Process optimisation for green synthesis of ZnO nanoparticles and evaluation of its antimacrofouling activity

Abstract

In recent years, considerable attention has been given to the plant‐mediated synthesis of nanoparticles because it is an eco‐friendly method compared to the synthesis by chemical route. This study aims to optimise the biosynthesis of zinc oxide nanoparticles (ZnO‐NPs) mediated by coconut water using response surface methodology (RSM). The effects of the individual variables (concentration of coconut water, temperature and time) and their interactions during the biosynthesis of ZnO‐NPs were determined by RSM employing Box–Behnken design. The variables selected were tested by a 17‐run experiment and quadratic model was used for the analysis of the results. The accuracy of the model was confirmed by the coefficient of determination (R ²) value of 0.9968. The significance of the regression model was found to be high which is validated by the low probability value of P  < 0.0001. The ZnO‐NPs thus synthesised was evaluated for its antimacrofouling activity against mollusks using in‐vitro foot‐adherence bioassay. The results demonstrated the potential of biosynthesised ZnO‐NPs in inhibiting fouling induced due to the test organisms.

1 Introduction

Nanomaterials have been widely used in several fields owing to their exceptional properties. Due to their unique properties, metal oxide nanoparticles have driven substantial interest in various fields including physics, chemistry, biotechnology, material science and environmental technologies [1, 2]. Zinc oxide nanoparticle (ZnO‐NP) one of the widely used metal oxide nanoparticles with exclusive physico‐chemical properties, is considered as a potential material for biological applications. Green synthesis of ZnO‐NPs employing plants has developed as an innovative, environmentally friendly alternate to the current conventional methods. To achieve better yield, increased stability and smaller particles in the synthesis of nanoparticles, optimisation of process parameters are considered to be one of the most significant steps.

Response surface methodology (RSM) is the most accepted technique used in the optimisation of important variables accountable for the production of biomolecules. It finds successful application in chemical, food and biological processes to optimise the conditions [3]. The advantages of using RSM are saves space, time and raw material. Also, it yields the mathematical model that precisely defines the complete process apart from investigating the influence of independent variables. Optimisation of different parameters in the synthesis of nanoparticles using RSM has been stated by several other researchers [4, 5, 6]. Physicochemical parameters such as pH, temperature, incubation time have been identified as significant parameters in our previous research for the microbial‐mediated synthesis of silver nanoparticles using RSM [7].

Nanotechnology could considerably solve several technological and environmental issues especially in the areas of catalysis, water treatment, solar energy conversion, and medicine. Especially metal oxide nanoparticles such as ZnO‐NPs find wide applications in several fields of science because of their unique properties. In this report, the biosynthesised ZnO‐NPs were evaluated for antimacrofouling activity against mollusks (marine macrofoulers) using a reliable and rapid in‐vitro ‘foot‐adherence bioassay’. Mollusks firmly adhere to the rocky surfaces or hard substratum using their broad flat foot and thus causing widespread fouling and biodeterioration to the submerged structures. Two mollusks viz. Patella sp. (limpet) and Trochus sp. were chosen as model organisms for determination of antimacrofouling activity of the biosynthesised nanoparticles. Further, the larvae of brine shrimp (Artemia salina) were chosen as model crustacean fouling organism and the anti‐crustacean assay was performed.

In the previous research study, we have synthesised and characterised ZnO‐NPs using coconut water as an eco‐friendly reducing agent and studied the antimicrofouling activity against marine biofilm forming bacteria [8]. The objective of this study is to use Box–Behnken design (BBD) for optimising the process parameters to increase the synthesis of ZnO‐NPs. The antimacrofouling assay for the synthesised ZnO‐NPs was studied against marine macrofoulers.

2 Experimental section

2.1 Synthesis of ZnO‐NPs using coconut water

Our previous study reported the biosynthesis of ZnO‐NPs using coconut water [8]. The biosynthesis of ZnO‐NPs was confirmed by various characterisation such as UV spectroscopy, XRD and transmission electron microscopy, which showed that the nanoparticles were in size range of 20–80 nm. As a continuation of the previous study, the optimisation of process parameters for the biosynthesis of ZnO‐NPs has been carried out in this study.

