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Research Article

Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method

Atena Shadmehr, Payman Nouroozi, Ghasemali Garousi and Ali-Reza Ahmadi
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After optimization of sterilizing of cyst and larva second stage of Heterodera schachtii, possibility of using nematode on seedlings of sugar beet (Beta vulgaris L.) in in vitro conditions were studied using sterilized larvae of beet cyst nematode. For this purpose, non sterile cysts were extracted from infected soil and hatched into zinc chloride solution with concentration of 0.5 g L-1. Then, for preparation of sterile second stage larvae, several sterilizing treatments were used. Mean comparisons were performed between sterilized live larvae number by Duncan's method. Results showed that 70% ethanol for 1 min followed by 2.5% hypochlorite sodium for 5 minutes and 0.1% hypochlorite sodium for 20 min were best treatments for disinfecting cysts and larvae, respectively. In parallel, two nematode susceptible sugar beet varieties were applied to produce seedlings in in vitro culture. PGoB medium containing different hormonal compositions was used for producing of hairy roots and inoculation of seedling with sterilized larvae. After nematode inoculation tests, daily observations were done by counting cysts and stained roots and larvae under stereomicroscope. Between 5-12 cysts formed on the roots of each seedling from two varieties 40 days after inoculation. As a result, it seems that this technique can be used for sugar beet germplasm evaluation to screen nematode resistant genotypes in in vitro controlled condition.

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Atena Shadmehr, Payman Nouroozi, Ghasemali Garousi and Ali-Reza Ahmadi, 2007. Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method. Pakistan Journal of Biological Sciences, 10: 2910-2914.

DOI: 10.3923/pjbs.2007.2910.2914



A large amount of water is used in oil industry for a wide variety of purposes. A large portion of it leaves this industry as wastewater that contains oil, phenols, many solvents and toxic substances (Zhao et al., 2006; Toril, 2001). Such wastewater can pollute water bodies, soil and even the air if it is not treated (Yang et al., 2000; Chen et al., 2002; Vegueria et al., 2002).

Biological treatment processes are economical and efficient methods that can be used for treating wastewater from oil industry (Jou and Huang, 2003). In many refineries, suspended growth systems, such as Conventional Activated Sludge (CAS) process, are applied to treat refinery wastewater (Tellez et al., 2002; Stepnowski et al., 2002). However, CAS process has some operational problems, such as the inability to settle the sludge, formation of excessive scum and foam and sludge bulking and requires operators' skill and large space, which is limiting factor in oil industries because these are located in populous areas without enough space for expansion (Park et al., 1996; Loukidou and Zouboulis, 2001; Xianling et al., 2005). Therefore, it is important that biological treatment systems can be easy to operate and can treat a large amount of wastewater in a space which is as small as possible (Park et al., 1996; Xianling et al., 2005). As a consequent, some novel biological treatment methods have been developed during recent years.

Attached growth bioreactors such as trickling filters and rotating biological contactors have been used for treatment of wastewaters for over a century. However, during the past two decades new versions of attached growth bioreactors that apply totally submerged media with high specific surface areas have been developed. These systems are known as Submerged Attached Growth Bioreactors (SAGB). Currently, submerged attached growth systems have attracted attention due to the high biomass concentrations that can be gained, leading to short Hydraulic Residence Times (HRTs). Short HRTs cause these systems to be compactly constructed (Leslie Grady et al., 1999). In comparison to suspended growth biological treatment systems, such as (CAS) process, this system can have some advantages, such as presence of higher concentrations of biomass in bioreactor, short hydraulic resistance time, resistance to toxic loading and adverse environmental conditions, easy operation, handling shock loads with high efficiency, lower consumption of energy and producing less waste sludge, (Park et al., 1996; Jianlong et al., 2000; Loukidou and Zouboulis, 2001; Jou and Huang, 2003; Guimarães et al., 2005):

Aerated Submerged Fixed-film (ASFF) process is a novel attached growth biological treatment system that uses totally submerged fixed media to support biomass growing as a thin biofilm on their surfaces (Hamoda and Abd-El-Bary, 1987; Park et al., 1996; Hamoda and Al-Ghusain, 1998, 1999; Al-Sharekh and Hamoda, 2001). Also, diffusers provide bubbles of diffused air for both aeration and turbulence. The turbulence created by this way prevents the excessive biofilm growth (Hamoda and Al-Sharekh, 1999). ASFF process has been successfully applied for treatment of both urban and industrial wastewaters by several researchers (Park et al., 1996; Hamoda and Al-Sharekh, 1999; Gálvez et al., 2003; Nabizadeh and Mesdaghinia, 2006).

This pilot-scale study was conducted to examine the performance of Aerated submerged fixed-film bioreactor in treatment of artificial wastewater containing crude oil under normal operating conditions.


Experimental set-up: Figure 1 shows a schematic diagram of the pilot plant that was set up during 11 months in 2006 at Health Environmental Engineering Department of Tehran University of Medical Sciences, in Tehran, Iran, for this study. The pilot plant included: one experimental ASFFR, one peristaltic pump in order to pump wastewater into ASFFR, one 400 L holding tank and one air compressor. Bioreactor consisted of a Plexiglas cylindrical column with 14.1 cm internal diameter, 64 cm effective height and a total volume of 10 L, a suitable round diffuser located at the bottom of the bioreactor which produced air bubbles of medium size and Bee-Cell 2000 as support media having porosity of 87% and a specific surface area of 650 m2 m-3.

