Production of a novel biomacromolecule for nanoelectronic devices and artificial retinas utilizing textile wastewater as carbon source: Optimization by Taguchi experimental design

Abstract

In this study, the ability of Haloarcula sp. IRU1 isolated from Urmia lake, Iran, for production of cruxrhodopsin as a biomacromolecule with nanofunction from textile wastewater was evaluated and optimized by Taguchi experimental design. According to Taguchi method, the optimum conditions at which high cruxrhodopsin production could be achieved were observed to be in the presence of textile wastewater at 0.25% (volume/volume), yeast extract at 0.025% (weight/volume) and KH₂PO₄ at 0.005% (weight/volume). In conclusion, Haloarcula sp. IRU1 is capable of cruxrhodopsin production from textile wastewater in different conditions.

Introduction

Retinal proteins are pigment–protein complexes, which contain retinal (vitamin A aldehyde) as the chromophore, and are invariably bound to biological membranes. The first knowledge retinal protein was the visual pigment rhodopsin, which exists in the rod photoreceptor membranes of the vertebrate eyes. Invertebrates also utilize retinal proteins as the visual pigment [1]. However, retinal proteins are not unique to the animal kingdom. In the 1970s, Oesterhelt and Stoeckenius discovered a new retinal protein in Halobacterium salinarium, which was named bacteriorhodopsin [1,2]. This biomacromolecule is an integral membrane protein with seven transmembrane α-helices and a chromophore group, retinal [3,4]. Although the primary function of bacteriorhodopsin is to pump protons, it is the ability of this biomacromolecule to absorb and convert light into energy that is of particular importance for use in photonic devices [5]. The light-driven photoreaction of bacteriorhodopsin is very similar to that of rhodopsin. Furthermore, the biomacromolecule shows highly efficient quantum conversion and excellent stability compared with other light-sensitive biological pigments [6]. Bacteriorhodopsin is used in the fabrication of micrometer and nanometer scale devices, structures and patterns for a wide range of applications such as biosensors [7–10]. The purple membrane with bacteriorhodopsin consisted of various intrinsic photochemical and photoelectric properties are well suited for exploitation in the artificial retinas [9].

Cruxrhodopsin (CR; MW ≈ 27,500) is a homologue of bacteriorhodopsin found in the species of genus Haloarcula (halophilic archaeon) [11,12]. In the present study, we report for the first time, the ability of Haloarcula sp. IRU1 isolated from hypersaline Urmia lake, Iran, in production of CR from textile wastewater as carbon source.

Experimental

Textile wastewater

The textile wastewater (from Kerepnaz textile factory, Kermanshah, Iran) contains multiple dyes and auxiliary chemicals such as Red PN3B, Red RB, Red 2G, Yellow GR, Yellow 4GNL, Orange 2R, Orange 3R, Scarlet GR, Blue GG, Blue GR, Blue RB, Black HFGR, Black NB Extra, Brown GR, Brown B, Na₂CO₃, NaCl and NaC₆H₇O₆. The pH and chemical oxygen demand (COD) of wastewater were 9.5 and 700 mg/ml, respectively.

Microorganism and growth conditions

The microorganism used in this study was Haloarcula sp. IRU1 isolated from hypersaline Urmia lake, Iran, cultivated in 100 ml Erlenmeyer flasks containing 20 ml of a basal medium and incubated in a shaker at 42°C and 120 r/min for 5 days under aerobic conditions. The basal salt medium contained (g/l) NaCl, 250; MgCl₂.6H₂O, 34.6; MgSO₄.7H₂O, 49.4; CaCl₂.2H₂O, 0.92; NaBr, 0.058; KCl, 0.5 and NaH₂CO₃, 0.17 in distilled water. The growth medium supplemented with various nutrient compositions by varying textile wastewater as carbon source at 0.25–2% (volume/volume [v/v]), yeast extract as nitrogen source at 0.05–0.4% (weight/volume [w/v]) and KH₂PO₄ as phosphorus source at 0.0005–0.004% (w/v) according to the details following the experiment design (Tables 1 and 2).

Table 1.

Setting of factors and their levels in experiment for CR production.

Factor	Parameter	Level 1	Level 2	Level 3	Level 4
A	Textile wastewater % (v/v)	0.25	0.5	1	2
B	Yeast extract % (w/v)	0.025	0.05	0.1	0.2
C	KH2PO4 % (w/v)	0.0005	0.001	0.002	0.004

CR: cruxrhodopsin; v/v: volume/volume; w/v: weight/volume.

Table 2.

The orthogonal array of Taguchi experimental design and corresponding CR production.

