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Biodegradation of wastewater to be safely use for irrigation of plants & its influence on their performance & characterization of telomerase



 The present study will be focused to assess the chemical composition & effects of irrigation with both wastewaters i.e., agro-industrial effluents & livestock effluents and microalgae treated wastewaters on selected vegetables i.e., different varieties of shade family vegetables and on soil (iii) to assess the risk of selected vegetables grown on sewage, agro-industrial & livestock/ microalgae treated sewage wastewater, agro-industrial effluents & livestock effluents irrigated soils on human beings (iii) isolation, characterization of telomerase from protein profile of selected vegetables and its role in cancer & diabetes

What actions do you propose?

The scientific methodology will be distributed into three phases




1.1  Isolation of different strains of Chlorella


Samples of fresh water will be collected from different ponds from Faisalabad. This fresh water will be used as a source of the different strains Chlorella i.e., vulgaris, variabilis, autotrophica. Fresh water samples will be analyzed with the help of fluorescent microscope in order to verify the presence of unicellular algae in the samples. For the isolation of microalgal species from the fresh water samples, standard plating methods will be used to separate algal populations. Multiple media recipes will be utilized to isolate the colonies. The field samples will first dilute to aid in the isolation process. A number of different nutrient media i.e., Guillard's f/2 medium, Jaworski's medium, Von Stosch's medium and Bold's Basal medium will be utilized to isolate the maximum number of colonies from each fresh water sample. In order to optimize the isolation these media components will be modified. Microalgal cultures will be initially separated based on morphological examination of the colonies on an agar nutrient medium. This general classification method will only be used to distinguish isolates on the most basic level. Identification of these isolates to the genus level will be based on the morphology of the individual cells following microscopic examination. The strains will be identified using the methodology of Wehr and Sheath, (2003).

1.2 Characterization of Wastewaters

Wastewater samples will be collected from three different points i.e., from local sewage irrigating pond in 27JB, agro industrial effluents from Sargodha road FSD and livestock effluents from 19RB, Faisalabad, all wastewaters will be filtered. Ammonia nitrogen (NH3–N), nitrate nitrogen (NO3–N), nitrite nitrogen (NO2–N), total nitrogen (TN), total phosphorus (TP), and COD will be determined spectrophotometric method (Wang et al., 2009). The waste water samples will be analysed for different micro organisms like E. coli and fecal Enterococci enumeration etc., by the membrane filtration method according to procedure UNI EN ISO 19250:2013.

1.3 Bioremediation of waste waters using different strains of Chlorella

The construction of waste water treatment plant will be designed in such a way that the sample water which has to be treated will undergo different treatment steps in specially designed parts of the plant.

· Primary treatment column

· Secondary treatment column

· Algal mat tank

· Light source

1.4 Treatment process

A cell column of specific size will be prepared with culture of algae with inlet and outlet valves and the column will be packed with algae.

Batch treatment process

The packed bed column will be tested for its treatment of waste water. The coloumn will be filled with and allowed to remain as such for treatment for a total period of 96hours. At regular intervals samples will be drawn and analyzed for residual COD and BOD. Algal strains will also be removed while sampling and cell content will be estimated in terms of chlorophyll and protein.

 Continuous treatment

Waste waters will be subjected to continuous treatment by algal cells loaded in the packed column. The prepared column will be fed with waste water from bottom to the top at flow rate 25ml/h, 50ml/h and 75ml/h using peristaltic pump. At regular intervals the samples will be drawn over a total optimized period.

1.5  Spectrophotometric analysis for growth of different strains of Chlorella sp.

Samples will be taken from the culture media every day for measurement of optical density at 680 nm (OD680) using a spectrophotometer as the algal density indicator. The growth rate (GR, per day) will be calculated by fitting the OD for the first 3 days of culture to an exponential function: GR =  (lnODt - lnOD0 ) t

where ODo is the optical density at the initial day, ODt is the optical density measured on day t. Each recorded ODt will be corrected by taking away that of the corresponding blank sample.

1.5       Analysis of treated different wastewaters

Inorganic mineral profile of treated wastewaters will be determined by spectral techniques, Toxicity estimation in terms micro-organisms will be carried out following the standard protocol.

     PART II      

Multiple varieties of Solanum lycopersicum (tomatoes) will be grown in pots and will be irrigated as follow

a-     Control- irrigation with normal water

b-    T1- Irrigation with wastewaters (polluted untreated water form three sources i.e., agro industrial effluent, live stock effluent and sewage water )

c-     T2- irrigation with treated polluted water (treated waste water form three sources i.e., agro industrial effluent, live stock effluent and sewage water ) in order to evaluate the effects of waste water on the microbiological soil properties as well as on crop production, the plants will be grown in a vertical setting, using nylon threads disposed between plants collar and iron wires arranged longitudinally in the direction of the plant rows, and fixed to the upper part of the net house, from the ground.

