Post treatment of secondary wastewater effluent for irrigation ...

Al-Azhar University-Gaza

Deanship of postgraduate studies

Institute of Water and Environment

Master of Water and Environment science

Post treatment of secondary wastewater effluent for

irrigation purposes using Ulva lactuca algae

By:

Ola Mahmoud Alrawagh

Supervisor:

Dr. Emad Abou Elkhair Masoud

Associated Professor Microbiology

Al-Azhar University-Gaza

A thesis submitted in partial fulfillment of the requirements for the

Degree of Master of Science in Water and Environment

April 2018

i

Declaration

I hereby declare that this submission is my own work and that, to the best of my

knowledge and belief, it contains no material previously published or written by

another person or material which to a substantial extent has been accepted for

the award of any other degree of the university or other institute, except where

due acknowledgment has been made in the text.

Name Signature Date

Ola Al-Rawagh

Copy Right

All rights reversed © 2018: No part of this work can be copied, translated or

stored in any retrieval system, without prior permission of the authors.

ii

Acknowledgment

In the name of Allah, the Most Merciful, the most Compassionate all praise is to

Allah, the Lord of the worlds; and prayers and peace be upon Mohamed His

servant and messenger. First and foremost, I must acknowledge my limitless

thanks to Allah, the Ever-Magnificent; the Ever-Thankful, for his helps and

bless. I am totally sure that this work would have never become truth, without

his guidance. I would like to thank my thesis advisor Dr. Emad Abou Elkhair

Masoud Assoc. Prof. of Microbiology at Al-Azhar University-Gaza. The door to

his office was always open whenever I ran into a trouble spot or had a question

about my research or writing. He consistently allowed this paper to be my own

work, but steered me in the right direction whenever he thought I needed it. I

would also like to thank officials in the institute of water & environment

especially Dr. khaldoun Abu Alhin for his efforts , MEDRC institute for

financial and moral support; also I would like to thank officials in faculty of

science & ministry of agriculture for all support and facilities. Finally, I must

express my very profound gratitude to my mother, my whole family, my

colleagues who participated in this study, and my friends for providing me with

unfailing support and continuous encouragement throughout my years of study

and through the process of researching and writing this thesis. This

accomplishment would not have been possible without them. Thank you.

.

iii

Dedication

This thesis is dedicated to:

The sake of Allah, my creator and my master,

My great teacher and messenger, Mohammed (May Allah bless and grant him),

who taught us the purpose of life,

My homeland Palestine, the warmest womb,

My father's soul (May Allah bless him),

The great martyrs and prisoners, the symbol of sacrifice,

Al-Azhar University; my second magnificent home,

My great mother, who never stop giving of herself in countless ways,

My beloved brothers, sister and nephews; particularly my younger brother for

his efforts,

To all my family, the symbol of love and giving,

My friends who encourage and support me,

All the people in my life who touch my heart

iv

Abstract

Post treatment of secondary wastewater effluent for irrigation purposes

using Ulva lactuca algae

Ola Alrawagh

Background: Due to the continuous population increase and so the

quantities of produced wastewater and the failure of treatment plants in Gaza

strip, huge quantities of raw or partially treated wastewater is discharged into the

sea which increase the presence of the seaweeds like Ulva sp. The macroalgae

of the genus Ulva can have applications in the wastewater treatment.

Objective: To study the effectiveness of Ulva lactuca whole organism

and powder in post treatment of secondary wastewater effluent for irrigation

purposes.

Materials and Methods: Each liter of wastewater was treated by powder

and whole algae. Electrical conductivity (EC), power of hydrogen (pH), chloride

ions and nitrate were measured for raw and treated secondary wastewater .

Microbiological quality for both raw and treated secondary wastewater

effluent were measured. The treated wastewater with the best results was used

for irrigation of Arugula seeds (Eruca sativa) which were bought from

JUMARSTM

Company, as well as others were irrigated by medium salinity water

(5000 mS), and by filter water, and some were fertilized with powder of algae.

Results: The treated wastewater with whole algae and powder algae

showed slight increase in EC, Cl-, pH and decrease in nitrate concentration.

COD levels decreased after treatment with algae. Heavy metals analysis (Fe, Zn,

Pb, Mn, Sr) showed decrease in concentration after algal treatment. Bacteria and

fungi count in treated wastewater with algae decreased as well as Coliform

bacteria and Salmonella and Shigella spp. Arugula plants samples which

v

irrigated with algal treated wastewater and those that fertilized with powder of

algae showed increase in average area of leaves, leaves number as well as

average root length

Conclusion: Ulva lactuca whole and powder algae have a tangible

impact in tertiary treatment of wastewater and reuse in irrigation, also the

powder algae has a tangible impact as a biofertilizer .

