Preparation of cyanobacteria-enhanced poly(vinyl)alcohol-based films with resistance to blue-violet light / red light and water.

The harmful cyanobacteria blooms which usually form in spring and summer, cause global eutrophication of freshwater and coastal marine ecosystems. This study tried to utilize cyanobacteria as a raw material to produce biological poly(vinyl)alcohol-based films. Cyanobacteria was firstly modified with poly(ethylene glycol), guanidine hydrochloride, carboxymethyl cellulose and 3-glycidoxypropyltrimethoxysilane as plasticizer, modifier, toughening agent and coupling agent, respectively. And then the modified cyanobacteria was introduced to poly(vinyl)alcohol and cellulose nanofibers/poly(vinyl)alcohol matrix to improve the barrier properties of poly(vinyl)alcohol to light and water. Compared with poly(vinyl)alcohol and cellulose nanofibers/poly(vinyl)alcohol films, the obtained cyanobacteria/poly(vinyl)alcohol and the cyanobacteria/cellulose nanofibers/poly(vinyl)alcohol composites exhibit better resistance to light and water. More interestingly, we found that after adding cyanobacteria, the poly(vinyl)alcohol-based films present better barrier properties to blue-violet light and red light. In adddition, introducing cyanobacteria into poly(vinyl)alcohol or cellulose nanofibers/poly(vinyl)alcohol matrix increases the surface roughness and contact angle to water of the composites.

Introduction

The global bioplastics for packaging industry is forecast to grow from 2017–2022 at an annual average rate of 17% to a market value of almost $7.2 billion according to a report from Smithers P.[1]. As one of the important bioplastics, poly(vinyl)alcohol (PVA) has a broad spectrum of applications in fabricating coatings and films in food applications [2]. It is also a main ingredient in lubricant or temporary skin covers or wound dressings in pharmaceutical applications [2-5] because of its biocompatibility, biodegradability, non-toxicity, inexpensiveness [6], easy process situation, excellent transparency, surface activity [3], and suitable film-forming capabilities [4]. However, on the other hand, its poor barrier property especially to light or water and thermal stability greatly limit the application development of PVA. Some studies have shown that the presence of hydrogen bond groups in PVA structure and its ability of forming hydrogen bond make it suitable for PVA to mix with other materials to improve its functional characteristics [3,4,7]. For example, PVA/chitosan blend membranes produced by Gupta, B., et al [8] and PVA/carboxymethyl cellulose composite films manufactured by Kanatt, S. R., et al [9] presented better properties than PVA in the view of applications in packaging industry. The main reason why Kanatt chose nanocellulose as the strengthening phase of PVA was considering the excellent properties and availability of nanocellulose. Cellulose nanofibers (CNF) are renewable, biocompatible and biodegradable [10,11], and can be extracted from biomass such as wood [12], bamboo [13] and crop straw [14]. It has been widely used to improve the tensile and thermal properties of PVA [15-17].

As a type of prokaryote, cyanobacteria (CY) can absorb a large amount of nitrogen and phosphorus, and then rapidly proliferates to form harmful cyanobacteria blooms in spring and summer, resulting in a global problem, eutrophication of freshwater and coastal marine ecosystems [18,19]. Prevention and control of cyanobacteria pollution has become a common concern among scientists and entrepreneurs. It is an effective way to reduce the pollution of cyanobacteria to utilize it as resources. So far, there have been two kinds of methods to utilize cyanobacteria as resources. One is engineering cyanobacteria cell factories for manufacturing various chemical compounds, such as lactic acid [20], C3 platform chemicals [21], 2, 3-butanediol [22], fatty acid [23], isobutylaldehyde [24], n-butanol [25] and bioplastic PHAs [26,27]. However, this route cannot effectively solve the problem of cyanobacteria excessive reproduction. The other method of utilizing cyanobacteria resources is to try to directly use cyanobacteria as raw materials to produce biological composites [28], biofuel [29], biological fertilizer [30]. In addition, some researchers proposed that cyanobacteria could be used as a biological agent to improve the degradation ability of soil to various pollutants [31]. Whereas, these methods need to be explored further because they are still in trials and have not been widely reported.

According to our investigation, there had been no reports of cyanobacteria being directly used to prepare composite films. Considering the insufficient resistance to light and water of PVA as a packaging material, cyanobacteria was introduced into PVA matrix in this paper. In our early elemental analysis test, the contents of carbon, nitrogen and hydrogen in the cyanobacteria sample were 45.02%, 5.98% and 9.11%, respectively. It was found that in the preliminary experiment directly mixing cyanobacteria, PVA and CNF to prepare the PVA-based films led to a poor interface combination because of the microphase separation between cyanobacteria and PVA and CNF. In this paper, cyanobacteria/cellulose nanofibers/poly(vinyl)alcohol (CY/CNF/PVA) bioplastic films were fabricated with poly(ethylene glycol), guanidine hydrochloride, carboxymethyl cellulose and 3-glycidoxypropyltrimethoxysilane as plasticizer, modifier, toughening agent and coupling agent, respectively. The purpose of this study is to explore the possibility of direct utilizing cyanobacteria as raw materials to prepare CY/CNF/PVA films, and to improve the light and water resistance of PVA films.

