Bacterial Diversity in Different Positions in the Iraqi Marine Area

. Abstract: Microorganisms establish both structural and functional construction in the marine environment, despite scientific advances, the identification of marine bacterial species is still considered as a common challenge in microbiology. Nevertheless, the present study aims to make an effort, although it seems modest, but it could establish a basis for studying the bacterial diversity in the Iraqi marine area, because of what this aspect entails of the poverty of studies related to this aspect in the studied area. The current results show the marine studied area are classified within worming area, where the average temperature ranged from 23.17 to 26.17 ºC. The recorded number of bacteria was increased with temperature increasing (0.210, 0.250, 0.305, 0.517, 0.553 and 0.625) through the studied period (January, February, March, April, May and June) respectively. The results showed that the identified bacteria were related to three bacterial phyla(Firmicutes, Proteobacteria, and Actinobacteria),with the clear superiority to the phyla (Firmicutes).The most occurrence bacterial genera were( Staphylococcus lentus, Vibrio parahaemolyticus, Vibrio alginolyticus, Sphingomonus paucimobilis and Aeromonas hydrophila/caviae), which have different percentage of frequency ,(50,40,60,30 and 20%) respectively. The present discoveries provide initial evidence on the bacterial diversity in the Iraqi marine waters. Further studies are necessary to make a more comprehensive image of bacterial diversity within these marine regions.


Introduction:
When describing environments according to their bacterial diversity, it can be said that the marine environment is the most diverse ecosystem, that the situation in the marine environment is neither selective, nor discouraging to a specific group 1 . The bacterial diversity in the marine environment reflects their health status and wellbeing 2 . Bacteria are responsible for the largest percentage of marine primary productivity, and it is the key factor in the processes of energy flowing through biogeochemical process, and influencing the climate of our planet, as they constitute approximately 90% of the marine environment 3 . Determining the societal composition of bacteria as well as their distribution patterns has an effective role in forming an important vision about the ecological and biogeochemical performance of the marine ecosystem 2 .
There are many environmental factors to which bacteria in marine environment are exposed, such as temperature, oxygen concentration, and salinity 4 . Marine bacterial diversity is affected by different environmental factors, but the temperature is considered as the most important ones. The previous studies have shown that there is a decrease in bacterial diversity in the polar regions due to decrease in temperature. In high temperatures, the metabolic rate increases and the age of generation is shortened, also high temperature leads to accelerate environmental and physiological processes and finally increases the rate of mutation and speciation resulting in a large site diversity 5 ، 6 ، 7 . All microbes have complex proton pumps that are involved in bioenergetics, but it is not clear how microbes might respond to changes in environmental proton balance 8 . Studies indicated that although the bacteria have ability to adapt with the change in the 2 see water acidity and conserve their abundance, yet in response to such situation they lose their diversity 9 . Expensively, salinity is referred to as an important factor affecting microbial assemblies in both lakes, coastal waters and oceans 10 . Salinity has also been suggested to be the major determinant of microbial community composition, exceeding the influence of temperature and pH 11 . The effect of increase salinity on the microbial component is quite complex. The studies showed different responses in the moderate salinity, including a decrease in bacterial diversity 12 , no effect 10 ,and occurrence of a peak in diversity at moderate salinity 13 . The concentration of oxygen in the marine environment has an important role in shaping the ecosystem, as it controls the temporal and spatial distribution of all living organisms, whether microbial organisms or higher organisms. The differential solubility of oxygen and consumption via microbial-mediated oxidative processes are also known to affect the oxygen concentration in marine water. 14 Studies have shown that there are factors restricting microbial diversity in marine waters, but the gradient in the concentration of oxygen remains the limiting factor for that diversity 14 .
The viral presence in the marine environment is one of the controllers in the biomass of bacteria and has a role in maintaining the biological balance as well as its role in highlighting biological diversity as it is an intermediate factor in the genetic exchange between living organisms 15 , 16 The unlimited possibilities of services that can be gained for human society as a result of microbial diversity have not been exploited yet. And one of the most important obstacles to exploiting these potentials is the lack of a database on the bacterial community through which it is possible to assess and determine future changes. Given the importance of the availability of a database of the quantity and diversity of bacteria in Iraqi marine areas, the current study aims to determine the quantity and diversity of bacteria in the current study areas.

Study areas
The study area included three stations located within the Iraqi marine borders. Table1 shows the coordination and the deep of this station.

Sample collection
Samples were collected randomly from a depth of half a meter using 500 ml of glass bottles already sterilized. "The samples were transferred under refrigeration to the laboratory and the samples continued to be collected monthly during the period from the January to the July /2018.

Physical and chemical properties of water samples
Some physical and chemical properties of water samples like; Temperature, pH, Electrical conductivity, Dissolve oxygen, Total dissolved substance, and Salinity were measured during sample collection using Multimeter device (Sensodiract, Germany).

Enumeration of Bacteria
For bacterial enumeration 0.1 ml of each water sample aseptically inoculated in 10ml tube containing nutrient broth (Hi media). The medium was prepared according to the manufacturer's instructions and sterilized in an autoclave. A duplicate tube was used for each sample with one as a control. Tubes were incubated with shaking incubator at 120 rpm for 24hs at 25ºC 17 .