Keywords: Ulva lactuca, algae, wastewater treatment, Arugula

vi

ملخص

الري غراض أواستخذامها في بطحلب خس البحر معالجة المياه العادمة المعالجة ثانىيا

عال الرواغ

رجح انشادج انظرزج ف عذد انظكا وتانران سادج كاخ انا انعاديح ورجح نقص كفاءج

انعانجح ف قطاع غشج , رى صزف كاخ كثزج ي انا انعاديح انخاو )فشم(انعذذ ي يحطاخ

ونهطحانة ,(Ulva)وانعانجح جشئا ف يا انثحز يا شذ ي وجىد األعشاب انثحزح يثم انجض أنفا

.انعذذ ي انرطثقاخ ف يعانجح انا انعاديح (Ulva)ي جض أنفا

حهة انكايم أنفا الكرىكا وانظحىق انجفف ي انطحهةهذف انذراطح: هى دراطح كفاءج انط

.عانجح ثاىا الطرخذايها ف أغزاض انزعه يعانجح انا انعاديح ان

طزقح انعم: ذى يعانجح انا انعاديح تظحىق انطحهة وانطحهة انكايم ,ثى ذى قاص انىاد و

وانعانجحانعانجح ثاىا رزاخ ف انا انعاديح انرىصم انكهزتائ,درجح انحىضح,اى انكهىرذ,وان

انا انعانجح ف ر ثاخ اوثى اطرخذنها وي جىدج انكزوتحان, كا وذى اضا قاص تانطحانة

.انجزجز, كا وذى اطرخذاو يظحىق انطحهة انجاف ف ذظذ انجزجز

وذزكش أى انكهىر انقهىحانرائج: أظهزخ انرائج سادج ف ظثح انرىصم انكهزتائ وظثح

واخفاض ف يظرىي انرزاخ نها انعاديح انعانجح تانطحانة. أيا تانظثح نألكظج انظرههك كائا

ذحانم انعاد انثقهح فقذ اخفضد انظثح تعذ انعانجح تانطحانة. ي احح اخزي فقذ أظهزخ رائج

(Fe, Zn Pb, Mn, Sr) نها انعانجح اخفاضا ف يظرىي ذزكش انعاد انثقهح ف عاخ انا

اعذاد انثكرزا اخفضدانعاديح انعانجح. كا حذز اخفاض ف انعذد انكه نهثكرزا وانفطزاخ , كا

يظاحح واعذاد أوراق وأطىال جذور ثاذاخ انثذور ف وطجهد سادج انقىنىح وانظانىال وانشجال.

.انر ذى رها تانا انعانجح, وانثاذاخ انر ذى ذظذها تظحىق انطحهة انجاف

انخالصح : ك اطرخذاو انطحهة انكايم وانظحىق انجاف ف يعانجح انا انعانجح جشئا

.يظحىق انطحهة انجاف ف انرظذ الطرخذايها ف اغزاض انز .كا ك اطرخذاو

انكهاخ انفراحح

انطحانة, يعانجح انا انعاديح, جزجز:

vii

Table of Contents

Title Page

Declaration

Acknowledgment

I

ii

Dedication

Abstract (English)

Abstract (Arabic)

Iii

iv

vi

Table of contents vii

List of Tables

List of Figures

List of Abbreviations

xi

xiii

xv

1. Introduction

1.1 Background

1.2 Geography

1.3 Problem statement

1.4 Main goals of the study

1.5 Objectives of the study

1.6 Environmental Impact

1

1

1

3

4

4

4

2. Literature Review 5

3. Materials and Methods

3.1 Sampling

3.1.1 Sampling of U. lactuca algae

3.1.2 Sampling of secondary treated wastewater

3.2 Study duration

3.3 Tools and equipment of the study

3.4 Experimental and laboratory work

3.4.1 Physicochemical analyses of treated wastewater

3.4.2 Physicochemical, COD, and Heavy metals analyses

18

18

18

18

18

18

19

19

19

viii

3.4.3 Microbiological analyses

3.4.4 Arugula planting

3.5 Data entry and analyses

3.6 limitations

20

20

21

21

4.Results

4.1 Physicochemical analysis Results

4.1.1 Physicochemical analysis of wastewater samples treated with

powder algae for 24 & 48 hr.

4.1.1.1 Electrical conductivity (EC)

4.1.1.2 Power of hydrogen (pH)

4.1.1.3 Chloride (Cl-)

4.1.1.4 Nitrate (NO3-)

4.2 Physicochemical analysis of wastewater samples treated by whole

algae for 24 hr.

4.2.1 Electrical conductivity (EC)

4.2.2 Power of hydrogen (pH)

4.2.3 Chloride (Cl-)

4.2.4 Nitrate (NO3-)

4.3 Physicochemical and heavy metals analysis of wastewater samples

treated by whole and powder algae 24 hr.

4.3.1 Physicochemical analysis of wastewater samples treated by whole

and powder algae 24 hr.

