115), crude protein (CNS5035, N6116), crude fat (CNS5036, N6117) and crude fiber contents (CNS 5037, N6118) was analyzed. The reducing sugar analysis protocol was modified by the Edson and Poe method.³² Mix 0.5 mL of enzyme hydrolysate and 0.5 mL of 3,5-dinitrosalicylic acid (DNS) reagent, then react in a boiling water bath for 5 minutes. After cooling to room temperature, add 5 mL of deionized water and then measure the absorbance at 540 nm by the spectrometer. The specific sugar content was analyzed by HPLC-RID (Agilent technology 1260 series, USA) with Coregel-87H3 column at 60 °C and 0.005 M sulfuric acid as mobile phase with flow rate at 0.6 mL/min. The enzymatic saccharification was calculated as follows:

ES (%) = \frac{(Δ S \times 0.9)}{C} \times 100

(2)

ES is the efficiency of enzymatic saccharification (%), ΔS is glucose increase (g/L) during enzymatic saccharification after the pretreatment, and C is cellulose content (g/L) in green seaweeds, and 0.9 was the correction for C6 sugars.

Estimate of the total phenol compound

The analysis method was modified by Singleton et al.³³ The 20-μL of enzymatic hydrolysate and 400 μL of Folin's reagent were added to 96 well plates for react 10 minutes, then 400 μL of 7.5% Na₂CO₃ was added and incubated for 90 minutes at room temperature in the dark, finally an ELISA reader BioTek Synergy H1 (Agilent, USA) was used to measure the absorbance value at 725 nm. Gallic acid was used as standard, and results are expressed in milligrams of gallic acid per gram of hydrolysate (mg GAE/g DW).

The water-holding capacity of BC

Modified from the method of Urbina et al.,³⁴ the BC was added in deionized water until constant weight. The formula for calculating the water retention rate is as follows:

WHC (%) = \frac{(W_{wet} - W_{dry})}{W_{dry}} \times 100

(3)

where W_wet is the wet weight of BC after being immersed in deionized water (g); W_dry is the weight of BC after freeze-drying (g).

The antioxidant activity

DPPH analysis was modified the method of Zmejkoski et al.³⁵ BC was cut to have a 6-mm diameter to react with 1 mL of 100 μM DPPH solution at room temperature in the dark. The absorption intensity was measured at 517 nm by the spectrometer. The free radical scavenging ability of BC is calculated by the following formula:

DPPH radical scavenging activity (%) = \frac{(AC – ABC)}{AC} \times 100

(4)

Among them, AC and ABC represent the absorbance of blank and sample, respectively.

The cationic scavenging activities of extracted cellulose films were measured using the ABTS method. The ABTS reagent was prepared by diluting a 7-mM ABTS stock solution with methanol until the absorbance reached 0.7 at 734 nm.¹⁹ Following the protocol of Dicastillo et al.,³⁶ a 0.283-cm² surface area of each developed material was immersed in 0.472 μL of ABTS+· radical solution and incubated for 30 minutes in the dark at room temperature. The absorbance was then measured at 734 nm, and the scavenging activity was calculated using the following formula:

ABTS scavenging activity (%) = (\frac{(A_Control - A_Sample)}{A_Control}) \times 100

(5)

Where, A_Control is absorbance of reagent without any sample, A_Sample – absorbance of sample.

The physical characteristic analysis of BC

The SEM analysis was used Hitachi Tabletop TM-3000 (FESEM, Hitachi SU8010, Tokyo, Japan). The freeze-dried BC adhered to carbon tape and formed a conductive film on the surface by vacuum gold plating. Observe and take images with an electron microscope at 5000 magnifications at an accelerating voltage of 10 kV. FTIR spectroscopy analysis of BC was done using Nicolet iN10 (ThermoFisher Scientific, USA) and modified according to the method of Tseng et al.²³ in reflection mode with a wavenumber scanning range from 4000 to 650 cm⁻¹. The D8 DISCOVER with GADDS (Bruker AXS Gmbh, Karlsruhe, Germany) was used for X-ray diffraction (XRD) analysis of BC modified as the method of Diaz-Ramirez et al.³⁷ The crystallinity index of BC was measured with a 2θ scanning ranging from 5° to 40° at a voltage of 45 kV and a current of 40 mA. The crystallinity index (CrI) of BC was calculated based on the following formula.³⁸

CrI (%) = \frac{(I_{Cr} - I_{am})}{I_{Cr}} \times 100

(6)

I_Cr is the intensity of diffraction at 2θ = 22.6°, and I_am is the intensity of the amorphous part of BC at diffraction 2θ = 16°.

Statistical analysis

The experimental results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA statistical analysis was performed using SAS software, and Duncan's multiple range test was used to test the differences between independent samples in each group. Significant differences are considered statistically significant when the p-value is less than 0.05.