Materials and methods

Materials

The cyanobacteria (CY) collected in this experiment were collected from the algal and water separation station in Yixing Bafang port, which is located on the western bank of Taihu lake (moderately nutrient-rich water quality), with a straight-line distance of 1.5 km from the long-depth high-speed (G25) and 0.8 km from the dubian line (S230). Due to the bloom of cyanobacteria, they can be artificial collected directly on the surface of the lake. The algae pump and the associated diversion and containment equipment were used to collect cyanobacteria from the surface. The surrounding ecological environment was not destroyed during the process. The fresh cyanobacteria was transported to the material laboratory of nanjing forestry university on the same day, and then firstly dried by sunlight for 2 days. After that dried it for at less 48 hours at 60°C until the moisture content was less than 20%. After screening, 100 mesh cyanobacteria powder was sealed in plastic bags for later use.

This study was approved by the Cyanobacteria Office of Wuxi Water Resources Bureau. All cyanobacteria used were collected by the staff of the Cyanobacteria Office.

Polyethylene glycol (200 molecular weight) were from Yonghua Chemical Technology Co., Ltd (Jiangsu Province, China). Polyvinyl alcohol (750±50 degree of polymerization), guanidine hydrochloride, Sodium carboxymethyl cellulose and 3-(Glycidoxypropyl)triethoxysilane (97 wt%) came from Sinopsin Chemical Reagent Co., Ltd (Jiangsu Province, China). Nanocellulose (shown in Table 1) were obtained from Zhongshan NFC Bio-materials Co., Ltd,(Guangdong Province, China).

Table 1

Information of the purchased nanocellulose.

Name	Specification
Product model	NFC1802N
Raw materials	Coniferous wood pulp fibre
Concentration	2.5±0.5%
Carboxyl content	1.02 mmol/g
Length	1~2 μm
Diameter	30 nm
Aspect ratio	30~100
Net weight	250 g

Preparation of the PVA-based films

Experimental scheme

The experimental scheme and formula of the the PVA-based films are shown in Table 2. No. 1 (PVA film) and No. 7 (CNF/PVA film) is the control to No. 2-No.6 and No. 8-No.12 respectively for investigating the influence of cyanobacteria on the properties of PVA films and CNF/PVA films.

Table 2

Experimental scheme and formula of the PVA-based films.

Film No.	CMa		PVAa	CNFa	(Glycidoxypropyl)triethoxysilanea
1	0 g	0 wt%b	20 g	0 g	0 g
2	1 g	5 wt%b	20 g	0 g	0.5 g
3	3 g	15 wt%b	20 g	0 g	1.0 g
4	5 g	25 wt%b	20 g	0 g	1.5 g
5	7 g	35 wt%b	20 g	0 g	2.0 g
6	9 g	45 wt%b	20 g	0 g	2.5 g
7	0 g	0 wt%b	13.3 g	6.7 g	0 g
8	1 g	5 wt%b	13.3 g	6.7 g	0.5 g
9	3 g	15 wt%b	13.3 g	6.7 g	1.0 g
10	5 g	25 wt%b	13.3 g	6.7 g	1.5 g
11	7 g	35 wt%b	13.3 g	6.7 g	2.0 g
12	9 g	45 wt%b	13.3 g	6.7 g	2.5 g

a These are the weight of solution (the modified cyanobacteria, 10 wt% PVA solution, 1 wt% CNF solution and 3-(Glycidoxypropyl)triethoxysilane solution).

b The weight content of CM in film No.1-6 is CM to PVA. The weight content of CM in film No.7-12 is CM to CNF/PVA.

Preparation of the films

2g PVA was added to 18 ml deionized water and stirred for 2 hours at 95°C to obtain 10 wt% PVA solution for later use. Meanwhile, 8g 2.5 wt% CNF solution was added to 12 ml deionized water, stirred at room temperature for 1 hour and sonicated for 40 min (XO-1200, Nanjing Xianou Instrument Manufacturing Co., Ltd., China) to obtain 1 wt% CNF solution for later use. The prepared 10 wt% PVA solution and 1 wt% CNF solution were mixed by weight ratio of 2:1 to obtain CNF/PVA solution for later use.

