Bioaugmentation and Bioaugmentation – Assisted Phytoremediation of Heavy Metals Contaminated Soil By a Synergistic Effect of Cyanobacteria Inoculation, Biochar, and Purtolaca Oleracea


 In recent decades, soil contamination with heavy metals has become an environmental crisis due to their long-term stability and adverse biological effects. Therefore, bioremediation is an eco-friendly technology to remediate contaminated soil, that its efficiency requires further research. This study was conducted to comparatively investigate two strategies, including bioaugmentation by using Oscillatoria sp and bioaugmentation assisted phytoremediation by using Oscillatoria sp -portulaca oleracea for the bioremediation of heavy metal (Cr (III), Cr (VI), Fe, Al, and Zn) contaminated soil at 180 days. To facilitate the remediation process, various quantities of biochar (0, 0.5, 2, and 5% (w/w)) were used in the experiments. The results of the bioaugmentation showed a significant improvement in chlorophyll a, nitrogen, organic carbon contents of soil and decrease all heavy metal bioavailability and EC of soil. The remediation efficiency test using plants proved the success of remediation treatments. Moreover, the findings of bioaugmentation-assisted phytoremediation displayed an improvement in soil fertility and a substantial reduction in the bioavailable fraction of heavy metals, especially in soil amended with 5% biochar. Cyanobacteria inoculation and biochar amendment dramatically enhanced the root lengths and shoot heights of portulaca oleracea while it significantly decreased their heavy metal accumulation compared to the control. For all heavy metals, TF and BAC (except Zn) values ​​were found to be less than 1.0 at all treatments, illustrated the successful phytoextraction by the P. In conclusion, cyanobacteria inoculation along with biochar addition enhanced the TI quantities while diminished BAC and BCF values, suggesting the feasibility their applying in heavy metal contaminated soil for the facilitation of phytoremediation and their ability in pollutant immobilization.


Introduction
Contamination with heavy metals is a dangerous environmental issue as the mining processing, and use of these elements has enhanced in different world regions (Dhal et al. 2013). Heavy metals are one of the primary pollutants, which exist in the soil for many years. These pollutants are toxic and pose a severe threat to food safety, human and environmental health due to their accumulation in the food chain (Song Pseudomonas sp., Enterobacter sp. ) enhanced the soil fertility and metal uptake by the plant due to its synergistic effects (Chen et al. 2019, Sanchez-Hernandez et al. 2019, Tu et al. 2020, Wu et al. 2019). However, the combination mechanism between microorganisms and biochar for the transformation or stabilization of heavy metal in the soil is not well understood. There is also a research gap in the effect of biochar on microbial assisted phytoremediation, especially from the aspect of plant growth and heavy metal bioavailability.
In this study, Oscillatoria sp, a lamentous cyanobacteria species, purtolaca oleracea, and biochar were employed as microbial, plant, and material agents to remediate soil contaminated with heavy metals (Cr (III), Cr (VI), Fe, Al, and Zn), respectively. However, to the best of our knowledge, interactions between biochar and cyanobacteria on heavy metal uptake by purtolaca oleracea grown on contaminated soil have not been reported. Hence, the speci c aim of this study is to: (1) evaluate the effect of cyanobacteria inoculation accompany with biochar amendment on the properties of soil and heavy metal bioremediation e ciency, (2) explore the in uence of Oscillatoria sppurtolaca oleracea -biochar partnerships on soil properties and heavy metal bioremediation e ciency, (3) assess the heavy metal accumulation in parts of purtolaca oleracea under metal stress and assisted remediated materials.

Contaminated soil description and characterization
The soil was collected from a contaminated industrial area in Mashhad, Iran (36° 29' 92" N, 59° 31'16" E). The sample was gathered from 0 to 40 cm of soil depth and air-dried at room temperature. After homogenizing and sieving (> 2 mm mesh), the soil texture was determined using a hydrometer method (Bouyoucos 1962), which was classi ed as loam (sand 50%, silt 28%, clay 22%). Soil pH and EC were characterized using pH meter (basic 20, Crison, Spain) and EC meter (4510, Jenway, England) with soil to distilled water ratio of 1: 25. The organic carbon (OC) and total nitrogen (N) contents of soil samples were determined by loss of ignition (Park et al. 2017) and the Kjeldahl method (Kirk 1950), respectively. Some of the important soil properties were as follows: pH; 9.05, EC; 121.8 dsm − 1 , OC; 0.022 g kg − 1 , N; 1120 mg kg − 1 , K; 101.7 mg kg − 1 , and P; 155 mg kg − 1 .
The concentration of heavy metals in soil was determined by the aqua regia digestion method (Alghanmi et al. 2015). 1 g of air-dried soil was mixed with 5 ml of HNO3 (65%) and 15 ml of HCl (37%) and stirred at room temperature for 12 h. After this, the obtained mixture was digested at 130°C for three h and then ltered. The heavy metal content of ltrate was measured by inductively coupled plasma-optical emission spectrometry (76004555, Spectro Arcos, Germany). The concentration of Cr (VI) was determined by using UV-VIS Spectrophotometer (DR5000-Hach, USA). The following results were acquired: Cr (III) ; 65.45 mg kg − 1 , Cr (VI) ; 12.71 mg kg − 1 , Zn; 104.07 mg kg − 1 , Al; 11287 mg kg − 1 , and Fe; 27405 mg kg − 1 .
Metal distribution in the soil samples was determined using the European Bureau of References (BCR) (Peña-Icart et al. 2011, Wuana et al. 2010). Three sequential extraction phases, including extractable (step1), reducible (step 2), organic-bound (step3), and residual (R), were conducted to attain various fractions of heavy metals in soil. According to this method, 1g of dried soil (105 ˚C for three h) was digested using sequential extractions (4 steps), which its detail as illustrated in Table 1. The heavy metal ions content of each fraction in soil was measured with the ICP instrument. Table 1 BCR sequential extraction procedure utilized for Cr, Zn, Al, and Fe speciation. Step Soil

