{'fraction_non_alphanumeric': 0.026643192488262912, 'fraction_numerical': 0.02171361502347418, 'mean_word_length': 4.954577218728162, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
STUDIES ON EFFECT OF COPPER (CU2+) ON PHOSPHATASE ENZYME ACTIVITY AND BIOMASS OF Aspergillus niger

The effects of metal on microbial ecosystems are primarily described in terms of the toxicity of heavy metals to microorganisms and their impact on microbial community, structure and function. Living organism requires certain metals for their growth and metabolism and they evolved an appropriate uptake mechanism for metals. It is particularly difficult to establish mutual relationship when soil is continuously contaminated with a variety of heavy metals. Under such conditions, the task of identifying which particular heavy metal predominantly destroys soil microbial properties become complicated, since the accumulated ABSTRACT


Introduction
The effects of metal on microbial ecosystems are primarily described in terms of the toxicity of heavy metals to microorganisms and their impact on microbial community, structure and function. Living organism requires certain metals for their growth and metabolism and they evolved an appropriate uptake mechanism for metals. It is particularly difficult to establish mutual relationship when soil is continuously contaminated with a variety of heavy metals. Under such conditions, the task of identifying which particular heavy metal predominantly destroys soil microbial properties become complicated, since the accumulated influence of several metals may not necessarily be the sum of individual effects (Lane and Morel, 2000).
Assimilation of phosphate from organic compounds by plants and microorganisms take place through the enzyme "phosphatases" which is present in wide variety of soil microorganisms. The principal mechanism for solubilization is the production of organic acids and acid phosphatases which play a major role in the mineralization organic phosphorous in soil.
It has been found that several cations inhibit soil phosphatase activity. Tyler (1976) has shown that Cu and Zn ions have a marked inhibitory effect on phosphatase activity. Acid phosphatase (orthophosphoric monoester hydrolases) are a group of enzymes that catalyze thehydrolyses of external phosphate esters (Joh et al., 1996). They played an important role in the mineralization of organic carbon, organic phosphate and low levels of free inorganic ions (Pi) . They are secreted by various fungi, including Aspergillus niger, a commercially important fungus in the production of fungal enzymes.

Materials and methods:
Three different concentrations of CuSO 4 viz. 0.5mM, 1.0mM and 3.0mM were selected for this experiment. Serial dilution plate method (Waksman, 1992;Parkinson et al., 1971) was followed for isolation of Aspergillus niger form Mizoram University campus soil. Pikovskaya solid medium (with agar) and Pikovskaya broth medium (without agar) was used to confirm whether Aspergillus niger has a capacity to solubilize phosphate or not. Aspergillus niger is a haploid filamentous fungus which is used for waste management and biotransformation in addition to its industrial uses, such as production of citric acid and extracellular enzymes. The fungus also plays a role in the solubilization of heavy-metal sulfides.

Enumeration, isolation and identification of fungal species:
Rose Bengal Agar medium (Martin, 1950) was used for the growth of fungal population. 1ml of the soil dilution (1:1000) was transferred into petridishes containing Rose Bengal Agar medium and were incubated upside down at 25±1⁰C for 7 days in a BOD incubator. Finally Aspergillusniger was selected for the experiment and they were again transferred into Potato Dextrose Broth Medium for maintaining stock culture medium.

Estimation of Phosphatase (Tabatabai and Bremner, 1969):
The culture filtrate was centrifuged at 5000 rpm for 10 minutes. 0.1ml of the filtrate was taken in each test tube maintaining three replicates for each treatment. 4ml of modified universal buffer (pH 6.5), 0.25ml of toluene and 1ml of 0.05M p-nitrophenyl phosphate (PNP) solution was added to the flask (Skyins, 1985). The flask is swirled for few seconds and then incubated at 37⁰C for 1hr in an incubator. After incubation, 1ml of 0.5M CaCl₂ and 4ml of 0.5M NaOH is added to the mixture. The optical density was measured at 430nm in Spectrophotometer. Blank is maintained similarly without the filtrate.