The PVA-based films were prepared by a solution casting method. According to the formulation in Table 2, a certain amount of 10 wt% PVA solution was poured into a petri dish, and put in an oven (101-2BS, Beijing Hengnuolixing Technology Co., Ltd., China) for drying at 40°C for 24 hours to get the PVA film (No. 1, shown in Fig 1). In the same way, the CNF/PVA film (No. 7, shown in Fig 1) was obtained.

Fig 1

Pictures of the PVA-based films.

Cyanobacteria powder (1g, absolute dry weight) was mixed with deionized water (46.75g) to obtain cyanobacteria homogenization. Poly(ethylene glycol) (1g), guanidine hydrochloride (0.75g) and carboxymethyl cellulose (0.5g) was added successively into the cyanobacteria homogenization. After stirring at room temperature for 2 hours, the modified cyanobacterial was obtained, as showm in Fig 1. According to the formulation in Table 2, a certain amount of modified cyanobacteria, referred to as CM, was mixed with PVA solution or CNF/PVA solution. After adding 3-(Glycidoxypropyl)triethoxysilane solution, the mixture was heated and stirred at 55°C for 1 hour, and casted in a petri dish, and then oven-dried (101-2BS, Beijing Hengnuolixing Technology Co., Ltd., China) at 40°C for 24 hours, resulting in the CY/PVA films (No.2 - No.6, shown in Fig 1) and CY/CNF/PVA films (No.8 - No.12, shown in Fig 1).

Characteristics of the PVA-based films

Fourier transform infrared spectroscopy (FTIR) analysis of the film samples

FTIR spectra of the film samples (PVA, CY/PVA, CNF/PVA, CY/CNF/PVA) have been acquired with a Fourier transform spectrometer (NICOLET IS10 Thermo SCIENTIFIC, Inc, USA) operating in the mode of Smart iTR diamond ATR and the range from 500 cm-1 to 4000 cm-1.The baseline correction was done using the accompanying built-in software.

Morphology analysis of the PVA-based films

The fracture morphology analysis of the PVA-based films was observed by a cold field emission scanning electron microscope (Hitachi Regulus 8200, Japan).

The section in the direction of film thickness was placed up and attached to the conductive tape on the sample stand. The gold plating on the section of the film sample was conducted under the condition of 20 mA of gold spraying current and less than 7 Pa of vacuum degree for 80~100 seconds. The working voltage of the scanning electron microscope was 5kv.

Barrier properties of the PVA-based films to light

A spectrophotometer U-4001 (Hewlett-Packard Co., Santa Clara, CA, USA) was used to analyze the barrier properties of the PVA-based films to ultraviolet and visible light. The transmittance of the films to light in the range of 200–800 nm was measured.

Surface contact angle to distilled water of the PVA-based films

After drying in an oven (Electric blast drying box, 101-2BS, Beijing Hengnuolixing Technology Co., Ltd, China) at 100°C for 24 hours, the PVA-based film was cut to pieces with a size of 20 mm × 20 mm (length×width). The surface contact angle measurement of the sample to distilled water was carried out in an automatic single fiber contact angle measuring instrument (OCA40, DataPhysics Intruments GmbH, German).

Water resistance of the PVA-based films

The swelling of a sample is usually used to indicate the water resistance of a material. Operating methods refer to ASTM d570-98. The PVA-based composite film with a size of 2 cm × 2 cm was firstly weighed (Wp), then oven-dried at 100°C for 24 hours and weighed (Wi). The degree of moisture content (X) of the composite films was calculated by Eq (1),

The sample after oven-dries was immersed in 50-ml distilled water for 24 hours at room temperature. Drying the moisture of the sample surface, the sample was weighed again (Wf). The degree of swelling (A) [32] of the sample was calculated by Eq (2),

Oxygen permeability of the PVA-based films

The film sample with a size of 10cm was firstly oven-dried at 100°C for 24 hours, and then tested its oxygen permeability with a differential pressure gas permeameter (PERME VAC-V2, Jinan Labthink Electromechanical Technology Co., Ltd., China). The analysis conditions were 50% relative humidity, room temperature (23°C), 10% proportion mode, GTR≥1 and 12 hours.

Results and discussions

Moisture content and thickness of the PVA-based films

Table 3 lists the moisture and thickness of the prepared PVA, PVA/CNF, CY/PVA and CY/CNF/PVA films. The data of the moisture content and thickness of the film with each formulation are the average of the 3 replica. It can be seen that the moisture content and the thickness of the composite films vary within the range of 3–6% and 0.2–0.5 mm, respectively.

Table 3

Moisture content and thickness of the PVA-based films.

