Effective removal of fly ash by Penicillium chrysogenum and determination of direct fly ash toxicity with Daphnia magna

This study demonstrates the removal of fly ash with Penicillium chrysogenum, a newly isolated species of fungus, and acute toxicity assessment with Daphnia magna. In the study, two different removal mechanisms were compared, both bio-removal and bio-sorption. Six different ash and three different biomass concentrations were used simultaneously. Although other fungal species in the literature failed at such a high concentration of fly ash, P. chrysogenumwas able to tolerate it even at 10% concentration. The highest bio-removal yield was recorded as 100% at 0.5% fly ash concentration. Maximum bio-sorption yield was 95.27% after 24th hour. The evaluation results of fly ash bio-toxicity by D. magna showed that the no observed effect level (NOEL) was 0.2 mg/L and the low observed effect level (LOEL) was 0.5 mg/L. The element analysis, determined by ED-XRF, clarified that Ca, Si, Fe and S were the common elements in this ash. This is the first study in the literature where fly ash removal was carried out using P. chrysogenum for both bio-removal and bio-sorption and needs to be developed in the future.

Cost effective biological methods are used to reduce hazardous materials. One of the most effective microorganisms used in biological treatments are fungi, which are very effective microorganisms in heavy metal accumulation (Leung et al. ; Tasţan et al. ). Therefore, they are frequently preferred in biotechnological applications.
The use of fungi in the bio-removal process of heavy metals from fly ash is one of the most popular research areas in recent years (Xu & Ting ). Penicillium chrysogenum, a species of fungus mostly found in damp environments, is prefered in treatment studies due to its ease of culture, availability, low cost and adaptation to adverse conditions (Aranciaga et

Fly ash
Incineration fly ashes of different sizes (116,232,248,265,348 and 809 μm) were obtained from power plants in the Central Anatolia Region in Turkey. Fly ashes were quantified by preparing 10,000 ppm stock solutions continuously mixed with a magnetic stirrer, after which they were autoclaved at 121 C for 15 min before use (Tasţan ).

Culture conditions
The aim was to use a low cost culture medium in this study. Therefore, fungal biomass was inoculated into 250 mL Erlenmeyer flasks containing sterile fly ash in 100 mL T6 nutrimedia (Maysa, Turkey Rev: NMT6.01.0113) as a low cost industrial medium (Tasţan ). The pH of the growth medium was adjusted to 6.0 by adding diluted (0.01 M) and concentrated (1 M) sulfuric acid or sodium hydroxide solutions. The flasks were incubated at 25 ± 2 C on a rotary shaker (VWR 5000 Model Orbital Shaker) at 100 rpm for 7 days. In order to quantify the initial fungal biomass concentration, 0.5 g/L dry weight of fungal biomass was inoculated in 100 mL culture medium.
In some treatment studies, a more effective removal process was tested by using two types of culture stages (Wang et al. ). In the first type of culture stage, the fungus was inoculated into culture media at the start time (t ¼ 0 day) and at the same time sterile fly ash was added to the media (t ¼ 0 day), then they were incubated for 7 days. In the second type of culture stage, first the fungus was preincubated for 5 days (t ¼ 5 days) without fly ash and then sterile fly ash was added on the fifth day (t ¼ 5 days) and incubated for another 2 days. A schematic of the experimental setup is shown in Figure 1.

Isolation of microorganism
Cheese from Erzurum (Turkey) was selected as the inoculum and it was spread on potato dextrose agar (PDA) in Petri plates and incubated at 30 ± 1 C. After 7 days of incubation, fungal colonies were isolated and then purified by streaking the cells repeatedly on the PDA. The pure cultures were kept at 4 C and were transferred to fresh PDA every 3 months (Tasţan ).

Identification of new isolate with PCR and sequencing
Whole cells from an exponentially growing culture of the isolate were used for 5.8S rRNA gene amplification. The 5.8S rRNA region was amplified with primers as designed Taq DNA polymerase was used in the amplification. Amplification by PCR technique was carried out by an initial denaturation at 95 C for 4 min, followed by 35 cycles of denaturation at 94 C for 30 seconds, annealing at 57 C for 30 seconds, an extension at 72 C for 1 min, and a final extension of 72 C for 5 min. Applied Biosystems Genetic Analyzer 3130 was used in sequencing.