Results and discussions

The effect of hydrothermal pretreatment and enzymatic hydrolysis on the sugar profile

The crude composition of seaweed

The composition analysis of aquaculture waste of C. crassa, and C. linum is shown in Table 1. The crude protein was 7.57 ± 0.00 g/100 g and 8.87 ± 0.06 g/100 g in C. crassa and C. linum, respectively. The crude fiber of C. crassa was 27.77 ± 0.08 g/100 g, and in C. linum it was 30.82 ± 0.48 g/100. The high content of carbohydrates in green seaweed ranged from 45.5% to 58.7% and protein was 11.1% to 26.8%.^39,40 The high carbohydrate content of green seaweed contributed to fiber (cellulose) and other polysaccharides.²² The high polysaccharide content and lack of lignin in the membrane structure in seaweed increase the conversion of monosaccharides via enzymatic hydrolysis.³⁹

Table 1.

The crude composition analysis and sugar profile of green seaweed.

	Crude composition of seaweed (g/100 g)
	Moisture	Protein	Lipid	Ash	Fiber	Carbohydrate^a
C. crassa	10.26 ± 0.09	7.57 ± 0.00	2.57 ± 0.13	13.29 ± 0.35	27.77 ± 0.08	66.31 ± 0.21
C. linum	10.47 ± 0.10	8.87 ± 0.06	3.10 ± 0.21	12.03 ± 0.04	30.82 ± 0.48	65.53 ± 0.08

Carbohydrates = 100 – Moisture – Protein – Lipid – Ash.

The sugar profile of enzymatic hydrolysates

The sugar contents of hydrolysate were affected by the biomass and enzymatic hydrolysis. After hydrothermal treatment at 121 °C for 20 minutes didn’t have a significant effect on breaking the polysaccharide linkage of seaweed's cell wall, and the reducing sugar of C. crassa and C. linum was 0.05 ± 0.01 g/100 mL and 0.07 ± 0.01 g/100 mL (Table 2). The monosugar was found to be less than 0.01 g/kg, and acetic acid was 4.8 g/kg in brown macroalgae, Nizimuddinia zanardini after being treated at 121 °C for 30 minutes.⁴¹ The reducing sugar of enzymatic hydrolysate by cellulase and viscozyme of C. crassa (CCH, CVH) and C. linum (LCH, LVH) was 1.40 ± 0.02 g/100 mL, 1.17 ± 0.03 g/100 mL, 0.78 ± 0.01 g/100 mL, and 0.82 ± 0.04 g/100 mL, respectively. Hydrothermal treatment combined with enzymatic hydrolysis treatment has improved 2717%, 2495%, 1416%, and 1530% reducing sugar yield in CCH, CVH, LCH, and LVH compared to the result of hydrothermal treatment. The hot water pretreatment on red, brown, and green seaweed at 121 °C for 45 minutes enhanced enzymatic conversion of biomass into sugars.⁴² The release of acetic acid during the course of pretreatment is an indication of the progress of the hydrolysis of hemicellulosic-like carbohydrates.⁴¹ In hydrothermal pretreatment, the water will generate hydronium ions at subcritical temperature and acts as an acid to degrade the lignocellulosic biomass, thereby enhancing subsequent enzymatic hydrolysis^43,44 to degrade the polysaccharide structure of the macroalgal cell wall into the monosaccharide. Glucan, xylan, rhamnan, and mannan were the main extracted polysaccharides from hydrothermal pretreatment on seaweeds. Glucose is a monomer unit of cellulose that is difficult to get extract by hydrothermal treatment, and 2 to 3% glucose concentrations based on glucan content were found in C. linum.²¹ It insist enzyme to be utilized for hydrolysis of cellulose.

Table 2.

The sugar profile of green seaweed.

	Reducing sugar concentration of supernatant (g/100 mL)			Sugar and organic acid profile (g/100 mL)
	Hydrothermal treatment	Enzymatic hydrolysate		Hydrothermal treatment	Enzymatic hydrolysate
		Enzymatic hydrolysate		Hydrothermal treatment	Cellulase			Viscozyme
		Cellulase	Viscozyme	Acetic acid	Glucose	Rhamnose	Acetic acid	Glucose	Xylose	Acetic acid
C. crassa	0.05 ± 0.01	1.40 ± 0.02	1.17 ± 0.03	2.30 ± 0.21	0.95 ± 0.05	0.39 ± 0.01	2.79 ± 0.15	0.75 ± 0.03	0.02 ± 0.00	2.61 ± 0.13
C. linum	0.07 ± 0.01	0.78 ± 0.01	0.82 ± 0.04	2.35 ± 0.12	0.54 ± 0.02	0.20 ± 0.02	2.49 ± 0.10	0.50 ± 0.01	0.02 ± 0.00	2.62 ± 0.20