Briefly, coconut water (50% solution) was used to prepare a 100 mM zinc nitrate by constant stirring using a magnetic stirrer. The solution was stirred vigorously at 150°C and for a period of 5–6 h under reflux. The solution was then cooled to room temperature and the solid white precipitate obtained was centrifuged, washed and dried for 48 h in a hot air oven.

2.2 Process optimisation for the green synthesis of ZnO‐NPs

A 17‐run experiment of BBD with three variables and three levels was used to evaluate the influence of the variables and the interaction between these variables which influence the synthesis of ZnO‐NPs. The concentration of coconut water (A); temperature (B); time (C) are the three variables which have been identified to have the potential effect on the response function. Table 1 depicts the Box–Behnken matrix of 17‐run experimental design. The three different factors selected in this study were represented as A, B and C and were analysed at three levels coded −1, 0 and +1 for the low, medium of intermediate and high value, respectively. All the experiments were carried out in duplicates and the average of the yield of ZnO‐NPs was considered as a response. A second‐order polynomial model is utilised to correlate the functional relationship between the response function and the independent variable and the equation is expressed as follows:

$$\begin{array}{rl}Y& =\beta 0+\beta 1A+\beta 2B+\beta 3C+\beta 1\beta 1A2+\beta 2\beta 2B2\\ & +\beta 3\beta 3C2+\beta 1\beta 2AB+\beta 1\beta 3AC+\beta 2\beta 3BC\end{array}$$ (1)

where Y is the measured response, β 0 is the intercept, β 1, β 2, β 3 are the linear coefficients, β 1β 1, β 2β 2, β 3β 3 are the quadratic coefficients, β 1β 2, β 1β 3, β 2β 3 are the interactive coefficient, ABC are the independent variables. Design expert statistical software (Version 7.0, Stat‐Ease, USA) was used to calculate and analyse the second‐order polynomial coefficients.

Table 1.

Actual design matrix presented in terms of coded units for the biosynthesis of ZnO‐NPs

Std	Run	Factor 1	Factor 2	Factor 3	Response 1
		A:A concentration of coconut water, %	B:B temperature, °C	C:C time, h	R1 ZnO yield, mg/l
2	1	1.000	−1.000	0.000	160
15	2	0.000	0.000	0.000	660
1	3	−1.000	−1.000	0.000	110
13	4	0.000	0.000	0.000	660
8	5	1.000	0.000	1.000	360
10	6	0.500	1.000	−1.000	140
11	7	0.500	−1.000	1.000	300
12	8	0.500	1.000	1.000	270
4	9	1.000	1.000	0.000	140
9	10	0.500	−1.000	−1.000	100
5	11	−1.000	0.000	−1.000	90
7	12	−1.000	0.000	1.000	140
17	13	0.000	0.000	0.000	660
6	14	1.000	0.000	−1.000	140
3	15	−1.000	1.000	0.000	170
16	16	0.000	0.000	0.000	660
14	17	0.000	0.000	0.000	660

2.3 Antimacrofouling activity of tetraethoxysilane (TEOS) sol–gel (TESGs) coatings

2.3.1 Test organisms

The test organisms used in this study viz. Patella sp. and Trochus sp. These common fouling organisms which are found on the rocky surfaces were collected from Mahabalipuram coast of Kanchipuram District in Tamil Nadu, India. The cysts of Artemia salina (brine shrimp) cysts were obtained from a local aquarium and allowed to hatch in nutrient‐enriched sea water in aerated condition for 24 h. From the hatching tank, the active first instar larvae were collected using a capillary tube. These larvae were used for the anti‐crustacean assay.

2.3.2 Preparation of TESGs coatings on substrates

TESGs were prepared and used as the matrix to incorporate the biosynthesised ZnO‐NPs in our previous study [8]. These coatings showed the potential of ZnO‐NPs in controlling the biofilm formation by bacteria isolated from the marine environment. In this study, ZnO‐NPs doped TEOS sol–gels (ZNSG) synthesised by a similar method was employed to study the antimacrofouling activity. Initially, glass Petri plates (test substrates) of 100 mm diameter were washed and surface sterilised before coating. The prepared TESGs at different concentrations were spin coated at 1000 rpm onto Petri plates and subjected to curing at a temperature of 70°C for 24 h. These test substrates (coated Petri plates) were employed to evaluate the antimacrofouling activity by mollusk foot‐adherence assay and anti‐crustacean assay.

