Simultaneous phycoremediation of petrochemical wastewater and lipid production by Chlorella vulgaris

A novel strategy of using microalgae Chlorella vulgaris for simultaneous bio-treatment of petrochemical wastewater and lipid production was developed in the present study. Phycoremediation was carried out in 30 days. The profile of fatty acids was identified, and the specifications of biodiesel including saponification value, iodine value, cetane number, long-chain saturated factor, cold filter plugging point, cloud point, allylic position equivalent and bis-allylic position equivalent were predicted by BiodieselAnalyzer® software. Besides, polycyclic aromatic hydrocarbons were determined in both wastewater samples and produced lipid. The observed data showed that biodiesel from C. vulgaris was superior to petrodiesel in terms of suitability in diesel engines. Moreover, contamination of petrochemical wastewater can influence the expression of a variety of genes in algae. To investigate the effectiveness of contamination on the expression of lipid synthesis as well as three photosynthesis genes, a real-time polymerase chain reaction assay was used to quantify transcript levels of PsaB (photosystem I reaction center protein subunit B), psbC (an integral membrane protein component of photosystem II), and rbcL (a large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase). Furthermore, the gene expression level of accD (acetyl-coenzyme A carboxylase carboxyl transferase subunit beta, chloroplastic) was studied to discover the effect of wastewater on lipid production. The results showed that when diluted petrochemical wastewater (50%) was used as a media for C. vulgaris cultivation, these genes expression significantly increased. For 50% diluted wastewater, the maximum removal of BOD, COD, total nitrogen, and total phosphor has been 30.36%, 10.89%, 69.89%, and 92.59%, respectively.


Introduction
Most of the industrial sectors cause severe negative impacts on the environment. Petrochemical industries are one of the major polluting industries [1]. In the phycoremediation method, algae are used to treat environmental contaminants [2]. Phycoremediation technology is economical, sustainable, energy-efficient, and highly proficient in removing pollutants from the water in comparison with the physical and chemical treatment lipid production as a feedstock for biodiesel and study the expression of the main genes involved in photosynthesis and lipid synthesis in the C. vulgaris cultivated in the petrochemical wastewater, including the large subunit of ribulose 1 and 5-bisphosphate carboxylase/oxygenase, psbC, psaB, and accD genes. The quality of produced lipid and biodiesel was also assessed in detail.
Wastewater samples were obtained from a petrochemical complex in Asaluyeh, Iran [26]. The composition of petrochemical wastewater is shown in Table 1. Four diluted wastewater levels of 25%, 50%, 75%, and 100% were tested to explore the optimum concentration of wastewater and the nutrient removal efficiency for C. vulgaris. Two different wastewater levels (50%, 100%) were used to investigate the genes' expression (the large subunit of ribulose 1 and 5-bisphosphate carboxylase/oxygenase, psbC, psaB, and accD genes) in the C. vulgaris cultivated in the wastewater.
The experiments were carried out at room temperature in 250 ml Erlenmeyer flasks, each bioreactor containing 200 ml of BG11 and wastewater samples, for daily cycles of 16 h light and 8 h dark [12]. The algae were grown for 30 days in triplicate Erlenmeyer flasks.

Biomass production and lipid extraction
At the end of the experiments, algal biomass was harvested by centrifugation at 3500 rpm for 15 min, and in order to determine dry weight biomass, the samples were lyophilized at-60 ° C to reach a constant weight (g L −1 day −1 ). The method of Bligh and Dyer [27] was used for the extraction of total lipids. The lipid of algae was extracted by transferring the sample into a glass tube, and then methanol: chloroform 2:1 was added to that and mixed for 30 min. This process was followed by shaking and spinning. After that, the lower layer separated and dried under nitrogen flow, and the total lipid concentration was determined gravimetrically [27].