4.3.1.1 Electrical conductivity EC



4.3.1.4 Chemical oxygen demand (COD)

4.3.1.5 Nitrate (NO3-)

22

22

22

22

22

23

23

27

27

28

28

28

32

32

32

32

33

33

34

ix

4.3.2 Heavy metals analysis results of treated wastewater after different

durations and concentrations of powder and whole algae

4.3.2.1 Lead (pb)

4.3.2.2 Ferrous (Fe)

4.3.2.3 Zinc (Zn)

4.3.2.4 Manganese (Mn)

4.4 Microbiological results

4.4.1 Bacterial count of treated wastewater samples after 12 and 24 hr.

4.4.2 Detection of Salmonella & Shigella spp. and fungi treated in

wastewater after 12 & 24 hours

4.4.3 Effect of shaking conditions on bacterial count after 2, 24 hr.

4.4.4 Effect of Shaking on Salmonella & Shigella spp., Coliform & Fungi

occurrence in the 2hr powder algae treatment process


occurrence in the 2 hr. whole algae treatment process




after 24 hr. of treatment with whole algae

4.5 Arugula results

4.5a Area leaves average, leaves number and root length of Arugula

planted in the field with different irrigation sources and fertilizers

4.5b: Area leaves average, leaves number and root length for Arugula

planted in the laboratory with different irrigation sources and fertilizers

5. DISCUSSION

6. Conclusion and Recommendations

38

38

39

39

40

44

44

46

49

52

56

58

61

65

65

68

71

75

x

6.1 Conclusion

6.2 Recommendations

7. Reference

75

75

76

xi

List of Tables

Number Title Page

Table 4.1 Chemical analysis results of treated wastewater by powder

algae after 24 & 48 hours of different concentration

24

Table 4.2 Chemical analysis results of treated wastewater by whole

algae after 24hours of different concentrations

29

Table 4.3 Chemical analysis results of treated wastewater after

different durations and concentrations of powder and whole

algae

35

Table 4.4 Heavy metals analysis results of treated wastewater after

different durations and concentrations of powder and whole

algae

41

Table 4.5 Bacterial count of treated wastewater after 12 and 24 hours 45

Table 4.6 Detection of Salmonella & Shigella spp. and fungi in

treated wastewater after 12 & 24 hours

47

Table 4.7 Bacterial count after 2&24 hours’ treatment under static and

shaking conditions

51

Table 4.8 Detection of Fungi, Salmonella & Shigella spp. and

Coliform after 2hr treatment by powder algae under

Shaking and static conditions

54

Table 4.9 Detection of Salmonella & Shigella spp., Coliform & Fungi

after 2hr treatment by whole algae under Shaking & Static

conditions

57

xii

Table4.10 Detection of Fungi, Salmonella & Shigella spp., and

Coliform after 24hr treatment by powder algae under

Shaking and static conditions

60

Table4.11 Detection of Salmonella & Shigella spp., Coliform & Fungi,

after 24hr treatment by whole algae under shaking and static

conditions

63

Table4.12a Area leaves average, leaves number and root length for

Arugula planted in the field with different irrigation sources

and fertilizers

66

Table4.12b Area leaves average, leaves number and root length for

Arugula planted in the laboratory with different irrigation

sources and fertilizers

69

xiii

List of Figures

Number Figure Page

Fig 1.2 location of El-Sheikh Ejleen wastewater treatment plant 2

Fig 4.1 The mean EC of treated wastewater by powder algae 25

Fig 4.2 The mean pH of treated wastewater by powder algae 26

Fig 4.3 The mean Cl- of treated wastewater by powder algae 26

Fig 4.4 The mean NO3- of treated wastewater by powder algae 27

Fig 4.5 The mean EC of treated wastewater by whole algae 30

Fig 4.6 The mean pH of treated wastewater by whole algae 30

Fig 4.7 The mean Cl- of treated wastewater by whole algae 31

Fig 4.8 The mean NO3- of treated wastewater by whole algae 31

Fig 4.9 The mean EC of treated wastewater by whole & powder algae 36

Fig 4.10 The mean pH of treated wastewater by whole & powder algae 36

Fig4.11 The mean Cl- of treated wastewater by whole & powder algae 37

Fig 4.12 The mean COD of treated wastewater by whole & powder algae 37

Fig 3..4 The mean NO3- of treated wastewater by whole & powder algae 38

Fig 4.14 The mean Pb of treated wastewater by whole & powder algae 42

Fig 4.15 The mean Fe of treated wastewater by whole & powder algae

42

xiv

Fig 4.16 The mean Zn of treated wastewater by whole & powder algae 43

Fig 4.17 The mean Mn of treated wastewater by whole & powder algae 43

Fig 4.18 The mean Sr of treated wastewater by whole & powder algae 44

Fig4.19 Detection of Salmonella & Shigella spp. 48

Fig 4.20 Detection of fungi after 12h treatment 48

Fig 4.21 Detection of fungi after 24h treatment 48

Fig 4.22 Detection of fungi after 2hr treatment 55

Fig 4.23 Detection of Salmonella & Shigella spp. 55

Fig 4.24 Detection of coliform after 2hr treatment 55

Fig4.25 Detection of coliform after 24hr treatment 64

Fig 4.26 Detection of fungi after 24hr treatment 64

Fig 4.27 Detection of Salmonella & Shigella spp. 64

Fig 4.28(a) Arugula in the field 67

Fig 4.28(b) Arugula in the field 67

Fig 4.29 Arugula in the laboratory 70

xv

List of abbreviations

AWWTP Algae Wastewater Treatment Plant

BOD Biochemical Oxygen Demand

COD Chemical Oxygen Demand

EC Electrical conductivity

G Gram

hr.