Glucose production from cellulose was achieved via enzymatic saccharification, resulting in the degradation of fiber in C. crassa and C. linum after hydrothermal pretreatment. The glucose contents were 0.95 ± 0.05 g/L, 0.54 ± 0.02 g/L, 0.75 ± 0.03 g/L, and 0.50 ± 0.02 g/L in CCH, LCH, CVH, and LVH. The rhamnose was only found in cellulase treatment of CCH and LCH. However, xylose only existed in viscozyme treatment. The rhamnose and xylose found in the enzymatic hydrolysates of C. crassa and C. linum were the main constituents of ulvan.⁴⁵ Viscozyme was the mixed carbohydrase of arabinase, cellulase, β-glucanase, hemicellulase, and xylanase, which was utilized to degrade the polysaccharide structure of macroalgal cell walls to enhance the protein extraction efficiency by proteinase.²² The glucose saccharification of enzymatic hydrolysate in CCH and CVH was 75.4% and 59.6% which were both higher than LCH and LVH. The sugar-rich hydrolysate of Ulva lactuca containing glucose, rhamnose, and xylose was obtained from the commercial cellulase GC220.⁴⁵ The enzymatic hydrolysate of U. rigida by viscozyme was obtained from 9.74 g/100 g DW carbohydrate extract.²² The monosaccharides (mainly glucose) of thermal acid hydrolysate of green macroalgae Enteromorpha intestinalis was 15.10 g/L and increased to 19.60 g/L and 19.00 g/L followed by enzymatic hydrolysis of cellulase and viscozyme.⁴⁶ The high concentration of acetic acid obtained in the hydrothermally pretreated hydrolysate of two green seaweeds might exert an inhibition effect on the enzymatic hydrolysis process.⁴⁴

The total phenol compound of enzymatic hydrolysate from green seaweed

Polyphenols refer to compounds containing multiple OH groups in their molecular structure, and their antioxidant capacity depends on the position and number of their OH groups. The total polyphenol content is calculated using gallic acid as a standard substance to calculate the total polyphenols content per gram of hydrolysate. The different carbohydrases have affected the yield of polyphenolic compounds in enzymatic hydrolysate in green seaweed. The total phenolic compound of green seaweed enzymatic hydrolysates was 4.89 mg GAE/g, 5.77 mg GAE/g, 20.42 mg GAE/g, and 17.17 mg GAE/g in CCH, CVH, LCH, and LVH (Figure 1). Phenolic compounds are rich in in seaweed's cell walls and attached mainly to proteins and polysaccharide moieties. Therefore, carbohydrases can break the links of phenolic compounds to polysaccharides to increase their extraction yield.²² The concentration of total phenolic compounds estimated in C. linum after enzymatic hydrolysis by carbohydrase was almost two-fold as observed in C. crassa. The enzymatic hydrolysate of green seaweed by viscozyme contained 0.241 g GA/100 g DW of total phenolic compound, red seaweed and brown seaweed was 0.246 to 0.427 g GA/100 g DW and 0.514 g GA/100 g DW,²² the enzymatic hydrolysate of red seaweed by the cellulase obtained 14.6 mg GAE/g extract and 21.9 to 29.3 mg GAE/g extract in brown seaweed.⁴⁷ Microorganism, Aspergillus oryzae, was able to ferment seaweed, Kappaphycus spp. to enhance the yield of antioxidant of phenolic compound.⁴⁸ The content of the total phenolic compound in the enzymatic hydrolysate of seaweed was affected both by specific carbohydrase and seaweed species.

Figure 1.

The total phenols of enzymatic hydrolysate from C. crassa and C. linum.