Trial	Factor A	Factor B	Factor C	CR (mg/l)
1	1	1	1	15.7
2	1	2	2	10.5
3	1	3	3	9.8
4	1	4	4	7.2
5	2	1	2	14.3
6	2	2	1	13.2
7	2	3	4	8.2
8	2	4	3	8.8
9	3	1	3	11.3
10	3	2	4	8.4
11	3	3	1	13.9
12	3	4	2	7.6
13	4	1	4	7.9
14	4	2	3	11.4
15	4	3	2	10.1
16	4	4	1	8.3

CR: cruxrhodopsin.

Determination of CR production

Cells from 10 ml culture broth were harvested by centrifugation and were lyzed by resuspending in equal volume (10 ml) of deionized water containing (0.01 mg) DNase (Fermentase). The lysate was homogenized and mixed with 4M NaOH and 4M NH₄OH in the ratio of (9:0.5:0.5, v/v) in the dark. The absorbance at 568 nm (A₅₆₈) was first measured in the dark (A₅₆₈⁰ ). The mixture was exposed to light for 24 h to remove retinal from membrane, and again the absorbance was measured (A₅₆₈²⁴). As the molecular weight of CR is 27.5 kDa and the molar extinction coefficient is 58,000 per M cm, the concentration of CR was determined by the following equation [13,14]:

$\text{CR}(g/l)=\mathrm{27\; 500}\times {\text{A}}_{568}^0-{\text{A}}_{568}^{24}/\mathrm{58\; 000}.$

Taguchi methodology experimental design

A factorial design using Taguchi method with three variables (carbon source, phosphorus source and nitrogen source) was examined in shake-flask cultivation. All the combination experiments using the assigned parameter values were conducted with the aim of obtaining the final optimum conditions. The Qualitek-4 software was used to design and for analysis of Taguchi experiments.

Results and discussion

The Taguchi experimental design differentiates between control factors and noise or uncontrollable factors and treats them separately by means of special design matrices called ‘Orthogonal Arrays’ (OA). Columns and rows of an OA are arranged in a fixed way indicating the combination of factor levels in each experiment to be run and allowing the simultaneous evaluation of several parameters with the minimum number of trials [15,16]. The Taguchi experimental design is a good option for optimization of biotechnological processes for microbial synthesis [17].

In order to attain maximum CR yield, three important variables including the carbon, nitrogen and phosphorus sources were simultaneously optimized by applying Taguchi statistical design. The matrix of the experiments, with factors levels and the response in terms of CR production are presented in Table 2. The range of CR was 15.7 mg/ml in run No. 1 (maximum) and 7.2 mg/ml in run No. 4 (minimum). Results indicate that Haloarcula sp. IRU1 give the highest CR production in the presence of textile wastewater 0.25%, yeast extract 0.025% and KH₂PO₄ 0.0005% (Table 2).

Analysis of variance (ANOVA) was carried out based on data from Table 2 in order to determine the significance of the control factors on CR production. The sum of square, variance and F-Ratio showed the highest value for KH₂PO₄ (104.54, 34.85 and 5.95, respectively), but textile wastewater and yeast extract had low sum of squares (4.25, 31.40), variances (10.47, 34.85) and F-Ratios (1.79, 5.95), respectively (Table 3). Then, KH₂PO₄ is the most significant in CR production.

Table 3.

Analysis of variance (ANOVA) of Taguchi experiments results.

Factor	DOF (f)	Sum of sqrs. (S)	Variance (V)	F-Ratio (F)	Pure sum (S)	Percentage (P; %)
Factor A	3	4.246	1.415	0.241	0	0
Factor B	3	31.401	10.467	1.788	13.84	7.894
Factor C	3	104.541	34.847	5.952	86.98	49.613

DOF: degrees of freedom.

The CR production under optimal conditions was estimated using the factors. Table 4 shows the optimum conditions for achieving highest CR production in terms of contribution for levels of factors. Among all the factors studied, KH₂PO₄ was important for CR production. The results indicate that the expected result and the total contribution from all factors under optimal conditions are 16.36 and 5.93, respectively. With these selected factors and levels, the grand average performance is 10.73%.

Table 4.

Point prediction for optimum conditions of CR production.

Factor	Level description	Level	Contribution
Factor A	3	3	0.693
Factor B	1	1	1.843
Factor C	1	1	3.093

CR: cruxrhodopsin.

Total contribution from all factors = 5.93.

Current grand average of performance = 10.73.

Expected result at optimum condition = 16.36.

Conclusions

This work demonstrated that Haloarcula sp. IRU1 can be a potential microorganism for production of CR from textile wastewater in extreme conditions, which makes the use of this microorganism even more promising for the industrial applications. In conclusion, by the production of CR from textile wastewater via this microorganism, the production cost of CR would markedly reduce, enhancing the economical feasibility of commercial applications of this potent biomacromolecule.