IAA

Hour

Indole-3-acetic acid

MOH Ministry Of Health

N Number

NPK complex fertilizer

P

pH

Powder

Power of hydrogen

Sh Shaken

SLF Seaweed Liquid Fertilizer

SS agar Salmonella Shigella agar

Stww secondary treated wastewater

TGP Glutamic Pyruvic Transaminase

TSB

TSS

Trypticase Soy broth

total suspended solids

U. lactuca Ulva lactuca

un-sh un-shaken (Static)

W Whole

1

1. Introduction

1.1 Background

Ulva lactuca, (sea lettuce), is an edible green alga in the division Chlorophyta,

species of the genus Ulva. It's a thin flat green alga growing from a discoid

holdfast; the margin is somewhat ruffled and often torn. It may reach 18

centimeters or more in length, though generally much less, and up to 30

centimeters across "Ulva lactuca". "Green to dark green in color, this species

in the Chlorophyta is formed of two layers of cells irregularly arranged, as seen

in cross-section. The chloroplast is cup-shaped in some references but as a

parietal plate in others, with one to three pyrenoids. There are other species of

Ulva which are similar and not always easy to differentiate. The distribution is

worldwide: Europe, North America (west and east coasts), Central America,

Caribbean Islands, South America, Africa, Indian Ocean Islands, South-west

Asia, China, Pacific Islands, Australia and New Zealand (Burrows, 1991;

"Ulva lactuca Linnaeus").

1.2 Geography

Gaza Strip is situated on a relatively flat coastal plain. Temperatures average in

the mid-50s F (about 13 °C) in the winter and in the upper 70s to low 80s F

(mid- to upper 20s C) in summer. The area receives an average of about 12

Living conditions in the Gaza Strip are typically poor for a number of reasons:

the region’s dense and rapidly increasing population (the area’s growth rate is

one of the highest in the world); inadequate water, sewage, and electrical

services; high rates of unemployment; and, from September 2007, sanctions

imposed by Israel on the region. Agriculture is the economic mainstay of the

employed population, and nearly three-fourths of the land area is under

cultivation (Gaza Strip). As the Israeli military occupation of the Palestinian

territory (oPt) enters its 50th consecutive year, humanitarian needs remain high.

2

Poverty and unemployment have driven more than a quarter of all households

into food insecurity; an estimated one million people are in need of health and

nutrition interventions; and 1.8 million people require some form of protection

assistance. Overall, nearly half of all Palestinians living across the West Bank,

including East Jerusalem, and in the Gaza Strip – some two million people in all

– will need some form of humanitarian assistance in 2017(Humanitarian

Response Plan).

Fig.1.2 location of El-Sheikh Ejleen wastewater treatment plant

3

1.3 Problem statement

The Gaza Strip is facing immense challenges related to water, sanitation and

hygiene (WASH), which pose significant health risks to its 1.8 million residents

and constrain socioeconomic development. Groundwater from the coastal

aquifer is the only water resource available in the Gaza Strip. However,

abstraction from the aquifer stands at four times the aquifer‘s recharge rate at

200 MCM/year, and is expected to rise to 260 MCM/year by 2020. In addition,

more than 96 per cent of abstracted water is polluted and not fit for human

consumption due to high salinity levels from sea water intrusion and high nitrate

levels from excessive use of agrochemicals and wastewater infiltration. The

water supply in the Gaza Strip is estimated at 90 l/c/d, below acceptable water

quantity standards of 100 l/c/d recommended by the World Health Organization

(WHO). Furthermore, the absence of sufficient wastewater treatment facilities

results in approximately 35 MCM/year of untreated/partially treated wastewater

discharged into the sea along the Gaza coast (reliefweb).

4

1.4 Main goals of the study

To study the effectiveness of Ulva lactuca whole organism and powder in post

treatment of secondary wastewater effluent for irrigation purposes.

1.5 Objectives of the study

1. Study the capacity of Ulva lactuca as biofilters for post treatment of

secondary wastewater effluent.

2. Evaluate of efficiency of Ulva lactuca powder and whole algae for

post treatment of secondary wastewater effluent.

3. Introduce a method to reduce the risk of marine environment when

pumping wastewater into the Sea.

4. Contribute to safe re-use of wastewater for maintaining the farmer's

health when they use treated wastewater for irrigation.

1.6 Environmental Impact

The proposed project will work on the positive impact on the environment from

several aspects as follow:

Reduce the risk to the marine environment when pumping wastewater into

the Sea.

Improvement of reuse wastewater quality in irrigation.

Keep health of human, animals, plants and soil.

5

2. Literature review

Seaweeds, one of the important marine living resources could be termed as the

futuristically promising plants. These plants have been a source of food, feed

and medicine in the east as well as in the west, since ancient times. Although,

seaweeds in India are used for industrial production of agar and alginate and as a

fertilizer, it is yet to be utilized on a large scale for various purposes, which is

not being done, due to lack of its awareness among the Indian populace. In order

to harness the rich potential of seaweeds in India, the present limited use needs

to be diversified into other contemporary areas of application. Being a plant of

unique structure and biochemical composition, seaweed could be exploited for

its multi-functional properties in the form of food, energy, medicine and

cosmetics. In addition to the comprehensive view on its uses, the article also

calls for the need to implement biotechnological tools for sustainable

management of seaweed resources. All in all, an attempt has been made to

highlight the prospects of seaweed in India in the modern context (Dhargalkar

and Pereira, 2005). Also, a study of cultivation of Ulva lactuca with manure

for simultaneous bioremediation and biomass production was carried out in

Denmark results in that the potential of liquid manure as sole nutrient source for

cultivation of Ulva lactuca was investigated with the perspective of utilizing the

produced biomass for feed and/or energy. Algae grown with manure

demonstrated equal growth rates to algae grown with standard food to mass ratio

"F/M"- medium. The optimum manure concentration, expressed as ammonium

concentration, was 25μM. At these conditions, the biomass produced was

potentially suitable for anaerobic digestion, due to a relative high

carbon/nitrogen ratio (approximately19). At higher manure concentrations,

tissue concentrations of nitrogen, phosphorus, proteins, and amino acids

increased, making the biomass less suitable for anaerobic digestion but

potentially interesting as a feed. Cultivating U. lactuca with manure as nutrient

6

source has potential in terms of bioremediation as well as production of

bioenergy and protein-feed. U. lactuca has a capacity for high rates of nutrient

assimilation, especially ammonium (NH4+), and grows well in eutrophic waters

that qualify this species for bioremediation purposes (Nielsen et al., 2012).