Effects of enzymatic hydrolysates of green seaweeds on BC production

The different enzyme hydrolysates from green seaweed were used as the carbon source in the HS medium for BC production. The yield of BC by K. europaeus 14,148 from different enzymatic hydrolysate of green seaweed was 0.26 ± 0.02, 0.51 ± 0.07, 0.48 ± 0.06, 1.77 ± 0.12, and 1.69 ± 0.03 g/L in HS medium, CCH, CVH, LCH, and LVH, respectively (Table 3). LCH and CCH treatment, which contained glucose and rhamnose as the main monosugar (Table 2) enhanced the yield of BC compared to the sugar content of glucose and xylose in LVH and CVH. Besides sugars, acetic acid is one common by-product generated during the pretreatment/hydrolysis process and shown to support enhanced BC production.^49–51 The enzymatic hydrolysate from the macroalgal source of C. linum at LCH and LVH with sufficient carbon source and high total phenol compound showed an enhanced effect on BC production compared to less total phenolic compound treatment in LCH and LVH. Tseng et al.²³ reported the yield of BC of 3.48 g/L from K. europaeus 14,148 which was cultivated in cellulase hydrolysate of hydrothermal treated pineapple leaf waste. The mandarin peel hydrolyzed by 0.6 M sulfuric acid obtained the lowest total phenolic content of 5.42 (g GAE/L) and got BC yield at 3.92 g BC/ 100 g peel.⁵² The green algae contains bioactive substances such as phenolic compounds, phycobiliproteins, carotenoids, alkaloids, terpenes, sulfated polysaccharides, and phytosterols which might inhibit the growth of bacteria,^53,54 but the macroalgal prebiotic also showed positive effect on stimulating bacterial fermentation.⁵⁵ The enzymatic hydrolysates from C. linum and C. crassa generated another re-utilizable carbon source for BC production by K. europaeus 14,148, but the antibacterial and bacteria-beneficial compounds in the enzymatic hydrolysate need more research to evaluate the advanced effect on the mechanism of BC production. Utilizing the enzymatic hydrolysis to treat the green seaweed was an eco-friendly bioprocess to decrease the usage of chemically purified carbon sources to prevent environmental pollution and decrease the cost of BC production.

Table 3.

The effect of enzymatic hydrolysates of C. crassa and C. linum on the BC production by cultivated K. europaeus on these mediums.

Treatments	Reducing sugar of cultivated medium (g/L)		Yield of BC (g/L)	Sugar utilization efficiency (%)	BC conversion ratio (%)
Treatments	Beginning	Final	Yield of BC (g/L)	Sugar utilization efficiency (%)	BC conversion ratio (%)
HS	2.14 ± 0.01	0.49 ± 0.06	0.26 ± 0.10	77.25 ± 2.72	0.02 ± 0.01
LVH	2.28 ± 0.02	0.47 ± 0.02	1.39 ± 0.03	79.52 ± 0.69	0.09 ± 0.00
LCH	2.35 ± 0.05	0.41 ± 0.03	1.77 ± 0.12	82.67 ± 0.80	0.02 ± 0.00
CVH	2.10 ± 0.01	0.48 ± 0.02	0.48 ± 0.06	77.24 ± 0.77	0.03 ± 0.00
CCH	0.53 ± 0.01	0.11 ± 0.01	0.51 ± 0.07	79.88 ± 0.87	0.24 ± 0.02

CCH: C. crassa cellulase hydrolysis; CVH: C. crassa viscozyme hydrolysis; LCH: C. linum cellulase hydrolysis; LVH: C. linum viscozyme hydrolysis.

Antioxidant ability of bacterial cellulose

DPPH is a stable free radical, and the hydrogen or electrons provided by antioxidants are the main factors affecting the scavenging of DPPH free radicals. The result of DPPH radical scavenging activity and ABTS cation radical scavenging ability were showed in Figure 2. The BC obtained from enzymatic hydrolysates of C. linum was found to have higher DPPH radical scavenging activity than C. crassa, however, C. crassa showed higher ABTS cation radical scavenging ability than C. linum. The antioxidant activity of BC was affected by the different concentrations of specific total phenolic compounds in the enzymatic hydrolysate of seaweed. The DPPH radical scavenging activity of BC might be attributed to the high total phenol compound (Figure 1) observed in LCH and LVH. The total phenolic compounds of seaweed enzymatic extract showed a marked correlation to the DPPH free radical scavenging activity.^22,56 The phenolics-rich medium consisted of the enzymatic hydrolysate of win pomace not only enhanced BC yield but also increased the phenolic contents and antioxidant activity, such as of BC.⁵⁷

Figure 2.

The effect of different enzymatic hydrolysate of C. crassa and C. linum on the (a) DPPH and (b) ABTS of BC obtained from K. europaeus.

The thickness and water retention rate of bacterial cellulose

In addition, Figure 3(a) also shows the thickness of BC. The thickness of BC produced by cellulose hydrolysate is about 0.20 to 0.30 mm. The thickness of 0.5% CCH was 0.52 mm, which is the highest among all hydrolysates by viscozyme. The thickness of BC produced is about 0.25 to 0.48 mm, which is average higher than that of hydrolysate produced by cellulase.

Figure 3.

The effect of different enzymatic hydrolysate of C. crassa and C. linum on the BC thickness and water-holding capacity.