Film No.	Moisture content (%)	Thickness (mm)
No.1	4.29±0.92	0.22±1.05
No.2	4.24±1.17	0.25±0.29
No.3	5.26±1.01	0.34±0.97
No.4	5.76±1.18	0.37±0.33
No.5	3.97±0.92	0.42±0.06
No.6	5.07±0.64	0.49±1.12
No.7	3.26±0.85	0.21±1.07
No.8	5.89±1.10	0.28±1.02
No.9	3.20±0.91	0.28±1.29
No.10	5.03±0.12	0.37±1.02
No.11	5.56±1.53	0.45±0.08
No.12	3.98±0.09	0.48±0.74

FTIR analysis of cyanobacteria and the PVA-based films

The FTIR spectra of the cyanobacteria used in this experiment, the obtained the PVA-based films are shown in Fig 2. The main characteristic peaks of PVA and PVA/CNF are consistent with those of the composites in the previous reports [33-36]. In the spectra of the PVA film and CNF/PVA composite, the peaks at 3276 cm-1 are attributed to the stretching vibration absorption peak of hydrogen bonding group. Although there exist a large amount of hydrogen groups in the structure of PVA, CNF and cyanobacteria, all vibration peaks at 3276 cm-1 in the spectra of the CY/CNF/PVA composites are weaker than that of PVA film or CNF/PVA composite. It may be due to the fact that the active amine in guanidine hydrochloride reacts with the inter- and intra-molecular hydrogen bonding of the lipids and starch in cyanobacteria [18].

Fig 2

FTIR spectra of the CY, PVA, PVA/CNF, CY/PVA and CY/PVA/CNF films.

In FTIR spectra of all samples, the absorption peaks at 2927 cm-1, 1661 cm-1, 1408 cm-1 and 1200–1000 cm-1 respectively belong to the anti-symmetric stretching vibration of hydrogen bond, the stretching vibration of carbon-oxygen double bond, the bending vibration of methylene group, and the characteristic spectral region of polysaccharide [33-36]. Each sample presents a characteristic spectral region of polysaccharide, which is attributed to the cyanobacteria or CNF in the sample. In addition, with the increase of cyanobacteria content, the absorption peak at 1661 cm-1 in the spectrum of the sample enhances owing to the peptide bonding of phycocyanin in cyanobacteria [37].

The peak at 1090 cm-1 is the characteristic absorption peak of PVA, which still appears in the spectra of all CY/CNF/PVA samples. The peak at 1020 cm-1 which is the characteristic absorption peak of cyanobacteria, still appears in the spectra of the CY/CNF/PVA samples with lower modified cyanobacteria content (No. 2, No.4, No.5, and No.8-No.10), indicating the successful blending of the modified cyanobacteria with the PVA and PVA/CNF. However, for the samples (No.3, No. 6, No.11, and No.12) with more modified cyanobacteria content, the spectra does not present the absorption peak at 1020 cm-1, showing that too much modified cyanobacteria leads to poor mixing of modified cyanobacteria with PVA and PVA/CNF.

Besides, a series of peaks appear at 800–400 cm-1 in the spectra of all CY/CNF/PVA samples, which are ascribed to the out-of-plane bending vibration of hydroxy groups[33-36], demonstrating that there exist some inter-molecular hydroxy groups in the obtained CY/CNF/PVA samples.

Barrier properties of the PVA-based films to ultraviolet and visible light

The transmittance curves of the PVA-based films for ultraviolet and visible light at a wavelength of 200–800 nm are shown in Fig 3. It can be seen that introducing CNF to PVA decreases the transmittance of the PVC for ultraviolet and visible light, and further adding cyanobacteria to CNF/PVA matrix further decreases the transmittance of the CNF/PVA. The higher the cyanobacteria content, the lower the light transmittance of the CY/CNF/PVA films. It means that the barrier property of PVA to ultraviolet and visible light can be enhanced by CNF and CY. This phenomenon can be explained by the fact that the particle in CNF and cyanobacteria sizes larger than the corresponding wavelength would obstruct light[38]. In addition, all spectral lines of CY/PVA and CY/CNF/PVA samples in Fig 3 show two strong absorption peaks at 403 nm and 666 nm. Table 4 lists the transmission the of the PVA-based films at two critical wavelengths. Light with wavelength less than 400 nm is ultraviolet light, and light with wavelength between 400nm and 760nm is visible light, in which the wavelength of 620-760nm belongs to red light, and that of 400–464 nm belongs to blue violet light. Therefore, a conclusion it can be made that the prepared CY/PVA and CY/CNF/PVA films have strong barrier properties to red and blue violet light. It is due to the chlorophyll a in cyanobacteria[37], which mainly absorbs red and blue violet light [39]. This property is particularly important in the field of food packaging, which can block the effect of ultraviolet and visible light on food, such as fresh-cut broccoli, soliddrinks, etc., to increase their life.

Fig 3

Transmittance of the PVA-based films at 200–800 nm.

Table 4

Transmission (%) the of the PVA-based films at two critical wavelengths.