The marine environment is home to a taxonomically diverse ecosystem.

Organisms such as algae, molluscs, sponges, corals, and tunicates have evolved

to survive the high concentrations of infectious and surface-fouling bacteria that

are indigenous to ocean waters. Both macroalgae (seaweeds) and microalgae

(diatoms) contain pharmacologically active compounds such as phlorotannins,

fatty acids, polysaccharides, peptides, and terpenes which combat bacterial

invasion. The resistance of pathogenic bacteria to existing antibiotics has

become a global epidemic. Marine algae derivatives have shown promise as

candidates in novel, antibacterial drug discovery. The efficacy of these

compounds, their mechanism of action, applications as antibiotics, disinfectants,

and inhibitors of food borne pathogenic and spoilage bacteria were intensively

investigated. As with all areas of drug discovery, extensive clinical trials will be

required to determine the in vivo fate of marine antibacterial extracts on

mammalian cells in terms of first pass metabolism and possible toxicity. The

marine environment is home to an immense taxonomic diversity that has

remained relatively unexplored in drug discovery by terrestrial standards. In

order to overcome the challenges to marine natural product development a

multi-disciplinary strategy can be adapted which utilizes nascent technologies

and tools for developing novel antimicrobial agents (Shannon and Abu-

Ghannam, 2016).

In a study by Loukil who studied biochemical parameters in Annaba, Algeria,

which were measured to assess the effects of exposure after fertilizers handling

in complex fertilizer "NPK" unit workers all the recorded changes in the

biochemical studied parameters, the obtained results after the assay of serum

7

biochemical parameters in workers are all in the standards. However, a highly

significant increase in the concentration of creatinine in both groups and a

significant increase of Glutamic Pyruvic Transaminase "TGP" were noted in

employees aged between 41-50 years compared to the control group. It was

concluded in the light of their results that the health effects such as perturbation

in the biochemical parameters can be associated with exposure to high

concentrations of the atmospheric pollutants and suspended particles in

workplace (Loukil et al., 2015).

Seaweed extract and powder are a new generation of natural organic fertilizers

containing highly effective nutritious and promotes faster germination of seeds

and increase yield and resistant ability of many crops. Unlike, chemical fertilize

nonpolluting and non-hazardous to humans, animals and birds (Dhargalkar,

2014). Fertilizers derived, from natural sources like seaweeds are found to be

viable alternatives to fertilizing input for agricultural crops due to its high level

of organic matter, micro and macro elements, vitamins, fatty acids, also rich in

growth regulators (Crouch et al., 1993). The growth promoting effect of liquid

extracts of seaweeds on seed germination (Venkataraman et al., 1993; El

Sheekh and El-Saied, 1999), vegetative growth (Sekar et al., 1995), and

biochemical characteristics (Thirumalthangam et al., 2003), in agricultural

crops have reported. Ecosystem is the treasure place for many natural resources

Anandhan and Sornakumari, 2011). Although seaweed extracts are widely

advertised for use as fertilizers, agricultural scientists have only rarely

investigated their effects. Many different beneficial effects have been described

following the application of seaweed extracts to crops (Abetz, 1980). Seaweed

extracts have been shown to increase the crop yield, improve growth, induce

resistance to frost, fungal and insect attacks. In modern agriculture, extensive

application of chemical fertilizers caused imbalance of soil nutrients. So, search

for natural organic inputs for sustainable crop productivity has been emphasized.

8

Bio-fertilizers are a 100% natural organic fertilizer that helps to provide all the

nutrients to the soil required for the plants. Bio-fertilizers based on renewable

energy sources are most effective supplement to chemical fertilizers. Seaweeds

are rich source of growth promoting substances (Sylvia et al., 2005), such as

Indole-3-acetic acid "IAA", kinetin, zeatin and gibberellins (Zodape et al.,

2009), auxins and cytokinins (Zhang and Ervin, 2004), metabolic enhancers

(Zhang and Schmidt, 1997), macro and micro elements (Strik et al., 2003)

amino acids, vitamins and beneficial results from their use in crop plants like

early seed germination and establishment, improved crop performance and

yield, elevated resistance to biotic and abiotic stress and enhanced post-harvest

shelf life of seeds (Hankins and Hockey, 1990, Guiry and Blunden, 1991;

Booth, 1965). Students at the University of Wisconsin, Madison, discovered the

effects of chemical fertilizers are compounded when mixed with a single

pesticide. They discovered altered immune, endocrine and nervous system

functions in mice, as well as influence on children's and fetus's developing

neurological, endocrine and immune systems. These influences "portend change

in ability to learn and in patterns of aggression (Kristensen et al., 2016).