Figure 3(b) shows the results of the water retention capacity of BC synthesized by cultivated K. europaeus 14,148 in different enzymatic hydrolysates. The water retention capacity of BC from macroalgal enzymatic hydrolysate ranged from 1600% to 1100%. When adding different concentrations of CCH and CVH, the water retention rate does not increase or decrease with the increase in concentration. The above results showed that the enzymatic hydrolysate from green seaweeds enhanced the water retention capacity of BC compared to the control HS medium. The water-holding capacity of BC produced from K. hansenii GA2016 cultivated in citrus peels hydrolysate medium ranged from 595.8 to 886.0% (w/w).⁵² 4870% water-holding capacity of BC from K. medellinensis 13,488 cultivated in grape pomance medium statically.³⁷

SEM analysis of BC

The SEM analysis on the surface of BC showed the different nanoscale structures produced from the enzymatic hydrolysate of green seaweed. BC which was obtained from CVH within a low total phenolic compound, found the crossed network of nanofiber with clear pore size structures compared to the compact cellulose sheet in CCH (Figure 4). The different BC structures found in SEM between CCH and CVH showed that the viscozyme could degrade the polysaccharide of macroalgal biomass to provide the re-utilizable carbon source for BC production. The BC pellicle produced from enzymatic hydrolysate of green seaweeds showed a large number of microfibril ribbons, macro-characteristically exhibiting a layer of transparent gel film structure. The high concentration of acetic acid in enzymatic hydrolysate medium produced thinner but denser packing of nanofibrils,⁵⁰ and the rice-like particles deposited on the surface of microfibril ribbons that might be the alkali not completely washed off from the BC membrane.⁴⁹

Figure 4.

The SEM analysis of BC obtained from (a) HS, (b) CCH, (c) CVH, (d) LCH, and (e) LVH.

FTIR analysis of BC

The specific peaks of characteristic functional groups of BC were analyzed by FTIR spectroscopy (Figure 5). The purity and the crystallinity of BC strengthen the peak of the characteristic functional group in the FTIR spectrum. The peak in the spectrum ranges from 3500 to 3200 cm⁻¹ represents the stretching vibration from the hydroxyl group (–OH). The peak at 2921 cm⁻¹ is typical of free cellulose. The absorption peak located at 1427 cm⁻¹ is the CH₂ symmetrical bending bonding functional group, which is related to the crystallinity of cellulose. The absorption peaks at 1160 and 898 cm⁻¹ are the C–O–C antisymmetric stretching structure of β-1,4-d-glucoside⁵⁸ and the C-O-C stretch bonding of β-1,4 bonded glucose polymer.⁵⁹ There was no significant difference from the spectrum analysis of FTIR on BC produced from enzymatic hydrolysis of macroalgal sources.

Figure 5.

FTIR spectra of BC films produced from HS medium, CCH, CVH, LCH, and LVE.

XRD analysis of BC

Figure 6 shows the XRD spectrum of BC from K. europaeus 14,148 by cultivating in HS medium added with enzymatic hydrolysates from green seaweeds. The characteristic peaks of BC were observed at 2θ = 14.4°, 16.8°, and 22.6°, respectively. The two diffraction angles 2θ = 14.4° and 22.6° reflect that all BC crystal forms belong to cellulose type I.^34,37

Figure 6.

XRD analysis of BC films produced from HS medium, CCH, CVH, LCH, and LVE.

The Segal method was then used to calculate the crystallinity index (CrI%) of BC (Table 4).⁶⁰ The crystallinity of BC from enzymatic hydrolysates of two green seaweeds are all above 70%, and the highest was 78.1% in LCH. The crystallinity of BC ranged from 46.7% to 92.0% obtained from carbon sources from agro-waste and fruit,^52,61 65% to 83% was found in commercial microcrystalline cellulose.⁶²

Table 4.

The crystallinity index (CrI) of bacterial cellulose obtained from different enzymatic hydrolysate.

Treatments	HS	CCH	CVH	LCH	LVH
CrI	63.04	75.62	77.17	78.11	74.44

Conclusions

This study developed an eco-friendly method for degrading aquaculture waste/macroalgal biomass and utilizing the sugars as carbon source to produce high crystalline BC. The hydrothermal pretreatment without acid addition supported the enzymatic saccharification of C. crassa and C. linum, producing glucose, rhamnose, and xylose which were utilized for BC production by K. europaeus 14,148. Cellulase and viscozyme efficiently broke down the polysaccharide structure of green seaweed. High concentrations of total phenol compounds in the enzymatic hydrolysate from green seaweed (C. linum) did not inhibit bacterial activity but enhanced BC production and improved radical scavenging activity. The high antioxidant ability of BC from the enzymatic hydrolysate makes it suitable for cosmetic, medical, and food packaging applications. Thus hydrothermally pretreated and enzymatically hydrolyzed waste macroalgal biomass serves as suitable carbon source for BC production.

Highlights

Green seaweed's enzymatic hydrolysate contained glucose, rhmanose, and xylose.