Film No.	1	2	3	4	5	6	7	8	9	10	11	12
in 403 nm	90.5	75.5	30.2	19.5	12.0	4.6	87.8	71.9	32.6	26.2	11.4	1.3
in 666 nm	91.7	84.7	64.5	53.9	46.1	37.6	89.4	82.3	64.6	64.6	51.7	25.5

c The data in the table is the data of a test.

Barrier properties of the PVA-based films to deionized water

Water resistance and swelling ratio

The water resistance and swelling ratio with the surface hydrophobicity can show the barrier properties of the PVA-based films to deionised water. Fig 4 shows the water resistance and swelling ratio of different PVA-based films. The swelling ratio is an important index to characterize the water resistance of a material, and the smaller the swelling ratio, the better the water resistance for a material. The swelling ratio of the PVA film and PVA/CNF film are both higher than 100%, which is due to the fact that they not only adsorb water on the surface, but also can form hydrogen bond with water with a large number of hydrophilic hydroxyl groups. Compared with the PVA and the PVA/CNF films, the swelling ratio of the CY/PVA and CY/CNF/PVA films (No.3-6 and No.9-12) is almost reduced by half, indicating that the water resistance of them is remarkably enhanced. Moreover, the swelling ratio decreases slowly with the increase of cyanobacteria content in the composite. This is attributed to two aspects. One is that the active amine in guanidine hydrochloride reacts with lipids in cyanobacteria and PVA, as well as with hydroxyl groups in starch, thereby reducing the amount of hydrophilic hydroxyl in the composite film. Second, the cyanobacteria cell wall has a limiting and blocking effect on the entry of water molecules into the complex membrane [36].

Fig 4

Water absorption and thickness swelling of different PVA-based films.

Surface hydrophobicity of the PVA-based films

The contact angle is usually used to evaluate the surface hydrophobicity of a material. The contact angles of the prepared PVA-based films are shown in Fig 5 and Table 5. Compared to the control (PVA film and PVA/CNF film), the CY/PVA and CY/CNF/PVA films present higher contact angles. Furthermore, a small increase in modified cyanobacteria content leads to a significant increase in the contact angle of the composite. When the modified cyanobacteria content increases from 0 wt% to 5 wt%, 15 wt%, 25 wt%, 35 wt% and 45 wt%, the contact angle of the CY/PVA film increases from 12° to 55°, 62°, 69°, 72° and 82°, respectively; and that of the CY/PVA/CNF film ascends from 23° to 67°, 74°, 75°, 76° and 77°, respectively. This phenomenon is attributed to the higher surface roughness as a result of introducing modified cyanobacteria into the matrix, which can be observed from the scanning pictures of the PVA-based films in Fig 1.

Fig 5

Contact angles of the prepared PVA-based films to deionized water.

Table 5

Contact angles to deionized water of the prepared PVA-based films.

Film No.	1	2	3	4	5	6	7	8	9	10	11	12
Contact angle/°	12	55	62	69	72	82	23	67	74	75	76	77

d The data in the table is the data of a test.

Morphology of the PVA-based films

The SEM images of the PVA film (No.1-a), CY/PVA films (No.3-b and No.5-c), CNF/PVA film (No.7-d), and CY/CNF/PVA films (No.9-e and No.11-f) are shown in Fig 6. It can be seen that the PVA film and CNF/PVA film are relatively smooth. After adding modified cyanobacteria, the cross-sections of the CY/PVA films and CY/CNF/PVA films become rougher and unevener. This phenomenon can be used to explain our founding that the contact angle of the composite film goes up with the increase of the modified cyanobacteria content. In addition, as shown in Fig 6(B), 6(C) and 6(E), the modified cyanobacteria is wrapped in the PVA matrix or the CNF/PVA matrix, and the interface is blurred, indicating good compatibility of the modified cyanobacteria with the matrix. However, in Fig 6(F), the modified cyanobacteria is phase-separated from the CNF/PVA matrix, showing that too much cyanobacteria leads to poor compatibility with the matrix. The observed phenomenon that the internal structure of the films becomes looser after adding of the cyanobacteria can be used to explain the increased oxygen permeability of the PVA-based film after adding cyanobacteria. It is consistent with the results of our FTIR test.

Fig 6

SEM images of the PVA film No.1 (a), the CY/PVA film No.3 (b) and No.5 (c), the CNF/PVA film No.7 (d), and the CY/CNF/PVA film No.9 (e) and No.11 (f).

Oxygen barrier properties analysis

The oxygen transmission rate of the PVA-based films is shown in Table 6. As the modified cyanobacteria content increases, the oxygen permeability coefficient of the composite presents an ascending trend. It may be due to the looseness and porosity of the inner space of the film after adding cyanobacteria into the matrix, as shown in the SEM images of the PVA-based films in Fig 6. It needs to explore new experimental techniques to improve the oxygen barrier properties in our future research.