Bio-removal process
Four different culture media compositions were prepared for the fungal ash bio-removal process. (i) T6 nutrimedia (12 g/L), was calculated using Equation (1) as described below. An ash-free control group was used as the blank.
Acute immobilization and bio-toxicity assessment EC 50 is the concentration estimated to immobilize 50% of D. magna (daphnids) within a stated exposure period.
Immobilization is taken to mean those animals that are not able to swim within 15 seconds, after gentle agitation of the test vessel (OECD ). To detect the accute immobilization of fly ash, daphnids were used as the test species (Balusamy et al. ). The daphnids were cultivated at a stable temperature of 20 ± 1 C in 16:8 hours light:dark periods. Increasing concentrations of fly ash at 0, 0.2, 1, 5 and 10 mg/L were tested in the test medium. Bio-toxicity studies were conducted as three replicates and five daphnids were used for each concentration and for the control group.
After the 24, 48 and 72 hours, daphnids in each group were tested for immobilization.

Analytical methods
In the experiments 3 mL samples were taken daily for bioremoval experiments and taken hourly for bio-sorption experiments during the incubation period. Erlenmeyer flasks not containing fly ash were used as the control media.
The fungus to be used during calculations was passed through a series of steps as follows: i. After the incubation period was completed, the fungi were filtered through coarse filter paper.
ii. The fungi were washed to allow cells to separate from medium residues.
iii. The fungi containing ash and the fungi without ash (control media) were weighed separately according to their purpose as wet or dry weight and tared.
Fungal ash bio-removal and bio-sorption yields (Y %) and maximum specific fly ash uptake (q m ) was calculated using Equations (1) and (2); In these equations, q m is the maximum amount of fly ash taken up per unit dry weight of fungal cells (mg/g), X is dry biomass (g/L), C 0 is the initial fly ash concentration (mg/L) and C t is the final fly ash concentration (mg/L), respectively (Tasţan et al. , ).
The biomass was calculated by determining the dried cell bulk. The cell bulk was calculated by measuring the centrifuged pellets, which were dried at 80 C overnight (Tasţan et al. ). The dried cell bulk was expressed as the ash-free control group (X c ) and the ash-containing group (X s ). The fly ash concentration was measured as 0.5, 1, 5, 7.5 and 10 g fly ash in 100 mL culture media and expressed as 0.5, 1, 5, 7.5 and 10%.
An energy dispersive X-ray fluorescence spectrometer (ED-XRF, Shimadzu) was used for element analyses. Schimadzu reference materials (standards) were used for calibrating the ED-XRF instrument. The samples were dried at 110 C for 12 hours before the analyses and detected with a silicon drift detector (Tasţan ).
All of the tests were carried out three times. All data were calculated with corresponding standard error as formulated and described by Kenney & Keeping (), Equation (3); σ is the square root of the estimated error variance of the quantity.