Groundwater contamination has been linked to gastric cancer, goiter, birth

malformations (Khandare, 2013) and hypertension testicular cancer

(Kristensen et al., 1996), and stomach cancer (Zaldívar and Robinson, 1973).

Nitrogen groundwater contamination also contributes to marine "dead zones".

The increase in the water-soluble nitrates creates an influx of plant-life, which

eats up oxygen and starves out fish and crustaceans. This has an impact not only

on the aquatic ecosystem, but also on local societies who depend on food

sourced from those areas (Venkataraman, 2008). Another aspect determined by

Divy et al., who concluded that seaweeds extract of Ulva lactuca have an

ameliorating effect on Abelmoschus esculentus seeds under salt stress in India;

because of the presence of growth hormones, nutrients and other important

physiochemical compounds. So, the supplementation of Seaweed Liquid

9

Fertilizer ''SLF'' could be used as a biological amendment in soil reclamation

technique which can boost food production not only in cultivated lands but also

in barren soils accumulated with salt. Further study needed to test the influence

of SLF on later growth and yield of Abelmoschus esculentus cultivated in salt

stress (Divya et al., 2015). Besides their use as food, the macroalgae of the

genus Ulva can also have applications in the removal of nutrients from effluent

waters of sewage, industry and mariculture. Studies showed that some Ulva

species have been tagged as pollution indicator due to their biomass

accumulation when they inhabited in highly polluted waters (Lahaye, 1998;

Largo et al., 2004; Wolf et al., 2012) For instance, U. lactuca has proven to be

a good seaweed biofilter in the treatment of fishpond effluents (Neori et al.,

2003) The opportunistic growth ability of these seaweeds makes them good

candidates for water recycling in integrated invertebrates or fish aquaculture

systems and of urban waters (Costa et al., 2010). For decades, macroalgae

species of the genus Ulva (Chlorophyta) have received interest as biomass

sources for food and feed purposes, due to its high contents of vitamins, trace

metals, and dietary fibers (Lahaye and Jegou, 1993). Recently, global warming

issues and limited supply of fossil fuels has drawn attention to algae as energy

crop as well. A promising and yet realistic estimate of the production potential

of Ulva lactuca cultivated in a northern latitude land-based facility is by Bruhn

who found this to be 45 t DW ha−1 y−1 corresponding to three to five times the

production of conventional energy crops, such as wheat straw, willow,

Mischanthus, or maize (Bruhn et al., 2011). An attempt has been made by

(Dhargalkar, 2005) to highlight the prospects of seaweed in India in the

modern context; they concluded that firstly, they should look for popularizing

seaweeds as health food because they are rich and easily available source of

vitamins, minerals and trace elements for poor people. In this regards, research

institutes/agencies and private entrepreneurs should come forward with

scientific and technical knowledge and marketing expertise. The value added

11

product is emerging recently as an area of high growth, wherein a small amount,

of seaweed material that produces high quality product needs to be

manufactured. Secondly, use of seaweeds in recuperating the human body from

various ailments needs to be emphasized. In spite of the fact that many

government institutes, agencies and private entrepreneurs are screening

seaweeds and other marine organisms for drugs, we have not yet made any

substantial, seaweeds and other marine organisms for drugs, we have not yet

made any substantial breakthrough in this field. Preliminary clinical trials have

shown the effectiveness of seaweeds on human health. There exists great

potential for developing drugs to treat cancer, AIDS and other diseases that are

killing thousands of people every year (Tease and Nutri, 1981; Tease, 1981).

Marine resources represent an interesting source of active ingredients for the

cosmetics industry. Algae (macro and micro) are rich in proteins, amino acids,

carbohydrates, vitamins (A, B, and C) and oligo-elements such as copper, iron

and zinc. All those active principles play roles in hydration, firming, slimming,

shine and protection. There are many properties that will be put forward by the

cosmetic industries (Guillerme, 2017). Besides its high growth potential, U.

lactuca has a capacity for high rates of nutrient assimilation, especially

ammonium (NH4+), and grows well in eutrophic waters which qualify this

species for bioremediation purposes. (Gevaert et al., 2007) has reported

assimilation rates of NH4+ in the range of 50−90 μ mol N g−1 DW h−1 among

different Ulva species, and these species have been verified as successful

biofilters of wastewaters from aquaculture (Martinez-Aragon et al., 2002).

Aquaculture effluents are rich in NH4+ and therefore highly suitable as nutrient

source for Ulva species. Integration of Ulva in multitrophic aquaculture serves a

binary purpose: the production of biomass simultaneously with a removal of

nutrients from the effluent waters of the aquaculture, thereby reducing the load

of dissolved nutrients to the environment. In modern society we have an

increasing need for re-thinking waste streams in order to sustainably manage

11

natural resources (Clark and Deswarte, 2015). Phosphorus (P) and nitrogen

(N) are both essential elements in agricultural fertilizers and both are causing

environmental concerns when washed out into the aquatic environment. P is a

limited resource and efforts are to an increasing extent being made to retain and

recycle this element in order to defer global P made to retain and shortage as

well as to limit eutrophication of the aquatic environment (Carpenter and

Bennett, 2011). One of these efforts is made in wastewater treatment plants,

where P is retained in the sediment sludge and recycled as fertilizer on

agricultural land (Van Loosdrecht et al., 1997). The liberation of N to the

aquatic environment and the atmosphere is also under increasing management

coastal waters (Compton et al., 2011). N is not as such a limited resource.