Table 6

Oxygen transmission rate of the PVA-based films.

Film No.	7	8	9	10
(cm3·cm/cm3·s·Pa)	1.23×10−16	6.18×10−16	7.38×10−16	3.47×10−15
	±9×10−18	±2.5×10−17	±5.7×10−17	±8.2×10−16

Conclusions

CY/PVA and CY/CNF/PVA films were prepared with a solution casting method. Compared with PVA and CNF/PVA films, the obtained CY/PVA and the CY/CNF/PVA composites exhibit better resistance to light and water. More interestingly, we found that after adding cyanobacteria, the PVA-based films present better barrier properties to blue-violet light and red light. In adddition, introducing cyanobacteria into PVA or CNF/PVA matrix increases the surface roughness and contact angle to water of the composites. This work explored a pathway for utilizing cyanobacteria as raw materials to prepare CY/CNF/PVA films with better resistance to light and water than PVA film.

(ZIP)

Click here for additional data file.

30 Oct 2019

PONE-D-19-24904

Preparation of cyanobacteria-enhanced poly(vinyl)alcohol-based films with resistance to blue-violet light / red light and water

PLOS ONE

Dear Mrs. Xu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Dec 14 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Antonio José Félix Carvalho, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript

A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

3. i) Please provide further details of how the cyanobacteria field sampling took place. Please clarify whether sampling was conducted specifically for this study, and by whom. Please clarify what 'orders' of cyanobacteria refers to. Was any attempt made to characterise and identify the cyanobacteria in the samples? Please provide the geographical coordinates for the sample site. Please also explain in the Methods section whether or not a field permit was obtained, and if not, a brief statement explaining why.

ii) Our internal editors have looked over your manuscript and determined that it may be within the scope of our Plastics in the Environment Call for Papers. The Collection will encompass a diverse range of research articles to better understand various aspects of the effect of plastics in the environment. Additional information can be found on our announcement page: https://collections.plos.org/s/plastics-environment. If you would like your manuscript to be considered for this collection, please let us know in your cover letter and we will ensure that your paper is treated as if you were responding to this call. If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments (if provided):

Please follow the reviewers' instructions and resubmit the manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PLOS ONE

Title: Preparation of cyanobacteria-enhanced poly(vinyl)alcohol-based films with resistance to blue-violet light / red light and water

The manuscript describes the preparation of composites based on cyanobacteria incorporated into poly(vinyl)alcohol and cellulose nanofibers/poly(vinyl)alcohol matrices. The scope is interesting once the authors suggest the use of the environmentally harmful cyanobacteria as to generate useful materials. The results and discussion are clear and well described. Some suggestions are listed below in order to improve the reading and reproducibility of some important points.

The title suggested the resistance to blue-violet light / red light and water are important properties, however it was not discussed in the text. I strongly suggest to include the importance of these features. It can be included in the introduction, or in results and discussion.

Line 69 – “In our early elemental analysis test, the contents of carbon, nitrogen and hydrogen in the cyanobacteria sample were 45.02 %, 5.98 % and 9.11 %, respectively.”. Do the authors have already published these data? If positive, please include the reference. If not, please discuss in the results section why it is important.

Table 2- It is need to include a column with the weight content (wt %) of each component of the composite to facilitate the understanding of the results.

Line 134- What is the meaning of CM?

Lines 281-282- please convert the mass values em wt % values, in agreement with the new suggested wt % column from table 2.

Please include a short text correlating the water resistance and swelling ratio with the surface hydrophobicity of the PVA-based films.

How difficult is to collect the cyanobacteria from the sea water? Could be interesting to insert a short text explaining as it could be feasible.

Typing errors:

line 67 -utilizerecycle

131- bsed

line 250 – wto

304 - filmgoes

Reviewer #2: In the present manuscript, the authors described the preparation of poly(vinyl alcohol) (PVA)/nanocellulose composites incorporating cyanobacteria in order to give the materials better light and water resistance. The study is of interest, but some modifications are needed before considering its publication in PLOS One.

- The writing must be reviewed, as some mistakes are present (p. 3, l. 57: “…utilizerecycle cyanobacteria…”; p. 3, l. 62: “The other method of resource utilizing cyanobacteria…”; p. 5, l. 87: “Polyethylene glycol (molecular weight, 200) were purchased…”, among many others).

- The introduction mentions some studies on the preparation of PVA films, but the utilization of cyanobacteria in the context of materials development is neglected. It is important to discuss strategies that were previously reported. In case there is nothing in the literature, the originality must be emphasized.