RESULTS AND DISCUSSION
Effect of growth medium on fungal ash bio-removal As a result of PCR and sequencing, the isolated new strain was identified as P. chrysogenum. The effect of different media compositions were tested for increasing the performance of fungal fly ash uptake and summarized in Table 1.
The bio-removal yield obtained in T6 nutrimedia was higher than the yield of T6 nutrimedia þ corn syrup media, but it was lower than the yields that were obtained in T6 nutrimedia þ whey powder. In this step, dry biomass amounts of the ash-free control group (X c ) and ash-containing sample groups (X s ) were compared. The lowest yield was obtained in T6 nutrimedia þ corn syrup þ whey powder media, while the lowest X s was obtained in T6 nutrimedia.
Therefore T6 nutrimedia þ whey powder media was more efficient in fly ash removal process and also the X s value (2.65 g/L) recorded in this medium was only 21.6% lower than of X c (3.38 g/L). By contrast, the maximum X c was obtained in T6 nutrimedia þ corn syrup media, while 30.8% decrease was observed in X s amount of fungus. The highest difference between X s and X c amounts was observed in T6 nutrimedia as 34.6%. In a summary, the highest bioremoval yield, highest X s amount and the minimum difference between X s and X c amounts were obtained in T6 nutrimedia þ whey powder media.
In the removal of various contaminants, it was found that different culture media had a direct effect on fungal removal efficiency (Marcial et al. ; Tasţan et al. ).
The use of this inexpensive and commercial T6 nutrimedia culture medium in the literature is very recent. Studies have shown that the medium is effective in ash removal by different types of fungi (Tasţan ). In this study, P. chrysogenum was able to tolerate ash up to 10% concentration using T6 nutrimedia culture medium as can be seen in Table 1. In a study using the same medium, Fusarium oxysporum and P. glabrum removed 6.36 and 18.65% fly ash at 7.5% pulp concentration, respectively (Tasţan ). In another study, standard sucrose medium was used as the culture medium and A. niger did not show any growth at 6% ash concentration (Xu & Ting ). The results show that this commercial medium is a highly productive medium that will contribute to the literature.

Effect of different fly ashes on fungal ash bio-removal
The fungus was cultivated in the two independent types of bio-removal process with six different fly ashes (Table 2). In the first type of culture stage, fungus was first cultivated  fly ash pulp density, but did not show an improvement at 6% (Xu & Ting ). In another study, more effective treatment was obtained by using two types of culture stages (Wang et al. ), and 45% decrease in lag phase was realized by pre-washing before the fungal bioleaching process and thus the bioleaching process was accelerated by 30%.
When all results are taken into consideration, 116 μm size of fly ash was selected for further testing in the second type of culture stage (t ¼ 5 days).

Effect of pH on fungal ash bio-removal
The effect of pH on fungal ash removal yield by P. chrysogenum is presented in Table 3. Although the same removal yields were observed at pH 4.0, 5.0 and 6.0, yields decreased up to 55.61% at pH 8.0. The maximum X (dry weight g/L) values were obtained as 3.08 g/L at pH 6.0 despite these equal removal yields. Therefore, the optimum pH was selected as 6.0 (Table 3).
In another study, two types of culture stages were used.
In the first one, fungus and ash were incubated at the same time, in the second, fly ash was added after the fungus devel-

Effect of increasing fly ash concentrations on fungal ash bio-removal
The results of increasing fly ash concentrations from 0.5 to 10% are summarized in Figure 2. As seen in the Figure 2, P. chrysogenum tolerated the fly ash well up to high concentrations. The maximum fungal ash removal yields were obtained at the lowest two concentrations of fly ash (0.5 and 1%). The fungus was able to completely remove low concentrations, and it was also able to tolerate high concentrations up to 10%. In another study the bioleaching kinetics of A. niger in the presence of fly ash at 1-6% concentrations in a bioleaching process was investigated.
According to the results, fungal growth was not observed at higher than 6% of fly ash concentrations (Xu & Ting ).  In a study by Xu & Ting (), different parameters were investigated on the bioleaching of fly ash by A. niger.
Results showed that fly ash concentration was more impor- According to Figure 2, the maximum specific fly ash uptake (q m ) was 4.08 mg/g at 0.5% fly ash concentration.
By contrast, the fungus was able to uptake fly ash at 0.08 mg/g at the highest fly ash concentration (10%). The mechanism of fungal cellular uptake was expressed in this stage of experiments. Therefore bio-sorption experiments were also conducted. Further studies are needed to better understand the mechanism, such as genetic, enzymatic and molecular analyses and advanced microscopic studies using transmission electron microscopy (TEM) and SEM.
Shortening the time factor in bio-removal studies affects profits at industrial scales. In the present study P. chrysogenum grew in 0.5-10% concentrations of fly ash with the help of adding fly ash after an incubation period, meaning that fungal growth was not inhibited by increasing concentrations of fly ash. Similar to the results of the present study, Xu & Ting () added fly ash after 2 days of fungal incubation and studied 1-8% concentrations. They noted that the fungus A. niger was able to grow in the presence of 4% fly ash.