Measurement of fungal growth and effect of heavy metal on rice:
Pikovskaya solid medium with different concentrations of Copper Sulphate was prepared and the pure culture was transferred into the hole in each three replicates of control, 0.5mM, 1.0mM and 3.0mM CuSO 4 . The plates were incubated at 25±1⁰C for about 3 days. After 3 days of incubation, colony diameter was measured. Rice seeds were taken and soaked in 5% mercury chloride for 10 minutes and washed with distilled water and then soaked overnight in the culture flasks that contain media supplemented with copper sulphate. After two weeks the height of the shoots, length of the roots and the biomass of rice plants was measured.

Statistical analysis:
Data obtained from the investigation will be analysed by using appropriate standard statistical methods and Statistica computer software.

Result and discussion:
1. Aspergillus colony in solid culture media: The colony size of Aspergillus was measured from four different treated plates viz. CTRL, 0.5mM, 1.0mM and 3.0mM CuSO₄. Control plate showed maximum colony size followed by 0.5mM and 1.0mM and the smallest colony size was observed on 3.0mM CuSO₄ plate. The one way ANOVA results show a significant variation on Aspergillus colony size in different treatment. Mycelium dry weight of Aspergillus was measured from four different treatments viz. Control, 0.5mM, 1.0mM and 3.0mM CuSO₄. It was noted that Control treatment showed maximum dry weight of mycelia and followed by 0.5mM, 1.0mM and 3.0mM CuSO₄.
Our experiments showed that control attained the highest growth rate, while application of varying concentrations of CuSO 4 hampered and retarded the growth of fungal mycelium correspondingly. This finding is in agreement with the result of Abu-Mejdad Najwa Mohammed Jameel (2013) using Aspergillus niger, Candida albicans and Cryptococcus neoformans for investigating the effect of heavy metals (Cu, Mg and Zn) on the growth in liquid and solid media under laboratory condition.

Phosphatase:
Phosphatase enzyme activity was measured from four different treatments viz. Control, 0.5mM, 1.0mM and 3.0mM CuSO₄.It was noted that Control treatment showed maximum phosphatase activity followed by 0.5mM, 1.0mM and 3.0mM CuSO₄. The effects of CuSO 4 upon the growth of fungal mycelium both in solid and liquid culture was observed after 3 days of incubation. CuSO 4 had negative effect on phosphatase activity and this finding is in agreement with the result of Huang and Shindo (2000) that phosphatase decreased significantly with the increase of Cu concentration.

Plant Biomass:
Plant biomass was measured from five different treatments viz. Control-1, Control-2, 0.5mM,1.0mM and 3.0mM CuSO₄. It was noted that Control-1 treatment showed maximum plant biomass and it goes on increasing from 0.5 to 3.0mM CuSO₄.

Height of rice shoots:
Height of rice shoots was measured from five different treatments viz. Control1, Control2, 0.5mM, 1.0mM and 3.0mM CuSO₄. It was noted that Control2 is higher than Control1 and it goes on decreasing from 0.5mM to 3.0mM CuSO₄.

Fig. 5: Height of Rice shoots
It is shown that the biomass of rice plant, height of shoots and length of roots gradually decrease from control to 3.0mM CuSO 4 . This finding is in agreement with Gupta et al. (2011) that the toxic effects of copper and cadmium on seed germination, seedling, root, shoot length and seedling dry biomass of maize was evaluated under laboratory conditions as compared to control values.

Conclusion:
The study revealed that CuSO 4 had negative effect on the growth of Aspergillus niger, phosphatase activity and plant biomass. Our results clearly showed that as the concentration of the metal increases, the degree of inhibition i.e. colony diameter and mycelium biomass in solid and liquid cultures increases correspondingly. This is in conformity with Ramya et al. (2011) findings upon effects of CuSO 4 on A. niger. Copper despite prevalent in our environment and was considered essential element, however is capable and in danger of showing toxic effects on plants and animals with their increased concentration in soil and water in our times because of heavy industrial establishments.