Experimental design setup
All experiments were conducted in a greenhouse at the Ferdowsi University of Mashhad, Mashhad, Iran, from July to September 2019. Generally, two experiment series were designated: (1) bioaugmentation by cyanobacteria and (2) bioaugmentation-assisted phytoremediation by cyanobacteria and purtolaca oleracea. Biochar was used as an amendment in both series. Table 2 illustrates the experimental design, a 4× 2 factorial design, with three republications with four quantities of biochar (0, 1, 2, 5 % w/w) and two types of treatments (Osillatoria sp as microorganism and purtolaca oleracea as a plant).
For bioaugmentation testing, the Petri dishes with a diameter of 8 cm and a height of 1 cm, were lled with 80 gr of sterilized contaminated soil, which previously mixed with various amounts (0, 1, 2, 5 % w/w) of biochar. Then, 1.4 g (wet weight) of cyanobacteria separated from the culture medium by centrifugation at 1500 rpm for 15 min, mixed and homogenized with 20 ml of distilled water at room temperature for 24 h, was added to the soil surface using a sterile syringe. The inoculated plates were placed in the greenhouse at 2 ± 27°C under the light intensity of 1600 lux and light to a dark ratio of 16 h: 8 h. They were irrigated with 24 mL of distilled water every 24 hours.
For bioaugmentation-assisted phytoremediation examination, 1 kg of sterilized contaminated soil mixed with different ratios (0, 1, 2, 5 % w/w) of biochar and placed into several polyethylene pots with a total volume of 2 L (area of 133 cm 2 and depth of 10 cm). The drainage holes were embedded in the bottom of the pots to guarantee the health of the roots. The plant seeds were disinfected with ethanol 99.8 % and planted directly in the soils. 0.8 g (wet weight) of the cyanobacteria were added to the surface soil in the pots when the seeds germinated. The pots were placed in the greenhouse at 30 ± 5°C with a light/dark ratio of 16 h: 8 h and irrigated with 50 ml of distilled water every 24 h. After four weeks, the surface soil of each pot was also inoculated with 0.8 g of cyanobacteria mat uniformly. Twelve weeks after planting, the plants were harvested. After washing thoroughly with deionized water, they were dried at 70°C for 24 h and store in a polyethylene bag for future analysis. Table 2 Design of experiment

Plant analysis
After three months of planting, rst, the purtolaca oleracea plant was cut, and its stem heights and root lengths were measured. The plant parts were washed thoroughly with deionized water and then dried at 70°C for 24 h. The concentration of heavy metals (Cr (III), Cr (VI), Zn, Al, and Fe) in the roots and shoots of purtolaca oleracea were determined using HNO 3  (1)

Statistical analysis
The Kolmogorov-Smirnov test was utilized to test data normality and homogeneity of variance. The statistically signi cant difference between data among control and treatments was determined using one-way analysis of variance (ANOVA) followed by Tukey's and Duncan's test. The signi cant difference (p-value < 0.05) among various treatments was displayed by using different letters. Data analysis was performed using IBM SPSS Statistics version 20.

Characterization of Biochar
The surface morphology of biochar was observed by SEM analysis (Fig. 1a). From this image, regular pores similar to honeycombs can be seen on its surface, which indicates its ability the adsorption of heavy metals. This honeycomb structure was related to the carbon skeleton of biochar (Ghani et al.  Figure 2 showed the application Oscillatoria sp. in the heavy metal contaminated soil amended with various biochar. After three months of inoculation, cyanobacteria were progressively distributed and covered on the soil s surface ( Fig. 2 (b)). The SEM image, taken from the surface of inoculated soil, displays mineral particles, and soil fragments were coated by exopolysaccharides (EPS) assumed to r  a  t  i  o  n  o  f  h  e  a  v  y  m  e  t  a  l  s  i  n  t  h  e  s  h  o  o  t  C  o  n  c  e  n  t  r  a  t  i  o  n  o  f  h  e  a  v  y  m  e  t  a  l  s  i  n  s     . They utilized the roots and hypocotyls lengths and the VI of lettuce as a biomarker for bioremediation e ciency of soil contaminated with Cr (VI) and lindane. In general, our nding con rms that the bioremediation effectiveness of cyanobacteria and biochar amendment.