Chemical analysis
Chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) were measured by a Hach DR 5000 Spectrophotometer (Hach Company, Loveland, CO, USA). QA/QC was carried out according to the analysis procedure. Moreover, five-day biochemical oxygen demand (BOD) was determined following the standard method [28]. The free fatty acid profile was analyzed using a Gas Chromatograph coupled with Flame Ionization Detector (GC-FID) (Agilent 6890; Agilent Technologies, Palo Alto, CA, USA). Polycyclic aromatic hydrocarbons (PAHs) were determined in both wastewater samples and the produced fatty acid. According to US-EPA (2018), samples were extracted by n-hexane. Determination of 16 US-EPA priority pollutant PAHs was performed by an Agilent 6890 gas chromatograph equipped with an Agilent 5970 mass selective detector. Besides, 48,953-U SUPELCO PAH Mix QA/QC was monitored as described by US-EPA procedures [26].

Characteristics of biofuel
Fatty acid content and composition were analyzed according to Breuer et al. [29]. As a gas carrier, helium was used at a constant flow rate of 1.1 mL/min. The temperature was programmed as follows: the initial temperature was held at 140 °C for 1 min, 4 °C per min ramp to 250 °C, and was held for 5 min. The temperature of the detector and the injection port was kept at 260 °C. Then, 1-µL of the sample was injected in spitless mode. SUPLCO standard of 18,919/18 was used to identify fatty acids.

Statistical analysis
Statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, IL, USA). In order to determine the statistical significance, a one-way analysis of variance (ANOVA) was used. When P was less than 0.05 or 0.01, the effects were significantly different. The Student's t-test was used for statistical significance between control and mean values of treated groups. Additionally, Duncan's Multiple Range tests were used to analyze the differences in relative expression levels of target genes.

Gene expression analysis
According to the manufacturer's instructions, total RNA was extracted from algae cells using TRIzol (Riboex GenAll, South Korea) and alcoholic deposition. Then, extracted RNA was verified by 1% agarose gel and measured via NanoDrop spectrophotometry (Thermo Scientific 2000c, USA) at 260, 280, and 230 nm. Afterward, extracted RNA was treated utilizing DNase I (Thermo Scientific, USA) to remove contamination of genomic DNA. Meanwhile, single-strand cDNA was synthesized by reverse transcriptase (Fast cDNA kit, Takara Japan) using 1 µg of total RNA. Herein, the oligo dT was performed as a primer to the synthesis of cDNA.
In this study, accD, psaB, psbC, rbcL were selected as target genes, and 18S rRNA (X13688) was selected as a housekeeping gene [30]. Sequences of these genes were collected from NCBI and aligned by Bioedit software [31]. The Oligo 7 software and Primer-BLAST tool were also utilized to design and check target primers. Primers designed for real-time PCR and annealing temperature are shown in Table 2.
Real-time PCR was carried out using the Stepone system (ABI stepone Real-Time PCR, USA), with the fluorescent dye SYBR ® Green (Takara SYBR Green Kit). The real-time PCR was run at 95 °C (10 min), 40 cycles at 94 °C (30 s), 58 °C (30 s), and 72 °C (20 s). Afterward, obtained data was analyzed using Livak and Schmittgen's [32] method (2 −ΔΔCt ) and normalized by multiplication efficiency. The mean comparison between different groups was tested based on the LSD test (P < 0.05).
To evaluate the cDNA quality, PCR was performed by primers of a housekeeping gene and query genes. Primers were designed based on exon-exon junction to avoid amplification of genomic DNA. According to Fig. 1, all of the studied genes illustrated the specificity of amplicons. Moreover, the melting curve analysis showed that all the primers are specific (Fig. 1, supplementary).

Phycoremediation
The ability of C. vulgaris for simultaneous growth in wastewater streams as well as total nitrogen and total phosphor removal has been assessed. The highest percentage of total phosphorus removal was observed at 25% wastewater. The best performance of C. vulgaris for BOD removal was observed at 50% diluted wastewater. The highest percentage of COD removal was detected at 75% diluted wastewater, as presented in Tables 3, 4. According to the results of the t-test, all the variables examined (except BOD at 75% concentration) had statistically significant differences at 99% and 95% confidence intervals level with control. The results of the Student's t-test and the initial concentration of pollutants can be found in the supplementary data.
Cai and co-authors [33] found out a total nitrogen removal efficiency of 79-100% by microalgae S. obliquus from municipal wastewater. Lim et al. [34] attempted to treat textile wastewater samples using C. vulgaris and removed N and P by 45% and 33%, respectively. A wide range of N (55-88%) and P (12-100%) removal was reported in the use of municipal wastewater as the waste stream [35][36][37][38].