Enzymatic hydrolysis of green seaweed enhanced the yield of phenolic compounds

Phenolic compounds boost BC yield and antioxidant activity.

Enzymatic hydrolysate of green seaweed improved BC radical scavenging activity.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Science and Technology Council (grant number 112-2222-E-992 -006 -MY2).

ORCID iDs

Anil Kumar Patel

Cheng-Di Dong

Reeta Rani Singhania

Yi-Sheng Tseng is an assistant professor at the National Kaohsiung University of Science and Technology, Taiwan.

Wen-Pei Hsieh is a project assistant at the National Kaohsiung University of Science and Technology, Taiwan.

Anil Kumar Patel is an associate professor at the National Kaohsiung University of Science and Technology, Taiwan.

Pei-Pei Sun is an associate professor at the Department of Seafood Science, National Kaohsiung University of Science and Technology, Taiwan.

Chung-Hsin Wu is a distinguished professor at the Department of Chemical and Materials Engineering, National Kaohsiung University of Science and Technology, Taiwan.

Cheng-Di Dong is a distinguished professor at the Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Taiwan.

Reeta Rani Singhania is an associate professor at the National Kaohsiung University of Science and Technology, Taiwan.

Mei-Ling Tsai is a professor at the Department of Seafood Science, National Kaohsiung University of Science and Technology, Taiwan.

References

Tsiaras

Tsapakis

Gkanassos

, et al. Modelling the impact of finfish aquaculture waste on the environmental status in an Eastern Mediterranean Allocated Ζone for Aquaculture. Cont Shelf Res 2022; 234: 104647.

Hou

Huang

Lee

, et al. Upcycled aquaculture waste as textile ingredient for promoting circular economy. Sustainable Mater Technol 2022; 31: e00336.

González-Gaya

García-Bueno

Buelow

, et al. Effects of aquaculture waste feeds and antibiotics on marine benthic ecosystems in the Mediterranean Sea. Sci Total Environ 2022; 806: 151190.

Wan Mahari

Waiho

Azwar

, et al. A state-of-the-art review on producing engineered biochar from shellfish waste and its application in aquaculture wastewater treatment. Chemosphere 2022; 288: 132559.

Lin

, et al. An effective biological treatment method for marine aquaculture wastewater: combined treatment of immobilized degradation bacteria modified by chitosan-based aerogel and macroalgae (Caulerpa lentillifera). Aquaculture 2023; 570: 739392.

Sebök

Hanelt

. Cultivation of the brackish-water macroalga Ulva lactuca in wastewater from land-based fish and shrimp aquacultures in Germany. Aquaculture 2023; 571: 739463.

Lavaud

Ullman

Venolia

, et al. Production potential of seaweed and shellfish integrated aquaculture in Narragansett Bay (Rhode Island, U.S.) using an ecosystem model. Ecol Modell 2023; 481: 110370.

Nie

Mubashar

Zhang

, et al. Current progress, challenges and perspectives in microalgae-based nutrient removal for aquaculture waste: a comprehensive review. J Cleaner Prod 2020; 277: 124209.

Zhang

Jiang

, et al. Long-term effects of three compound probiotics on water quality, growth performances, microbiota distributions and resistance to Aeromonas veronii in crucian carp Carassius auratus gibelio. Fish Shellfish Immunol 2022; 120: 233–241.

10.

Noman

Al-Gheethi

Radin Mohamed

RMS

, et al. Quantitative microbiological risk assessment of complex microbial community in Prawn farm wastewater and applicability of nanoparticles and probiotics for eliminating of antibiotic-resistant bacteria. J Hazard Mater 2021; 419: 126418.

11.

Pekkoh

Chaichana

Thurakit

, et al. Dual-bioaugmentation strategy to enhance the formation of algal-bacteria symbiosis biofloc in aquaculture wastewater supplemented with agricultural wastes as an alternative nutrient sources and biomass support materials. Bioresour Technol 2022; 359: 127469.

12.

Baden

Fredriksen

Christie

, et al. Effects of depth and overgrowth of ephemeral macroalgae on a remote subtidal NE Atlantic eelgrass (Zostera marina) community. Mar Pollut Bull 2022; 177: 113497.

13.

Xie

Shao

, et al. Algal growth and alkaline phosphatase activity of green tide Chaetomorpha linum in response to phosphorus stress. J Mar Sys 2024; 241: 103912.

14.

Fraga-Corral

Ronza

Garcia-Oliveira

, et al. Aquaculture as a circular bio-economy model with Galicia as a study case: how to transform waste into revalorized by-products. Trends Food Sci Technol 2022; 119: 23–35.

15.

Lopes

Braos

Cruz

MCP

, et al. Valorization of animal waste from aquaculture through composting: nutrient recovery and nitrogen mineralization. Aquaculture 2021; 531: 735859.