In uence of treatments on Portulaca oleracea growth
The effect of cyanobacteria inoculation and biochar amendment on Portulaca oleracea growth was measured (Table 4). By cyanobacteria inoculation in soils, the shoot and root lengths increased from 12.73 and 1.67 cm to 18.87 and 3.07 cm, respectively. This was attributed to the enhancement of nitrogen and organic carbon content of the soil in the presence of cyanobacteria, which promote plant growth (Rossi et al. 2017). Besides, with an increment of biochar dose from 0 to 5 percent in the soils, the shoot and root lengths grew 4.6 and 3 fold, respectively. This may be due to the decreased availability of heavy metals and improvement in nitrogen and organic carbon content (

Metal speciation in the contaminated soil
To explore the in uence of the cyanobacteria inoculation and biochar percentage on heavy metal speciation, the BCR method was carried out after bioremediation experiments. Their results for Cr (III), Cr (VI), Fe, Zn, and Al illustrates in Fig. 5. As shown in Fig. 5 (a), the concentration of Cr (III) in S1 fraction (water-and acid-soluble and exchangeable) reduced from 3.5 % in the sample with cyanobacteria inoculation (SCP) and to 90 % in the sample with the highest value of biochar (SCP5). Besides, the S1 fraction of Cr ( Figure 6 shows the concentrations of Cr (VI), Cr (III), Zn, Al, and Fe in shoots and roots of Portulaca oleracea depending on the treatment under different treatments. As shown in Fig. 6, the heavy metals concentrations in shoots and roots of Portulaca oleracea growing in the remediated soil were signi cantly (p < 0.05) decreased relative to the control. The metals accumulation in the root of Portulaca oleracea was higher than the shoot. In other words, the application of biochar and cyanobacteria inoculation signi cantly reduced the above-mentioned heavy metals concentrations in portulaca oleracea. The minimum concentrations of the shoot and the root metals were obtained when the 5 % biochar accompany with cyanobacteria (SCPB5) were used in the pots. Moreover, the highest decrease of -88.7 % and − 94.

Phytoremediation potential
The BCF, BAC, and TF were estimated for various treatments and were shown in Fig. 7. BCF indicates a plant's ability to accumulate heavy metals from soil, and TF shows a plant's ability to translate heavy metal from the roots to the shoots. The translocation of heavy metals from soil into the above-ground parts of the plant, such as the shoot, is called the phytoextraction process (Garbisu &Alkorta 2001). This process occurred in the plant when the BCF and TF > 1 (Nazir et al. 2011). In this study, for all heavy metals, TF values were found to be less than 1.0 at all treatments (Fig. 7). It has been proved that plants with TF more than 1 accumulate the metals in the aerial part while that with TF lower than 1 shows the lower ability for transferring the heavy metal from root to shoot (Arán et al . It was ranked as (1) excluder BAC < 1; (2) accumulator 1 < BAC < 10; and (3) hyperaccumulator BAC > 10 (Baker 1981). For Zn, the BAC was higher than 1 illustrates that the Portulaca oleracea has the potential to be an accumulator. For other metals, it was excluder due to having a BAC of less than 1.
Another factor was the tolerance index (TI), which analyzed the difference between the plant roots at the control and various treatments (Fig. 7). As shown in this gure, for all heavy metals, TI values were

Conclusion
In this study, two approaches, including bioaugmentation and bioaugmentation-assisted phytoremediation, were utilized for remediation of heavy metals (Fe, Cr (III), Cr (VI), Al, and Zn) contaminated soil. In bioaugmentation experiments, cyanobacteria inoculation accompany with an increment of biochar dose ( from 0 to 5 %) resulted in enhancement of the soil chlorophyll a, nitrogen, and organic carbon contents, reduction of the pH and EC, and also a signi cant decrease in the bioavailability of all heavy metals. Besides, evaluation of remediation e ciency with the help of lettuce and radish plants, after three months of treatment, con rmed the improvement of soil conditions. The results of bioaugmentation-assisted phytoremediation using biochar-cyanobacteria -Portulaca oleracea showed a substantial improvement in the above -mentioned properties of soil and a notable reduction in the bioavailability of all heavy metals, especially at a 5 % dose of biochar. Moreover, the Portulaca oleracea displays low translocation (TF < 1) for all heavy metals, immobilized, and accumulates the metals in root than shoot, which show their phytostabilization potential. According to the BCF values, the Portulaca oleracea showed an intensive tendency for Zn and Cr (III) uptake, a strong tendency for Cr (VI) absorption, and a weak tendency for Al and Fe. Although inoculation of cyanobacteria and the addition of biochar into contaminated soil promoted plant growth, it signi cantly reduced the phytostabilization potential of this plant. It can be concluded that the combined use of biochar and bioaugmentation with cyanobacteria  Figure 1