Lipid and biomass production
C. vulgaris has been widely applied in the lipid production process employed in producing biofuels in the last decade. Since the media utilized in this research is free and available media, this is a practical approach.
The algal lipid composition was analyzed by GC-MS chromatography to determine PAHs in algal oil. Since PAHs were not detectable in all the samples, the lipid was used as a raw material for biodiesel production without any health and environmental concerns. ANOVA indicated no significant differences between biomass productions in BG11 and various diluted wastewater levels to evaluate the effect of diluted wastewater on biomass and lipid production. Biomass production was in the range of 0.402-0.470 g/L.D. For comparison matters, Duncan's multiple range test was also used. The comparison of the mean of the lipid content and biomass production in C. vulgaris by Duncan's method has been shown in Table 5. According to the results, the highest lipid content was observed in 50% of wastewater. Then it was decreased by 75% and 100% of wastewater.
Zhou et al. [39] reported that Chlorella sp. produced biomass and lipid from municipal wastewater in the range of 231.40-241.70 mgL −1 d −1 and 30.91-33.53 DW%. Chinnasamy et al. [40] achieved 23.00 mgL −1 d −1 biomass and 18.10 DW% lipids by Chlorella saccharophila from industrial wastewater. Ammonia nitrogen inhibited microalgae growth and reduced the utilization of wastewaters [41,42]. The primary mechanism of ammonia was to inhibit the microalgae by poisoning their photosynthetic system [43,44]. The higher algal lipid content for 50% diluted petrochemical wastewater conditions resulted from the existence of NH4+ -N in pure wastewater or having lower toxicity than 75% and pure wastewater.
The lipid accumulation using C. vulgaris is essential due to reduced expenses while producing a lipid that is a useful source to be utilized in biodiesel production. This approach is economically significant and may be developed as an applicable BECCS. The same approach has been recently suggested by Rosli and co-authors [45].

Characteristics of biodiesel
The results of chromatography showed that since the fatty acid profile generated by C. vulgaris contained a significant amount of palmitic acid (C16:0), stearic acid (C18:0), and palmitoleic acid (C16:1), oleic acid (C18:1), it could be used as promising biodiesel feedstock. The biodiesel's predicted  properties from C. vulgaris showed that they reached the standards developed by ASTM (Table 6). C. vulgaris was introduced as a suitable candidate for biodiesel production [46,47]. Ji et al. [48] used a piggery wastewater sample (TN: 56 ± 2 mg/L and TP: 13.5 ± 0.6 mg/L) as a medium for cultivation of C. vulgaris to integrate biofuel production and the treatment of piggery wastewater. They reported that C. vulgaris had good potential for treating wastewater as well as producing high oil content for lipid accumulation.

Quality control of produced biodiesel
PAHs were recognized as a mutagenic, carcinogenic, and teratogenic class of organic compounds, which was available in most of the wastewaters in the petroleum-related industries. Since the medium of algae was petrochemical wastewater in this study, PAH concentrations were determined in the produced lipid using gas chromatography/ mass spectroscopy. Each time, 16 US-EPA priority pollutant PAHs were measured. The results indicated that the biodiesel had outstanding quality, and PAHs were not detectable in all biodiesel samples. Presumably, C. vulgaris used carbon to accumulate lipids mainly by the source of CO 2 . Therefore, this type of produced biodiesel can be strongly proposed with no environmental concerns.