2.1  Growth Parameters

The following germination parameters will be calculated during experimentation.

  • Germination rate
  • Germination percentage (G%)
  • Germination Energy
  • Germination Index
  • Mean Germination Time (MET)
  • Days to 50% Emergence (E50)
  • Coefficient of Uniformity of emergence (CUE)

2.2   Soil chemical analysis


The soil electrical conductivity and pH will be measured on 1:2 (w/v) and 1:2.5 (w/v) aqueous soil extracts, respectively. The available phosphorus will be determined using the sodium bicarbonate method (Olsen et al., 1954), and the total organic carbon (TOC) will be determined by oxidation with potassium dichromate titration of FeSO4, according to Walkley and Black (1934). The soluble NO3–N and NH4–N will be determined according to Keeney and Nelson (1982). All of the soil chemical parameters will be used together with the soil microbiological characteristics for multivariate analysis, to determine the effects of the two soil treatments (i.e., GW, TW) on the dynamics of the bacterial communities.

2.3 Yield and fruit qualitative analysis

During the harvest, the marketable and discarded vegetable will be counted and weighted, to estimate the different components of the pea yield. The following parameters will be measured: mean diameter (equatorial and longitudinal diameter) (D; cm), soluble solids content of the flesh (SSC; ◦Brix), titratable acidity (TA; g citric acid 100 ml−1 fresh juice) (AOAC, 1995), dry matter content (DM; % fruit fresh matter) (AOAC 1990), a*/b* ratio (CI) (Jiménez-Cuesta et al., 1981), and Ca2+, Na+, Mg2+, K+ and nitrate NO3− content.

2.3  Microbiological analysis

The bacteriological analysis of the soil, plant and fruit samples included determination of E. coli, fecal coliforms, and total heterotrophic counts (THCs). These analyses will be conducted by the spread plate method, as follows: 25.0 g of each sample will be weighed and added to 225.0 ml buffered peptone water, homogenized in a stomacher for 180 s, and stored at room temperature for 30 min to allow bacterial cell recovery. Then serial 10-fold dilutions in buffered peptone water will be spread onto plates containing TBX for E. coli, C-EC agar (Biolife) for fecal coliforms, and tryptic soy agar for THC. The plates will be incubated under different incubation temperatures (and times): 37 ◦C for E. coli (24 h), 44 ◦C for fecal coliforms (48 h), and 22 ◦C for THC (72 h).


2.5  Protein profiling


Protein profiling in the high-throughput mode is a most useful technique that allows formation of reference databases for cells and tissues and performance of comparative proteomics. In the proposed protocol protein extraction from plant will be followed by 2D gel electrophoresis (2DE) with subsequent in-gel digestion and identification of soluble proteins by two individual mass spectrometric techniques, tandem matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano-liquid chromatography (nano-LC)-MS/MS. The proposed combined use of these two MS approaches will lead to a very high identification rate of well-separated protein spots from a gel. In the first step 2DE separates high-abundance proteins (those visualized by nonsensitive Coomassie blue staining) that will be subsequently picked, digested and aliquoted for MS applications. Protein samples not identified by MALDI-MS or MS/MS (77% of all spots) will be finally unambiguously identified by nano-LC-MS/MS (total identification rate 94%).

2.5 Anti-oxidant activity determination

Antioxidant activity of plant material will be done by following different parameter i.e., determination of reduction potential, total phenolic and flavonoid content, determination of DPPH scavenging activity etc.

2.6 Risk assessment

Risk to human health (Hazard Quotient, HQ) for intake of heavy metals through consumption different varieties of peas (Pisum sp.) as vegetables grown on wastewaters soils will be calculated using the following relationship (Pierzynski et al., 2000) HQgv = (add/RfD), where HQgv is the hazard quotient to a human from consumption of green

vegetables, add: the average daily dose (mg metal/kg body weight/day) and RfD the reference dose. Theose values of RfD for heavy metals will be used as recommended by WHO, (1982). Daily intake of green vegetable is 200 g/person/day which is recommended amount from nutritional point of view (Hassan and Ahmed, 2000).  Average daily dose (add) will be computed using following relationship:


dose = mc × cf ×  di /bw


where mc is the metal concentrations in plant (mg kg-1) on dry weight basis, cf the fresh to dry weight conversion factor, di the daily intake of green vegetable (kg) and bw the body weight (kg).