16.

Mubashar

Zhang

Liu

, et al. In-situ removal of aquaculture waste nutrient using floating permeable nutrient uptake system (FPNUS) under mixotrophic microalgal scheme. Bioresour Technol 2022; 363: 128022.

17.

Cutajar

Falcone

Massa-Gallucci

, et al. Stable isotope and fatty acid analysis reveal the ability of sea cucumbers to use fish farm waste in integrated multi-trophic aquaculture. J Environ Manag 2022; 318: 115511.

18.

Tambat

Tseng

Kumar

, et al. Effective and sustainable bioremediation of molybdenum pollutants from wastewaters by potential microalgae. Environ Technol Innovation 2023; 30: 103091.

19.

Perumal

Huang

Singhania

, et al. In vitro evaluation of antioxidant potential of polysaccharides and oligosaccharides extracted from three different seaweeds. J Food Sci Technol 2023; 61: 1–11.

20.

Afrin

Ahsan

Mondal

, et al. Evaluation of antioxidant and antibacterial activities of some selected seaweeds from Saint Martin’s Island of Bangladesh. Food Chem Adv 2023; 3: 100393.

21.

Thygesen

Ami

Fernando

, et al. Microstructural and carbohydrate compositional changes induced by enzymatic saccharification of green seaweed from West Africa. Algal Res 2020; 47: 101894.

22.

Teixeira-Guedes

Gomes-Dias

Cunha

, et al. Enzymatic approach for the extraction of bioactive fractions from red, green and brown seaweeds. Food Bioprod Process 2023; 138: 25–39.

23.

Güzel

Akpınar

. Preparation and characterization of bacterial cellulose produced from fruit and vegetable peels by Komagataeibacter hansenii GA2016. Int J Biol Macromol 2020; 162: 1597–1604.

24.

Tseng

Singhania

Cheng

, et al. Removal of heavy metal vanadium from aqueous solution by nanocellulose produced from Komagataeibacter europaeus employing pineapple waste as carbon source. Bioresour Technol 2023; 369: 128411.

25.

Singhania

Patel

Tsai

, et al. Genetic modification for enhancing bacterial cellulose production and its applications. Bioengineered 2021; 12: 6793–6807.

26.

Singhania

Patel

Tseng

, et al. Developments in bioprocess for bacterial cellulose production. Bioresour Technol 2022; 344: 126343.

27.

Tseng

Patel

Chen

, et al. Improved production of bacterial cellulose by Komagataeibacter europaeus employing fruit extract as carbon source. J Food Sci Technol 2023; 60: 1054–1064.

28.

Khan

Saroha

Raghuvanshi

, et al. Valorization of fruit processing waste to produce high value-added bacterial nanocellulose by a novel strain Komagataeibacter xylinus IITR DKH20. Carbohydr Polym 2021; 260: 117807.

29.

Mittal

Raghavarao

KSMS

. Extraction of R-phycoerythrin from marine macro-algae, Gelidium pusillum, employing consortia of enzymes. Algal Res 2018; 34: 1–11.

30.

W-z

Zhang

, et al. Pretreatment of corn stover for sugar production using dilute hydrochloric acid followed by lime. Bioresour Technol 2014; 152: 364–370.

31.

Hestrin

Schramm

. Synthesis of cellulose by Acetobacter xylinum. II. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose. Biochem J 1954; 58: 345–352.

32.

Edson

Poe

. Determination of reducing sugars in food products. Comparative study of colorimetric methods. J Assoc Off Agric Chem 1948; 31: 769–776.

33.

Singleton

Rossi

. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Vitic 1965; 16: 144–158.

34.

Urbina

Hernández-Arriaga

Eceiza

, et al. By-products of the cider production: an alternative source of nutrients to produce bacterial cellulose. Cellulose 2017; 24: 2071–2082.

35.

Zmejkoski

Marković

Zdravković

, et al. Bactericidal and antioxidant bacterial cellulose hydrogels doped with chitosan as potential urinary tract infection biomedical agent. RSC Adv 2021; 11: 8559–8568.

36.

de Dicastillo

Bustos

Guarda

, et al. Cross-linked methyl cellulose films with murta fruit extract for antioxidant and antimicrobial active food packaging. Food Hydrocolloids 2016; 60: 335–344.

37.

Diaz-Ramirez

Urbina

Eceiza

, et al. Superabsorbent bacterial cellulose spheres biosynthesized from winery by-products as natural carriers for fertilizers. Int J Biol Macromol 2021; 191: 1212–1220.

38.

Shah

Ul-Islam

Khattak

, et al. Overview of bacterial cellulose composites: a multipurpose advanced material. Carbohydr Polym 2013; 98: 1585–1598.