Gene expression
Since lipid composition and starch content vary among microalgae, not all are suitable for biofuel production. Some species can naturally biosynthesize and accumulate large amounts of starch and intracellular lipids, even further improved by genetic modifications, making them more useful in producing biofuels [49]. Statistical analysis of Duncan (Table 7) showed a significant difference in the relative expression of the accD gene in 50% and 100% wastewater treatments compared to the control group. Treatment with 50% of wastewater had the highest expression, as can be seen in Fig. 2a. The expression of rbcL and psbC also showed a significant difference between treatments, with high transcript abundance in 50% of effluent treatment (Fig. 2b, c). Based on the Ct value, this difference always exists. Likewise, the expression rate of psaB gene also illustrated the highest expression in 50% of wastewater compared to other treatments (Fig. 2d), while the Ct value showed no consistent difference as observed in the rbcL gene. According to the results of Sect. 3.2, increasing the expression of genes involved in the photosynthesis pathway at 50% wastewater leads to the lipid content increased at this concentration. Increasing the expression of genes involved in the lipid synthesis pathway is well analyzed by the data obtained from the optimization and the corresponding mathematical models. According to the results of this study, nitrogen was found as a potential-limiting factor, which is confirmed by the statistical model. In addition, the affluent environment has no inhibitory effects on the expression of genes involved in the photosynthesis pathway as well as those involved in lipid synthesis. Micro-algae stabilizes a considerable amount of carbon during photosynthesis; for example, the carbon fixation efficiency of C. vulgaris in the membrane photobioreactor is consistently 260 mg/ha [50]. Wawrik et al. showed that the expression of the ribulose 1 and 5 carboxylase/oxygenase (rbcL) bisphosphate in species of diatoms living in the shallow-water is less pronounced than those living in deep water [51]. In addition, Wan et al. reported the expression of large enzyme 1 and 5 carboxylase/oxygenase bisphosphate in different species of microalgae (Dunaliella salina and Nannochloropsis oculata, sorokiniana, Chlorella) under mixed culture conditions [52]. In this study, they realized that the expression of rbcL decreased in mixed culture conditions when glucose was added to the medium, which reduces the activity of rbcL and, consequently, reduces the process of photosynthesis. Yan et al. studied the expression of rbcL, psaB, and psbC genes involved in the photosynthesis process in Chlorella vulgaris species by adding glucose, atrazine, and diclofop-methyl herbicides, leading to decreased expression of psbC gene when these three herbicides are present, however, this expression was not significant. Nevertheless, the expression of rbcL and psbC genes decreased significantly. Moreover, they found realtime PCR as a reliable technique for assessing the toxicity of components in aqueous media [53].
According to Fan et al. [54] the expression of the rbcL gene in C. pyrenoidosa species in food deficiency conditions, including nitrogen, phosphorus, and iron, was inversely proportional to lipid content, proving the key role of this gene in photosynthesis and cell growth processes. However, the expression of the accD gene had a negative correlation between the expression of this gene and lipid content. Lee et al. examined the effect of nitrogen deficiency on the transcript abundance of accD gene and lipid content, and it was reported that the gene expression was directly related to lipid content [55].

Conclusion
Green algal Chlorella vulgaris was successfully employed for phycoremediation of petrochemical wastewater and lipid productivity at the same time. Biodiesel's physical and chemical characteristics demonstrate that biodiesel is a high-quality fuel and can easily adapt to diesel engines due to the sufficient similarities with petrodiesel, and it can be confidently recommended for diesel vehicles without a further engine change. PAHs were not detectable in all samples, which ensured the use of industrial wastewaters as an applicable source of alternative microalgae. The expression of accD in the wastewater environment was 40 times higher than that in the BG11 medium. In addition, the expression of this gene in 50% of effluent was significantly increased compared to 100% effluent and the BG11 environments. For 100% non-diluted wastewater (pure wastewater), the maximum removal of BOD, COD, total nitrogen, and total phosphor has been 21.63%, 10.41%, 37.67%, and 66.67%, respectively. Since many pollutants are emitted in the petrochemical wastewater, its application as a media for algae cultivation may change its function as a biomass production and lipid accumulation, as well as its potential for remediation, lipid composition, and expression of photosynthesis genes functions. Future studies should be directed toward applications of C. vulgaris for the treatment of wastewater from other industries, including food, beverage, agriculture, aquaculture. The biodiesel and petrodiesel blend should be developed for diesel consumer engines, which are presumably useful in capturing CO2 from the atmosphere.
Acknowledgments The authors would like to appreciate Mr. Ali Ahmadzadeh from Pars Special Economic Energy Zone (PSEEZ) for his support in sampling on the field.

Conflict of interest
The authors have no conflicts of interest to declare that are relevant to the content of this article.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.