2.3.3 Mollusc foot‐adherence assay

The effect of ZnO‐NPs on the settlement of macrofoulers was determined by mollusk foot‐adherence assay. This study is based on the adherence of the mollusk foot to the substrate by the spreading and shrinkage concerning the ZnO‐NPs. The experiment was carried out in triplicates and the fouling percentage and regaining ability of mollusks when exposed to various concentrations of TESGs coatings were determined. The assay plates were filled with filtered seawater (to one‐third level) and the test organisms (five animals per plate) were introduced into it and placed over an illuminated glass surface. The organisms were observed for foot reflex and mobility till the foot was entirely shrunken. The fouling percentage was estimated depending on the extent of spreading and shrinkage of the foot. After introducing the treated organisms in fresh seawater, the spreading of their foot was observed and thus the percentage of fouling was determined.

2.3.4 Anti‐crustacean assay

The repelling effect of the prepared TESGs coatings was studied against the crustacean fouling organisms. The active first instar larvae of Artemia, the model organism, collected from the brighter part of the hatching tank were introduced into the test substrate plates filled with the brine solution (10 ml). The number of surviving larvae after 24 h of exposure was noted.

3 Results and discussion

3.1 Process optimisation for green synthesis of ZnO‐NPs

The results which were acquired in terms of the response, ZnO‐NPs yield (mg/l) for the synthesis of ZnO‐NPs have been provided in Table 1. The three individual parameters (concentration of coconut water, temperature, time) were optimised in the present BBD design with a total of 17 runs and the results were analysed using the quadratic model. From the BBD runs, it was found that the second‐order polynomial quadratic equation shows the best fit, and is expressed as follows:

$$\begin{array}{rl}Y& =1.50-0.31A-0.097B-0.32C+0.075AB\\ & -0.032AC-0.058BC-0.94A2-0.28B2-0.58C2\end{array}$$ (2)

An F ‐value of 240.89 obtained from the present model specified the significance of the analysed quadratic model. Table 2 shows the results of ANOVA analysis and the ‘Probability > F ‐value’ is indicative that the model terms are significant. Further, Pred R ² and Adj R ² were in the reasonable agreement which endorses the significance of the quadratic model used. Fractional factorial design such as BBD with a restricted number of experiments can give information concerning parameter interactions [9]. In a study, Varshosaz et al. [10] applied central composite design to minimise the particle size and maximise the drug loading efficiency of solid lipid nanoparticles for controlling the drug release in drug delivery systems. In another study, Ganea et al. [11] have optimised poly (d,l‐lactide‐co‐glycolide) (PLGA) synthesis by CCD. Similarly, the lactase enzyme immobilisation into the AuNPs was optimised with RSM by Dwevedi et al. [12]. Hence in this study, to enhance the synthesis of ZnO‐NPs, Box–Behnken experimental design have been utilised for optimisation of various process parameters.

Table 2.

Results of ANOVA for response surface model

Source	Sum of squares	df	Mean square	F value	P ‐value	Prob > F
model	8.977×105	9	99,742.60	88.46	<0.0001	significant
A–A‐concentration of fruit extract	10,512.50	1	10,512.50	9.32	0.0185	—
B–B‐temperature	888.89	1	888.89	0.79	0.4041	—
C–C‐time	30,160.56	1	30,160.56	26.75	0.0013	—
AB	1701.39	1	1701.39	1.51	0.2590	—
AC	7605.56	1	7605.56	6.75	0.0356	—
BC	1225.00	1	1225.00	1.09	0.3319	—
A2	2.314×105	1	2.314×105	205.22	<0.0001	—
B2	2.579×105	1	2.579×105	228.74	<0.0001	—
C2	1.857×105	1	1.857×105	164.68	<0.0001	—
residual	7893.06	7	1127.58	—	—	—
lack of fit	7893.06	3	2631.02	—	—	—
pure error	0.000	4	0.000	—	—	—
cor total	9.056×105	16	—	—	—	—

Fig. 1 shows the response surface plot for the green synthesis of ZnO‐NPs, concerning temperature and concentration of fruit extract whereas time remained unchanged at the central value. To attain the maximum yield, the predicted variables obtained from the BBD model are a concentration of fruit extract 50%, temperature 120°C and the time was 6 h. The interaction existing between time and concentration of fruit extract on ZnO‐NPs synthesis has been shown in Fig. 2. Initially, the OD value was found to increase with an increase in the concentration of fruit extract, and then it progressively decreased. Correspondingly, the influence of the boundary between temperature and time on the yield of ZnO‐NPs is shown in Fig. 3. The variation inclination of the 3D response concerning temperature and time showed an increase and later decrease. Also, the perturbation plot of the three independent variables is presented in Fig. 4. The results reveal that factor C (time) had more effect on the yield of ZnO‐NP than factors A and B.