- Materials and methods: p. 5, l. 86: “After screening, 100 orders of cyanobacteria powder…”. What kind of screening was conducted? What does 100 orders mean? The text must be clear enough to be reproduced by other researchers. Table 2 is not clear. What does CM, CY, CNF and PVA mean? What about the other compounds used in the films formulation (poly(ethylene glycol), guanidine hydrochloride, and carboxymethyl cellulose)? In fact, the reason why these compounds were added, and the amounts chosen must also be explained. The authors conducted water resistance tests, based on the statement: “The swelling of a sample is usually used to indicate the water resistance of a material.”. However, there is no ASTM or any kind of reference to support it. PVA is soluble in water, and therefore the concept of swelling is not correct in this case.

- Results: Table 3 includes results of moisture content, but the methodology for this test was not described in the appropriate section. FTIR of isolated and dried cyanobacteria must be included. The values present in Table 4, Table 5 and Table 6 come from only one measure of each sample? It is important to do the test in triplicate and to present the standard deviation. Why oxygen permeability was conducted only for samples 7-10?

- Reference list is not standardized.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

7 Jan 2020

PONE-D-19-24904

Preparation of cyanobacteria-enhanced poly(vinyl)alcohol-based films with resistance to blue-violet light/red light and water

PLOS ONE

Response to Reviewers

Dear Editors and Reviewers,

Thank you for the time and effort you have spent reviewing our paper. We are pleased to note that you have found our research work interesting and also pointed out some problems to help us improve the quality of our work.

Motivated by your comments, we have deeply reconsidered the architecture of our work and tried to fix all the problems you mentioned. In particular, the revised manuscript of our resubmitted letter has significantly been improved mainly as follows:

Improvement-1: We have modified our manuscript format based on the PLOS ONE style templates.

Improvement-2: i) We obtained a cyanobacteria field permit and commissioned the staff of the algal and water separation station to perform the field sampling for this study. The station is located on the western bank of Taihu lake. We provided further details of the cyanobacteria field sampling in line 86-100. We characterised and identified the FTIR spectra of cyanobacteria samples in Fig 2.

ii) We would like our manuscript to be considered for the Plastics in the Environment Call for Papers, as specified in the cover letter.

Improvement-3: Because all data and other information is already presented in the tset, there is no supporting information section.

Improvement-4: We have revised the manuscript based on the reviewers’ comments (repeated below in italics for your convenience). Our response to each point is as follows:

Reviewer #1: PLOS ONE

Title: Preparation of cynaobacteria-enhanced poly(vinyl)alcohol-based films with resistance to blue-violet light/red light and water

The manuscript describes the preparation of composites based on cyanobacteria incorporated into poly(vinyl)alcohol and cellulose nanofibers/poly(vinyl)alcohol matrices. The scope is interesting once the authors suggest the use of the environmentally harmful cyanobacteria as to generate useful materials. The results and discussion are clear and well described. Some suggestions are listed below in order to improve the reading and reproducibility of some important points.

The title suggested the resistance to blue-violet light / red light and water are important properties, however it was not discussed in the text. I strongly suggest to include the importance of these features. It can be included in the introduction, or in results and discussion.

Our response:

Thank you for your nice suggestion. We added the discussion of these important properties in line 264-267.

Line 69 – “In our early elemental analysis test, the contents of carbon, nitrogen and hydrogen in the cyanobacteria sample were 45.0 2%, 5.98 % and 9.11 %, respectively.”. Do the authors have already published these data? If positive, please include the reference. If not, please discuss in the results section why it is important.

Our response:

These data have not been published before, which came from the extra tests we did in the research. They were put into the introduction to provide more information about cyanobacteria. We decided to delete these data after careful conideration to avoid unnecessary misunderstandings.

Table 2- It is need to include a column with the weight content (wt %) of each component of the composite to facilitate the understanding of the results.

Our response:

The weight content (wt%) of the modified cyanobacteria (CM) was added to help understand the results in Table 2. The wt% of CM in film No.1-6 is CM to poly(vinyl)alcohol. The wt% of CM in film No.7-12 is CM to cellulose nanofibers/poly(vinyl)alcohol.

Line 134- What is the meaning of CM?

Our response:

Thank you for pointing out our negligence. The meaning of CM is the modified cyanobacteria, and a description was added to line 136.

Lines 281-282- please convert the mass values em wt % values, in agreement with the new suggested wt % column from table 2.

Our response:

Thanks for your suggestion. We have modified it.

Please include a short text correlating the water resistance and swelling ratio with the surface hydrophobicity of the PVA-based films.

Our response:

Thank you for pointing out the problem. We added a short text to illustrate this in line 278-279.

How difficult is to collect the cyanobacteria from the sea water? Could be interesting to insert a short text explaining as it could be feasible.

Our response:

Thanks for your suggestion. We inserted a short text explaining of collecting cyanobacteria in line 90-100.

Typing errors:

line 67 -utilizerecycle

131- bsed

line 250 – wto

304 - filmgoes

Our response:

Thank you for your careful attention to our typing errors. We have corrected these.