Absorbance of bacterial growth
Different assays for viability can be used, however the optical method is the best one to be adapted into high-throughput setups as it can be done on standard plate readers 18 . The OD. method is done by sending light at a specified wavelength, in this case 600 nm (OD600). The obtained optical density is a number that depends on the amount of diffracted light. The number is directly related to the amount of bacteria 19 . In the current study the absorbance of bacteria was measured using spectrophotometer (Uv-1800, double beam, Shimadzu, Japan) under scanning wave length. The readings of absorbance at 600 nm wave length were recorded and tabulated by taking only nutrient broth media as blank standard; (Absorbance readings at other than 600nm wave length recorded not relevant, hence were not shown in the data) 17 .

Isolation of marine Bacteria
For isolation of marine bacteria 1.0 ml from each water sample was mixed with 9 ml of distilled water, then serial dilution was performed up to 10 -5 . 0.1ml from the final dilution was spread on the surface of the nutrient agar, (Hi media). The inoculated plates were incubated at 30 o C for 24-48 hs. Colonies developed after incubation were purified by repeated sub culturing on nutrient agar and finally, bacterial colonies with distinct characteristics such as pigmentation, size, opacity, elevation, margin and surface appearance were chosen for further characterization 20

Identification of bacteria
Morphological and some biochemical test (Grams stain) were carried out for diagnosis the bacterial colony from nutrient agar media. The automated instrument for bacterial identification (Vitek II) has been used for accurate identification.

Results and Discussion: Physical and chemical properties of sea water
As the current study focus on the diversity of bacteria, so it takes care of assessing the most effectiveness physical and chemical properties of water in the studied area. Table 2. shows the results of these studied properties. The temperature ranges from, 23.17-26.17 ºC during the studied period (table.2). Temperature is an elementary water quality and it governs the suitability of water for the different forms in aquatic environment. Depending on the geographic location the mean annual temperature varies in the range of 10 to 21 ºC 21 . Effect of temperature on the bacterial diversity related to its effect on the rate of oxygen solubility and photosynthesis by algae and other aquatic plants, metabolic and production rate 22 . The present study recorded that the pH value is ranges from, 8.34-8.76 (Table.2). Usually any extreme changes in the hydrogen potential play a crucial role in changing the concentrations of other substances in the water bodies to a more toxic form, for example, the ammonia toxicity, chlorine decontamination effectiveness, and metal solubility are all dependent to changes in pH value 23 . Salinity of sea water usually related to the sodium chloride, generally water salinity effecting on the aquatic life, and every type of organism has an ideal salinity range that it is suitable for their presence and proliferation. The present study recorded that salinity ranges from, 46.00-43.41(ppt), and general aquatic life, occupation well in a salinity range of 6.0 to 9.0(ppt). 23 . Dissolved oxygen is a result of the diffusion from the air during rapid movement and as byproduct of photosynthesis, the amount of dissolved oxygen is crucial for all living organisms and eventually effecting their diversity 23 . The range of dissolved oxygen recorded in the present study was 3.61-2.93 mg/l (Table.2). The total dissolved solid is a term defining the amount of inorganic salts and small amounts of organic matter present in the water, the amount of TSS in the present study was within the range 44.85-39.09 g/l (Table.2).

The absorbance of bacterial growth
In the present study the obtained results (Table.3), shows the number of bacteria through the studied period and at the three studied stations. The statistical analysis shows no significant difference (P<0.05) among station, and significant difference (P> 0.05) presented among the studied months. Results also indicated that the means number of bacteria in the studied stations follow this order station 2< station 1< station 3 (Fig.1), The logical explanation for these results; is the high drainage and the influx into the Arabian Gulf in addition to the high influxes of nutrients provided by the Shatt Al-,Arab, additionally as a result of dilution in salinity by Shatt Al-Arab River or any near resource of fresh water 24 In relation to the number of bacteria during the study period, the order was June< May< Apr.< Mar.< Feb.< Jun. (Fig.2.). In temperate marine areas, the microbial community varies according to "changes in temperature and the length of the day 25 . 26 indicated that communities were separated into two main groups, corresponding to two main periods: from November to April (colder months), and from May to October (warmer months). Previous studies have shown that in temperate regions, the change in the bacterial community ranges between a gradual change throughout the year or that the change is continuous and rapid between cold and warm months 27 .

Types of bacteria identified
Totally, 100 bacterial isolates were isolated from the water of the three sampling stations by dilution-plating technique. After occurrent purification, 18 pure bacterial colonies, were subjected to morphological, and microscopic examination by Gram stain test, to make sure of the bacterial staining ability Finally, 18 pure isolates were identified using the VitekII instrument (Table  4). Among the 18 identifying bacteria five genera (Staphylococcus lentus, Vibrio parahaemolyticus, Vibrio alginolyticus, Sphingomonus paucimobilis and Aeromonas hydrophila/caviae), shows the respected occurrence in the studied stations, with different percentage of frequency (Table.4). Bacterial communities differ in different marine environments, while they are similar in marine environments that are similar but separated by long distances 27,28

Conclusions:
The results of the current study confirm that the Iraqi marine area is classified among the warm regions, as the temperature during the study period ranges between (23.17-26.17 ºC). In relation to the bacterial diversity in the study area, the results show the superiority of three bacterial phyla( Firmicutes , Proteobacteria, and , Actinobacteria),with the clear superiority to the phyla( Firmicutes). The most occurrence bacterial genera is( Staphylococcus lentus, Vibrio parahaemolyticus, Vibrio alginolyticus, Sphingomonus paucimobilis and Aeromonas hydrophila/caviae), which has different percentages of frequency ,(50,40,60,30 and 20%)