39.

Hong

Jeon

Lee

. Comparison of red, brown and green seaweeds on enzymatic saccharification process. J Ind Eng Chem 2014; 20: 2687–2691.

40.

Nagarajan

Oktarina

Chen

, et al. Fermentative lactic acid production from seaweed hydrolysate using Lactobacillus sp. and Weissella sp. Bioresour Technol 2022; 344: 126166.

41.

Yazdani

Zamani

Karimi

, et al. Characterization of Nizimuddinia zanardini macroalgae biomass composition and its potential for biofuel production. Bioresour Technol 2015; 176: 196–202.

42.

Parab

Khandeparker

Amberkar

, et al. Enzymatic saccharification of seaweeds into fermentable sugars by xylanase from marine Bacillus sp. strain BT21. 3 Biotech 2017; 7: 96.

43.

Saini

Chen

Patel

, et al. Valorization of pineapple leaves waste for the production of bioethanol. Bioengineering 2022; 9: 57.

44.

Saini

Singhania

Patel

, et al. A circular biorefinery approach for the production of xylooligosaccharides by using mild acid hydrothermal pretreatment of pineapple leaves waste. Bioresour Technol 2023; 388: 129767.

45.

Bikker

Krimpen

Wikselaar

, et al. Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels. J Appl Phycol 2016; 28: 3511–3525.

46.

Nguyen

Sunwoo

, et al. Acetone, butanol, and ethanol production from the green seaweed Enteromorpha intestinalis via the separate hydrolysis and fermentation. Bioprocess Biosyst Eng 2019; 42: 415–424.

47.

Habeebullah

SFK

Alagarsamy

Arnous

, et al. Enzymatic extraction of antioxidant ingredients from Danish seaweeds and characterization of active principles. Algal Res 2021; 56: 102292.

48.

Norakma

Zaibunnisa

Wan Razarinah

WAR

. The changes of phenolics profiles, amino acids and volatile compounds of fermented seaweed extracts obtained through microbial fermentation. Mater Today Proc 2022; 48: 815–821.

49.

Yang

Huang

Guo

, et al. Beneficial effect of acetic acid on the xylose utilization and bacterial cellulose production by Gluconacetobacter xylinus. Indian J Microbiol 2014; 54: 268–273.

50.

Kuo

Chen

Liou

, et al. Utilization of acetate buffer to improve bacterial cellulose production by Gluconacetobacter xylinus. Food Hydrocolloids 2016; 53: 98–103.

51.

Islam

Ullah

Khan

, et al. Strategies for cost-effective and enhanced production of bacterial cellulose. Int J Biol Macromol 2017; 102: 1166–1173.

52.

Güzel

Akpınar

. Production and characterization of bacterial cellulose from citrus peels. Waste Biomass Valorization 2019; 10: 2165–2175.

53.

Nalini

Sandy Richard

Mohammed Riyaz

, et al. Antibacterial macro molecules from marine organisms. Int J Biol Macromol 2018; 115: 696–710.

54.

Silva

Lourenço-Lopes

, et al. Antibacterial use of macroalgae compounds against foodborne pathogens. Antibiotics (Basel) 2020; 9: 12.

55.

Krangkratok

Chantorn

Choosuwan

, et al. Production of prebiotic ulvan-oligosaccharide from the green seaweed Ulva rigida by enzymatic hydrolysis. Biocatal Agric Biotechnol 2023; 54: 102922.

56.

Heo

Park

Lee

, et al. Antioxidant activities of enzymatic extracts from brown seaweeds. Bioresour Technol 2005; 96: 1613–1623.

57.

Azi

Dong

, et al. Green and efficient in-situ biosynthesis of antioxidant and antibacterial bacterial cellulose using wine pomace. Int J Biol Macromol 2021; 193: 2183–2191.

58.

Lotfy

Basta

Abdel-Monem

, et al. Utilization of bacteria in rotten guava for production of bacterial cellulose from isolated and protein waste. Carbohydr Polym Technol Appl 2021; 2: 100076.

59.

Reiniati

Hrymak

Margaritis

. Kinetics of cell growth and crystalline nanocellulose production by Komagataeibacter xylinus. Biochem Eng J 2017; 127: 21–31.

60.

Segal

Creely

Martin

, et al. An Empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text Res J 1959; 29: 786–794.

61.

Cao

Yang

. Production of bacterial cellulose from byproduct of citrus juice processing (citrus pulp) by Gluconacetobacter hansenii. Cellulose 2018; 25: 6977–6988.

62.

Goh

Rosma

Kaur

, et al. Microstructure and physical properties of microbial cellulose produced during fermentation of black tea broth (Kombucha). II. Int Food Res J 2012; 19: 153.