Fig. 1.

3D response surface curves of ZnO‐NPs yield, the interaction of temperature, the concentration of coconut water

Fig. 2.

3D response surface curves of ZnO‐NPs yield, of time, the concentration of coconut water

Fig. 3.

3D response surface curves of ZnO‐NPs yield, the interaction of temperature and time

Fig. 4.

Perturbation plot

3.2 Mollusc foot‐adherence assay

The results of this assay revealed that the fouling percentage caused by the Patella sp. reduced as the concentration of ZnO‐NPs increased from 100 to 160 µg/ml. Fouling was completely inhibited at 160 µg/ml concentration and 28% of the organisms showing regaining ability (Fig. 5). Analogous effects were witnessed when tested with Trochus sp., and complete inhibition of fouling was noticed at a concentration of 160 µg/ml. The regaining ability was found to be 22% (Fig. 6).

Fig. 5.

Percentage of fouling and regaining of Patella sp. due to ZnO‐NPs

Fig. 6.

Percentage of fouling and regaining of Trochus sp. due to ZnO‐NPs

3.3 Anti‐crustacean assay

Artemia salina, a crustacean model test organism was used to assess the repulsive property of TESGs coatings. It was observed from the results that the cytotoxic effect of TESGs coatings was significantly high. The mortality percentage of the larvae was found to increase with an increase in the concentration of ZnO‐NPs (Fig. 7). Marine macrofoulers including barnacles, mussels and mollusks have been reported to cause serious operational problems to maritime industries largely influencing the activities of thermal power plants, shipping, aquaculture systems, and other submerged structures.

Fig. 7.

Anti‐crustacean activity of biosynthesised ZnO‐NPs on the larvae of Artemia salina

Antifouling is the process of preventing the settlement of fouling organisms on submerged surfaces using various antifouling agents. Numerous compounds with antifouling property obtained from sponges, algae and other marine organisms have been studied [13, 14]. The biological activities of marine macrophytes against fouling organisms have been demonstrated by Chen et al. [15]. The antifouling properties of Syringodium isoetifolium and Cymodocea serrulata against micro and macrofoulers have been reported by Iyapparaj et al. [16]. In recent years, intense research is focused on nanoparticles due to their vast application in various fields. Although numerous studies have been carried out to elucidate the antifouling potential of nanoparticles, research studies concentrating on the use of nanoparticles as antimacrofouling agents are scanty. Dineshram et al. [17] have assessed the application of metal oxide nanoparticle coatings for prevention of macrofouling in marine environment. Chapman et al. [18] demonstrated the antifouling potential of period four metal nanoparticles at the early stages of development. Another study reported the antifouling activity of nano, micro and macro form of copper and concluded that a nano form of copper had the highest antifouling activity [19]. A recent study reported the possible application of two‐dimensional Ti₃ C₂ T_x (MXene) nanosheets for inhibition of fouling in water treatment. The coatings showed excellent antibacterial activity against E. coli and Bacillus subtilis [20]. In this study, antimacrofouling potential of green synthesised ZnO‐NPs was tested against mollusks (marine macrofoulers) using a reliable and rapid in‐vitro ‘foot‐adherence bioassay’. The results showed complete inhibition of fouling with the use of biosynthesised ZnO‐NPs.

4 Conclusions

In this study, process parameter optimisation using RSM was carried out to enhance the synthesis of ZnO‐NPs. The model showed an R ² value (coefficient of determination) of 0.9968. The high significance for the regression model is demonstrated by the probability value (P <0.0001). The antimacrofouling activity of the biosynthesised ZnO‐NPs was demonstrated with marine macrofouling organisms. The results showed that biosynthesised ZnO‐NPs could bring about complete inhibition of fouling caused by the test organisms. Thus this study gives room for the promising development of ZnO‐NPs based antifouling coatings to control marine biofouling.