Approximately 5 billion metric tons of N is contained on Earth in atmosphere,

ocean, soil, biota and sedimentary rock. However, the unlimited N resource is

the free N2 in the atmosphere not the reactive or biologically available N, which

constitutes less than two percent of the nitrogen on Earth. From being spread on

agricultural land and all through the food chain, reactive N is lost to the

representing a financial loss to agricultural ecosystems, and an environmental

threat to background terrestrial and aquatic ecosystems (Galloway, 1998). There

are good arguments for recycling the reactive nitrogen: conversion of the free N2

in the atmosphere into biologically available NH4+ for fertilizer via the Haber–

Bosch process requires huge amounts of energy, approximately 1% of the

world’s annual energy supply (Smith, 2002). At the wastewater treatment

plants, resources are spent removing the biologically available nitrogen to avoid

eutrophication of adjacent water bodies. Here the cycle is closed as reactive

inorganic forms of nitrogen are converted back into free N2 through microbial

activity and re-liberated to the atmosphere (Schmidt et al., 2003). Rethinking

this pathway, by recycling the biologically available nitrogen could offer a more

sustainable and less energy demanding resource flow, while still satisfying the

need for nitrogen fertilizer as well as for removal of nutrients from wastewater.

12

A literature survey of the marine macroalgae of the genus Ulva (Phylum

Chlorophyta) in Portugal covering the period of 1985 to 2012 by Silva et al.,

(2013) who discovered the secondary metabolites isolated from members of this

genus and biological activities of the organic extracts of some Ulva species as

well as of the isolated metabolites were discussed. The emphasis on their

application in food industry and their potential uses as biofilters are also

addressed (Silva et al., 2013). Bioremediation of different types of nutrient rich

waste could be–and had been suggested as– part of the solution (Neori et al.,

2004). The majority of macro algae grow submerged in water and all are

capable of taking up dissolved nutrients across the entire surface area. Ulva

(Chlorophyta) is one genus of opportunistic green macroalgae that owing to its

foliose morphology has efficient nutrient uptake and high growth rates, enabling

these organisms to proliferate fast upon fortunate, conditions (Pedersen and

Borum, 1996). Using Ulva species for extraction of nutrients from nutrient rich

wastewater is presently applied in land based aquaculture (Bartolia et al.,

2005). The protein rich algae biomass can be applied as a feed supplement for

cultivated finfish shrimps or shellfish. Green macroalgae have also been tested

for bioremediation of agricultural wastewater: marine species such as Ulva

lactuca (Nielsen et al., 2012), and multispecies cultures of freshwater algae, that

were following successfully tested as a slow release fertilizer (Mulbry et al.,

2005). The efficiency of Ulva to extract nutrients from urban wastewater has

also been documented (Tsagkamilis et al., 2010). In addition to the effect of

bioremediation Ulva has an antibacterial effect on the wastewater, thus reducing

the health related problems of the wastewater (Lu et al., 2008). Reject water is

another interesting type of wastewater from a phyco remediation point of view

at an increasing number of wastewater treatment plants; the sediment sludge is

utilized for biogas production through anaerobic digestion, before being spread

on agricultural land as a fertilizer.

13

A study in Australia using Ulva species for extraction of nutrients from nutrient

rich wastewater was presently applied in land based aquaculture by Castine who

discovered that integration of algal and macrophyte cultures can also be

optimized to increase wastewater treatment efficiency and profitability of the

farms, and be tailored to local flora and regional requirements for specific end-

products to engage with synergistic industrial ecology (Castine et al., 2013).

Also in Denmark, Sodea found out that, U. lactuca grew well on reject water.

Growth dynamics and biochemistry of Ulva cultivated with reject water were

not different from Ulva cultivated with NH4+

. Cultivating Ulva with N

concentrations of 50–100µm resulted in maximal growth rates and high uptake

rates of N and P. The biomass produced at these nutrient concentrations was rich

in protein, and the content of heavy metals did not exceed limit values for use

for animal feed or soil improvement. The challenges in future are the area needs

and balancing high production costs with high value utilization of the algae

biomass (Sodea et al., 2013). An alternative method for wastewater treatment

was developed in India, using a special constructed column treatment plant. The

microbial mats used for the study are dominated by the algal species like Ulva

sp., Cladophora sp. and Chlorella sp. Various parameters like Chemical oxygen

demand (COD), Biological oxygen demand (BOD) were observed after the

treatment process in three phases, free cell process, batch process and

continuous flow process. Better results in percentage of reduction were observed

with continuous flow process using chlorella sp. and the reduction rate was 52.1

(COD) and 50.8 (BOD) along with changes in dissolved oxygen (DO) and pH.

The results clearly enunciate the potentials of chlorella sp. for employing in

wastewater treatment. This was an innovative, economical and environmentally

safe alternative for treating wastewater (Gvns et al., 2011).