Artificial wastewater containing crude oil was continuously pumped into the bioreactor at the base, with a flow rate ranged from 8.5 to 102 L day-1. The air was supplied at the bottom of the bioreactor. The air flow rate was increased with increasing organic loading in the range 5-30 L min-1 in order to maintain dissolved oxygen level of more than 2 mg L-1 throughout the bioreactor. Table 1 gives a summery of the experimental mean hydraulic and organic loading rates.

Seed sludge: A return activated sludge from wastewater treatment plant of Behran oil refinery located in Tehran was selected as seed sludge in the experiment, because microorganisms within this sludge was acclimated with oily wastewater. The bioreactor was first inoculated by seed sludge and it was allowed to operate on batch mode for a few days before changing to a continuous mode.

The artificial wastewater containing crude oil: The artificial wastewater containing crude oil was prepared by adding 75 mg L-1 Sodium Dodecyl Sulphate (SDS), as an emulsifier, to a mixture of 40 L water and 10 L crude oil. Crude oil used was abstracted from crude oil storage tanks of Tehran petroleum refinery. Characteristics of the artificial wastewater containing crude oil are shown in Table 1. Because this wastewater was very strong, it was diluted. Table 2 shows composition of diluted oil-based wastewater.

Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method
Fig. 1: Schematic diagram of the pilot plant: (1) aerated submerged fixed-film bioreactor, (2) round diffuser, (3) air compressor, (4) activated carbon column, (5, 8 and 12) cutoff valve, (6) air rotameter, (7) liquid rotameter, (9) wastewater pump, (10) equalization tank, (11) floater, (13) reservoir

Table 1: The experimental mean hydraulic and organic loading rates
Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method
† Hydraulic retention time

Table 2: Characteristics of the artificial oil-based wastewater
Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method

Table 3: Composition of diluted oil-based wastewater
Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method

The wastewater was enriched with the inorganic nutrients by adding NH4Cl as nitrogen source and (NH4)3PO4 as phosphorus source based on a COD:N:P ratio of 100:5:1. Sodium bicarbonate (Na2 CO3) was used to maintain the pH at 7.56±0.25 (Table 3).

Analytical methods: Samples were analyzed for Total and Dissolved Chemical Oxygen Demand (TCOD and SCOD), Volatile Suspended Solids (VSS), alkalinity, Total Kajeldal Nitrogen (TKN), Total Phosphorus (TP) and Dissolved Oxygen (DO) according to the Standard Methods for the Examination of Water and Wastewater (APHA, 2005). Temperature was measured by a thermometer and pH was measured by a pH-meter E520 Metrohm Herisau. In order to determine soluble COD, all influent and effluent samples were filtered through Whatman membrane filters of 0.45 μm pore size.

Attached biomass analyses: In order to determine the amounts of biomass attached to media surface, a given number of media were randomly selected and taken from several depths of the bioreactor once a week. The media with biomass attached to them were put in a very dilute sulfuric acid solution for several days so as to detach biomass from surface of medium. Detached biomass was used for determining VS according to the Standard Methods for the Examination of Water and Wastewater (APHA, 2005).


There was a linear relationship between the COD percentage removal efficiency and surface area COD loading rate (Fig. 2). It is indicated that removal efficiency decrease with increasing the COD loading rate. While the surface area loading rate ranged between 1.310-15.797 g COD m-2 day-1, the COD removal efficiency was more than 70%. It can be seen that the COD removal efficiencies at the high COD loading rates of up to 10 g COD m-2 day-1 are reasonably satisfactory, which indicates that the process had been perfectly efficient.

Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method
Fig. 2: Effect of OLR on COD removal efficiency

Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method
Fig. 3: Relationship between the COD loading rate and the specific substrate utilization rate

Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method
Fig. 4: The effluent COD variation with OLR

Image for - Study of Sugar Beet Cyst Nematode Life Cycle Using Plant Tissue Culture Method
Fig. 5: Effect of specific organic loading rate on the effluent COD

The relationship was a straight line with a high correlation coefficient (R2 = 0.9949). This implies that the specific substrate utilization rate increases when the COD loading rate is increased although increases in the COD loading rate lead to the decreased percentages of COD removed (Fig. 3). Therefore, this shows that the process can utilize more organics at higher surface area loading rates.

It is indicated that the effluent COD concentration increased with increases in the surface organic loading rate. The effluent COD varied between 68.67 and 292.6 mg L-1 at the surface organic loading rates experienced in this study (Fig. 4).

For an effluent COD of 100 mg L-1, an applied food to biomass ratio of up to 0.271 is quite reasonable for satisfactory operation (Fig. 5).


The COD removal efficiencies ranging from 70.87 to 93.12% were achieved by aerated submerged fixed-film reactor. The biofilm growth considerably increases with the increases in the organic loading rate, because the system can retain significant amounts of attached biomass. For a wide range of organic loading rates, the process is cable of attaining high organic removal rates. Although the organic removal efficiency decreases with increased organic loading rate, the organic removal rate increases as the organic loading rate increases.


The authors wish to thank all the staff of the laboratories of the Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Iran, who collaborated with them in this study. The authors would also like to acknowledge gratefully the fact that the completion of this research would not have been possible without the support of Tehran University of Medical Sciences, Iran.


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