Effect of increasing biomass concentrations on fungal ash bio-removal
To find a suitable biomass concentration of P. chrysogenum, 0.25, 0.5, 1 g/L initial biomass concentrations were tested.  As seen in Table 4, the minimum fungal ash removal yield, 97.42%, was obtained at 0.5 g/L initial biomass concentration in the sixth hour of incubation. When the initial biomass concentration increased to 1 g/L, the yield increased to 100% in the sixth hour. All of the fungal fly ash removal yields increased to 100% after 24 hours at all biomass concentrations. As can be seen from the results, the fungus can be recorded as a very active species for ash removal. Regardless of the initial fungal biomass concentrations within the studied ranges, ash removal yields

Bio-sorption experiments
To better understand the mechanism of fly ash removal by fungi, the effect of bio-sorption on fungal ash uptake were tested. The data obtained after 24 hours of incubation with autoclaved (dead) fungus are summarized in Table 5. The maximum bio-sorption yield was 95.27% after 24 hours. Moreover half of fly ash was biosorbed in the first hour of the incubation. Therefore bio-sorption of fly ash by P. chrysogenum biomass can be recommended as fast way to remove fly ash, a harmful pollutant, from the environment.
In another study, the bio-sorption of heavy metals was carried out by A. niger biomass in bioleaching of fly ash.
The fungal biomass was in contact with a heavy metal solution extracted from fly ash. This research showed that the bio-sorption of metal ions decreased clearly in the presence of fly ash compared to that in the absence of fly ash (Yang et al. ). It was determined that the hours-long biosorption process was more effective in ash removal than the days-long fungal cellular uptake mechanism.

Element analyses
The element analysis results conducted by ED-XRF are shown in Table 6. In the samples, Ca, Fe and Si were the  (Table 6). Elemental analysis results also show that this application can be useful in bio-removal of fly ash. Therefore the isolated fungi, P. chrysogenum, could be suggested as efficient bio-material in the process of remediation of metals from fly ash when it is scaled up.
Bayat () examined the removal of Cr and Cd from two fly ashes. The adsorption of Cd was found to be higher than Cr. In another study, some properties of various fly ashes in Turkey were detected and high Ca levels were found (Bayat 1998). In the present study, the reason for high fungal tolerance to increasing fly ash concentrations may be to the high Ca levels in fly ash and in the growth medium.

Acute immobilization test
The EC 50 concentration was found to be 10 mg/L. However, no LD 50 value was determined by D. magna after 72 hours of fly ash exposure time. The no observed effect level (NOEL) was found at 0.2 mg/L and low observed effect level (LOEL) was found at 0.5 mg/L fly ash (Figure 3).
There are only a few studies in the literature on the toxicity tests of fly ash using crustaceans. In a study, the toxicity evaluation of municipal solid waste incineration fly ash was evaluated with the role of cadmium by a clone of D. pulex.
They explained that the leachate was more than 20 times more toxic than expected from its cadmium concentration  Therefore the present study is the first to reveal the biotoxicity evaluation of fly ash with above mentioned requirements.

CONCLUSIONS
This study highlights the potential usage of P. chrysogenum in the bio-removal of fly ash. Six different sizes of fly ash, two different culture stage techniques and three different fungal biomasses were used. In the first type of culture stage, the maximum removal rate was 82.32%, and in the second type of culture stage the maximum removal rate was 100%. The second type of culture stage allowed the rapid transformation of fungal spores into micelles. Thus, the fungal biomass developed rapidly. Fungal biomass could tolerate 10% fly ash concentration, which to the best of our knowledge, is the highest ash concentration in the literature. ED-XRF results helped to show the fate of metals in fly ash. In this study, in which both bio-sorption and bio-removal mechanisms were investigated, biosorption was found to be a highly effective mechanism, as well as bio-removal. P. chrysogenum could serve as an effective bio-agent for bio-removal and bio-sorption of fly ash from aqueous media. The present method was found to be advantageous as compared to other reported bio-removal studies, as it allowed the fungus to tolerate high concentrations of fly ash.