Part III

3.1 Isolation and Characterization of telomerase

Telomerase will be sliced out after staining with Coomasie blue. The sliced bands will be transferred in a mortar with little liquid nitrogen and bands will be crushed with the help of pestle till it becomes a fine powder. A viscous solution will be prepared after dissolving in a PBS. After centrifugation at 13000rpm, telomerase will be in the pellets. Telomerase will be characterized to using various bioinformatics tool

3.2 Kinetic and thermodynamic study

Kinetics of folding & unfolded G-quadruplexes, single molecule fluorescence resonance energy transfer method will be used by modifying the methodology of Ying et al., (2003). Thermodynamic study of irreversible thermal denaturation and thermal melting of telomerase will be determined by UV-Visible Spectrophotomeric methods, NMR and native electrophoresis. The denaturation constants (kd) will be calculated and perceptible half-life will be estimated. Arrhenius plot will be used for determination of kd, the activation energy (Ea) for thermal denaturation will calculated from the slope of the plot (Sharma and Sheardy, 2014). Activation enthalpy (ΔH*) for thermal denaturation was calculated according to the following equation:

ΔH* = Ea-RT………………. i)

ΔG* = - RT ln (kd,h/kb.T)………. ii)

ΔS* = (ΔH*- ΔG*)/T……………… iii)


3.4 Study the role of telomerase in cancer & diabetes

Reduced telomere length and impaired telomerase activity have been linked to several diseases associated with senescence and aging. Therefore after complete study of structure, kinetics and thermodynamics, link of telomerase in cancer and metabolic disorders will be studied using animals’ models. Telomerase detection, inhibition for cancer, and telomerase activation for degenerative diseases will also be performed using animal models. 

Who will take these actions?

Dr. Shagufta Kamal with team members of Industrial biotechnology lab

Where will these actions be taken?

Govt. College University, Faisalabad


What are other key benefits?

It will cause the improvement of quality in terms of toxicity reduction caused by effluents used for irrigation purpose.

it will protect environment, biodegradation of different industrial effluents will be the step towards water pollution control, fulfill water deficit of country and protect people from water borne diseases

Help to understand the link of telomerase in normal and abnormal conditions i.e., healthy, diabetic and  cancerous condition 

What are the proposal’s costs?

Item of Expenditure                 Year 1                   Year 2

 Salaries                                  2078.92$              2078.92$

 Operating                            15418.67$             15418.67$

 Equipment                             48123.2$

Sub-Total                            29631.81$               25704.47$

Incentives for Scientists (5% of the project cost)   4155.92$

 Incentive for PM (1% of the project cost)            872.7$

 Grand Total                                       88147.05$


Time line

2 years for experimental work and 1 for Output-1          


 Sample collection from various areas of Faisalabad + Isolation of different strains of Chlorella

 From start

3 months




Optimization of physico-chemical parameters for biodegradation + spectrophotometric study of algal growth




4 months












Quality assurance parameters study + inorganic components determination + microbiological analysis




5 months




Application of different treatments on crop under study + growth parameters analysis




17 months











Isolation, Characterization, Kinetic , thermodynamic study of telomerase to study the link of telomerase with tumour generation and metabolic disorders




22 Months



Component objective

All other analysis mentioned in activity # 1, 2  + Data compilation + statistical analysis + Report writing
















Soil chemical analysis + Yield and fruit qualitative analysis + Microbiological analysis





23 months




Microbiological analysis +

* Protein profiling + antioxidant activity determination




23 months




Results compilation and Final report writing








Data compilation + statistical analysis




23 months




Final report writing




24 months


Related proposals


1.     Chaolan Z., Zhongyi Li., Weiwei Y. Lipping P., Minghua G. and Dokyoung L. 2013. Assessment of metals pollution on agricultural soil surrounding a Lead-Zinc mining area in the Karst region of Guangxi, Chaina, Bull Environ Contam Toxicol, 90, 736-741

2.     Chibuike, G.U., and S.C. Obiora. 2014. “Heavy Metal Polluted Soils: Effect on Plants and Bioremediation Methods.” Applied and Environmental Soil Science.

3.     Harris, E. H. (1989). The Chlamydomonas sourcebook. San Diego: Academic.

4.     Hassan,N.,Ahmed,K., 2000. Intra familiar distribution off  ood in rural Bangladesh. Institute of Nutrition and food Science, University of Dhaka, Bangladesh

5.     Health Organizaion, Geneva.

6.     Kamal, Simi. "Pakistan’s Water Challenges: Entitlement, Access, Efficiency, and Equity." Running on Empty: Pakistan's Water Crisis. Ed. Michael Kugelman and Robert M. Hathaway. Washington, D.C: Woodrow Wilson International Center for Scholars, 2009. 28-44. Web. 16 May 2014.

7.     Keesoo Lee, K. M. L. Eisterhold, F. RindiS. Palanisami, and P. K. Nam. 2014. solation and screening of microalgae from natural habitats in the midwestern United States of America for biomass and biodiesel sources. Nat Sci Biol Med.  5(2): 333–339.