Reviewer #2: In the present manuscript, the authors described the preparation of poly(vinyl alcohol) (PVA)/nanocellulose composites incorporating cyanobacteria in order to give the materials better light and water resistance. The study is of interest, but some modifications are needed before considering its publication in PLOS One.

- The writing must be reviewed, as some mistakes are present (p. 3, l. 57: “…utilizerecycle cyanobacteria…”; p. 3, l. 62: “The other method of resource utilizing cyanobacteria…”; p. 5, l. 87: “Polyethylene glycol (molecular weight, 200) were purchased…”, among many others).

Our response:

Thank you for your careful attention to our writing mistakes. We have corrected these.

- The introduction mentions some studies on the preparation of PVA films, but the utilization of cyanobacteria in the context of materials development is neglected. It is important to discuss strategies that were previously reported. In case there is nothing in the literature, the originality must be emphasized.

Our response:

Thank you for your nice suggestion. We added thedescriotion of the originality of cyanobacteria used directly in the preparation of composite films.

- Materials and methods: p. 5, l. 86: “After screening, 100 orders of cyanobacteria powder…”. What kind of screening was conducted? What does 100 orders mean? The text must be clear enough to be reproduced by other researchers.

Our response:

Thank you for pointing out the problem. We screened cyanobacteria for particle size. “order” is a writing mistake and has been modified to “mesh”.

Table 2 is not clear. What does CM, CY, CNF and PVA mean?

Our response:

Thank you for pointing out the problem. We have modified Table 2. CM, CY, CNF and PVA are abbreviations of modified cyanobacteria, cyanobacteria, cellulose nanofibers and poly(vinyl)alcohol. Their meanings are mentioned in line 136, 51, 47 and 33, respectively.

What about the other compounds used in the films formulation (poly(ethylene glycol), guanidine hydrochloride, and carboxymethyl cellulose)? In fact, the reason why these compounds were added, and the amounts chosen must also be explained.

Our response:

The reasons why these compounds are putted in line 76-80, and the explanation for the amounts chosen is putted in line 131-133.

The authors conducted water resistance tests, based on the statement: “The swelling of a sample is usually used to indicate the water resistance of a material.”. However, there is no ASTM or any kind of reference to support it. PVA is soluble in water, and therefore the concept of swelling is not correct in this case.

Our response:

Thank you for pointing out the problem. We added an ASTM standard to support it in line 180.

- Results: Table 3 includes results of moisture content, but the methodology for this test was not described in the appropriate section.

Our response:

The methodology for this test was added in line 180-184.

FTIR of isolated and dried cyanobacteria must be included.

Our response:

FTIR of isolated and dried cyanobacteria is labeled “CY” in Fig 2,

The values present in Table 4, Table 5 and Table 6 come from only one measure of each sample? It is important to do the test in triplicate and to present the standard deviation.

Our response:

i)Thank you for your suggestion. The standard deviation have been added in Table 6.

ii) The transmission test is to show that the poly(vinyl)alcohol-based films greatly enhances the barrier properties to blue-violet light/red light after mixing cyanobacteria in Table 4. The contact angle test is to show that the poly(vinyl)alcohol-based films greatly enhances the surface hydrophobicity in Table 5. Because the results of these two tests are special effective and do not pursue overly detailed values, only one test was done.

Why oxygen permeability was conducted only for samples 7-10?

Our response:

We screened representative samples for oxygen permeability testing based on previous test results. In addition, because the oxygen permeability of samples 7-10 is regular, other samples were not tested.

- Reference list is not standardized.

Our response:

Thank you for your careful attention to our manuscript. We have corrected these.

We hope this paper is suitable for PLOS ONE and it can help a little bit for people studying similar fields.

We deeply appreciate your consideration of our manuscript. If there are any other modifications we could make, we would like very much to modify them. We look forward to receiving comments from the reviewers. If you have any queries, please don’t hesitate to contact me at the address below.

Thank you and best regards.

Yours sincerely,

Changyan Xu

Corresponding author:

Name: Changyan Xu, Sainan Chen

E-mail: changyanxu1999@163.com, csn@jshb.gov.cn

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

24 Jan 2020

Preparation of cyanobacteria-enhanced poly(vinyl)alcohol-based films with resistance to blue-violet light / red light and water

PONE-D-19-24904R1

Dear Dr. Xu,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Antonio José Félix Carvalho, Ph.D.

Academic Editor

PLOS ONE

31 Jan 2020

PONE-D-19-24904R1

Preparation of cyanobacteria-enhanced poly(vinyl)alcohol-based films with resistance to blue-violet light / red light and water

Dear Dr. Xu:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Antonio José Félix Carvalho

Academic Editor

PLOS ONE