ISOLATION AND SCREENING OF MICROORGANISMS
ISOLATION OF ASPERGILLUS NIGER
The sample was isolated from site in Malaysia which at Bukit tinggi, Pahang.This place have a maximum average temperature of about 38°C and a minimum average temperature of 16°C. The isolation was directly from the agricultural soils and decaying woods materials. The step was using shotgun approach. About 1 kg of soil sample collected using sterile spatulas and keep on polyethylene bags. Next, samples brought into laboratory to growth the microorganism.
About 1 gram of soil sample was mixed with 9 ml distilled water in a test tube. The mixture were shaken well using mixer. Then, various serial dilution were prepared from 10-2 until 10-6 using sterile distilled water to get a specific microorganism. Next, 0.1 ml of solution was transferred into petri dish containing a growth culture using aseptic technique and spread using L shaped glass rod to get pure culture. The sample were incubated for 3-5 days at 37 ° C for isolation of aspergillus niger. After 3-5 days, variety of fungal colonies were form on the media.
SCREENING OF ASPERGILLUS NIGER
Preliminary identification of fungi were conducted by differentiate their morphological characteristics referred on “A Manual of Soil Fungi”. After preliminary step, the desired traits will be chosen into next step. The desired traits were subcultured to purify the fungal and examined the pectinase activities.
If the traits were not satisfied, genetic engineering method will be conducted. This methods provide unlimited opportunities of production combination genes, very specific and can be controlled. The steps must start with cut the fragment of DNA with restriction enzymes and joins with vector which the host cell. The joining will be performed by DNA ligase. Next, recombinant molecules will be introduced via transduction or transformation. The fungi were autonomous replicate in the host cell which maintained the recombinant DNA. Lastly, DNA libraray will formed to save the different recombinant DNA molecule by different individual clones.
Screening for pectinase activities was performed on pectinase screening plate. The pectinase screening plate was contained of 1% pectin powder, 5 g/l of peptone, 15 g/l of agar, 5 g/l of sodium cloride, 3 g/l of beef extract and the distilled water up until 1000 ml. Then, before sterilization procedure, the pH of mixture must adjusted at 7.2. The mixture will be autoclaved about 30 minutes at 120 ?. The medium will be used in bacterial purification processed. One of important materials was Logule’s iodine solution which will proved the pectinase producers. The fungal colonies will appear a clear zones because of flooding with Logule’s iodine solution. After last step of screening, the chosen fungi will go through the optimization procedure.
CHARACTERISTICS OF ASPERGILLUS NIGER
HYPERLINK "https://www.caister.com/aspergillus" Aspergillus is HYPERLINK "https://www.britannica.com/science/genus-taxon" genus of HYPERLINK "https://www.britannica.com/science/fungus" fungi which a phylum of HYPERLINK "https://www.britannica.com/science/Ascomycota" Ascomycota, kingdom of Fungi and also the oldest within the other named as genera of fungi. It had become best-known mould groups which the most studied by 1926. There are many type of Aspergillus which one of them is Aspergillus niger. It is the most common species of genus HYPERLINK "https://en.wikipedia.org/wiki/Aspergillus" o "Aspergillus" Aspergillus in a fungi type. Furthermore, in the field of microbiology, Aspergillus niger is very important microorganism and is a fungi that have haploid filamentous.
Aspergillus niger produce colonies that covered by dark asexually produced fungal spores composed of white or yellow colour. Septum and transparent are functional to divided the mycelial or threadlike and hyphae. The asexually produced fungal spores by A. niger which is conidiophores usually the length is range from 900-1600 m and contain globose vesicles at the tip ranging from 40-60 m in diameter. The projections from conidiophore of A. niger arise biseriate phialides which covered each globose vesicle completely and have spines or conspicuous ridges that not arranged in rows. Conidiogenous cell is created on brown metulae and also a site where phialides appear. Blastic basipetal conidiogenesis process must go through by phialides to make a globose mitospores. The globose mitospores have ranges from 3 to 5m. of diameter. The surface of A. niger is smooth while the conidia surface is very rough.
Aspergillus niger is commonly found in soil, decaying vegetation, stored grain and plants debris which the environment is mesophilic. In indoor environments, black colonie of A. niger is reported which difficult to differentiate with HYPERLINK "https://en.wikipedia.org/wiki/Stachybotrys" o "Stachybotrys" Stachybotrys which species also known as black mould but conidial structure is distinct at microscopic level after identification. The spores of A. niger are widely spread and can be found anywhere in soil. In laboratory, it commonly isolated via chemostat cultures. Chemostat cultures can use as a test to fungi which to prove the positively or negatively.
Aspergillus niger called as a typical contaminated the food and can causes a black mould which is a diseases on ornamental plants, vegetables and fruits such as apricots, grapes, peanuts and onions. Onion seedlings embody only when conditions are helpful that infected by A. niger. The black mould can be observed on bulb when the post harvest disease appear on onions.
A niger is commonly causes ear infections on fungal which can cause hearing loss in temporary time and pain while HYPERLINK "https://en.wikipedia.org/wiki/Tympanic_membrane" o "Tympanic membrane" tympanic membrane and HYPERLINK "https://en.wikipedia.org/wiki/Ear_canal" o "Ear canal" ear canal damaged occurred in severe cases. However, it is not very dangerous to human because it rarely cause a disease to human rather than other Aspergillus species. In one of rare case, humans become ill due to a serious lung disease which called HYPERLINK "https://en.wikipedia.org/wiki/Aspergillosis" o "Aspergillosis" aspergillosis. It had been studied that Aspergillus spores rich in HYPERLINK "https://en.wikipedia.org/wiki/Peat" o "Peat" peat dust where HYPERLINK "https://en.wikipedia.org/wiki/Horticulture" o "Horticulture" horticultural workers who inhale it and they are a frequent people that infected by aspergillosis. It also formed in the mummies of ancient Egyptian tombs and when it being disturbed, people can inhale the dust.
Aspergillus niger has a metabolic system which are contained of the mitochondria, cytoplasm and peroxisome. Carbohydrate and amino acid metabolism are integrated in metabolic system that occurred in both catabolic and anabolic reactions. Amino acids alanine, proline and glutamine thrive in this surroundings by using method of logarithmic sensitivity analysis. A. niger is breaking down minerals into basic element to gain energy.
Large vesicle at end of broken conidiphore bearing metulae & phialides with black pigmented conidia already dispersed.
Micrograph of A. niger grown on HYPERLINK "https://en.wikipedia.org/wiki/Agar_plate" o "Agar plate" Sabouraud agar.
A.niger growing on onion
niger growing on HYPERLINK "https://en.wikipedia.org/wiki/Sabouraud_agar" o "Sabouraud agar" SDA
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Schuster E, Dunn-Coleman N, Frisvad JC, Van Dijck PW (2002). "On the safety of Aspergillus niger—a review". Applied Microbiology and Biotechnology. Retrieved from HYPERLINK "https://link.springer.com/article/10.1007%2Fs00253-002-1032-6" https://link.springer.com/article/10.1007%2Fs00253-002-1032-6 on May 9, 2018.
K Diba, Kordbacheh P, Mirhendi SH, S Rezaie, M Mahmoudi (October 2007). Identification of aspergillus species using morphological characteristics. Retrieved from HYPERLINK "https://www.pjms.com.pk/issues/octdec207/article/article9.html" https://www.pjms.com.pk/issues/octdec207/article/article9.html on May 9, 2018.