Algae Wastewater Treatment Plant (AWWTP) utilizes sunlight and local algae

species to remove nutrients and other contaminants from wastewater while

14

generating of large quantities of electricity. The AWWTP is an emissions-free

process. Recently, algae have become significant organisms for biological

purification of wastewater since they are able to accumulate plant nutrients,

heavy metals, pesticides, organic and inorganic toxic substances and radioactive

matters in their cells/bodies (Janus and van der Roest, 1997; Advanced Algae

Bioremediation System, 2018).

Industrial activities often produce wastewater containing large amounts of heavy

metals that are discharged into environment, and, that become an important

source of pollution. Due to their toxicity mobility and accumulation tendency,

the contamination of aqueous environments with heavy metals is an important

issue with serious ecological and human health consequences (Freitas et al.,

2008; Sulaymon et al., 2013). Therefore, it is, desirable to eliminate the heavy

metals from industrial wastewaters and this could be also important from

economical consideration (Montazer-Rahmati et al., 2011). Among heavy

metals, Pb(II), Zn(II) and Co(II) are the most common contaminants of

wastewater, due to their varied uses in different industrial activities, and have

priority for removal from aqueous waste stream (Mendoza et al., 1998). Heavy

metals toxicity and the danger of their bioaccumulation in food are the major

environmental and health problems of our modern society. Primary sources of

pollution are from the burning of fossil fuels, mining and melting of metallic

ferrous ores, municipal wastes, fertilizers, pesticides and sewage sludge (Peng

et al., 2006) with the most common heavy metals contaminants being cadmium

(Cd), chromium (Cr), copper (Cu), lead (Pb), nickel (Ni) and zinc (Zn). Most of

the organic pollutants are degraded or detoxificated by physical, chemical and

biological treatments before released into the environment. Although the

biological treatments are a removal process for some organic compounds, their

products of biodegradation may also be hazardous. Moreover, some non-

degradable compounds like the heavy metals ions discharged into the

15

environment along with the treated waste compounds can cause problems due to

non-degradability, bioaccumulation, biomagnification and transport to long

distances. Many agricultural wastes, including barks, manures and composts,

contain high levels of lignocellulosic materials. This paper gives an account of

the toxicity of Cd, Cr, Cu, Ni, Pb and Zn and assesses the performance of green

biosorbents (vermicomposts, fungal biomass, biomass of non-living, dried

brown marine algae, agricultural wastes and residues, composite chitosan

biosorbent prepared by coating /chitosan, cellulose-based sorbents and bacterial

strains) that have been tested for their removal by adsorption from contaminated

waters. Mini-review is an abridged version of the book chapter ‘Heavy metals:

Toxicity and removal by biosorption, published in the book Environmental

Chemistry for a Sustainable World (Lichtfouse et al., 2012). The removal of

heavy metals ions is now steadily shifting from the use of conventional

adsorbents to the use of biosorbents for reasons of degradability and

environmental sustainability. This shift is contextualized within the concept of

Green Chemistry (Mudhoo et al., 2012).

Bulgariu and Bulgariu detected in their study in Romania that, the biosorptive

characteristics of alkaline treated waste marine green algae (Ulva lactuca) have

been investigated for the removal of Pb (II), Zn (II) and Co (II) ions form

aqueous solution, in comparison with untreated waste biomass. The

experimental results have indicated that the alkaline treated waste marine green

algae have better biosorption characteristics than untreated waste biomass, and

have potential for serving as biosorbent for removal of heavy metals from

aqueous solution (Bulgariu and Bulgariu, 2014). Also in a study in Haryana,

India by Mudhoo, industrialization and urbanization have resulted in increased

releases of toxic heavy metals into the natural environment comprising soils,

lakes, rivers, ground waters and oceans. Research on biosorption of heavy

metals has led to identification of a number of microbial biomass types that are

16

extremely effective in bioconcentrating metals. Biosorption is the binding and

concentration of adsorbate from aqueous solutions by certain types of inactive

and dead microbial biomass. The novel types of biosorbents presently reviewed

are grouped under fungal biomass, biomass of non-living, dried brown marine

algae, agricultural wastes and residues, composite chitosan biosorbent prepared

by coating chitosan, cellulose-based sorbents and bacterial strains. The reports

discussed in the review collectively suggest the promise of biosorption as a

novel and green bioremediation technique for heavy metal pollutants from

contaminated natural waters and wastewaters (Mudhoo et al., 2012). Kalesh

and Nair reached to the context of use of marine algae as biological indicators of

heavy metal pollution in coastal waters, six species of marine algae collected

from the southwest coast of India were analyzed for the levels of heavy metals

(Ni, Cr, Sr, and Ag). Interspecies and interclass variations were determined on a

spatial and temporal scale. The metal contents varied in the ranges (Kalesh and

Nair, 2005). Tsagkamilis used seaweeds for phosphate absorption which was

examined as a tertiary treatment in sewage treatment plants, to improve the

water quality and reduce eutrophication risks. The data came from both

laboratory and field experiments that took place on Ios Island sewage treatment

plant. Three different macroalgae were tested and Ulva lactuca was finally

chosen thanks to its high survivability in low salinity waters. Since the main

restrictive factor was low salinity, workers initially established the ratio of

seawater effluent that combined satisfactory viability with maximum phosphate

absorption. The biomass growth under these conditions was also examined.

Based on the above results, workers designed a continuous-flow system with a

1/4 volume per hour water turnover, in a mixture of 60% sewage effluent: 40%

sea water and 30 g L-1

initial biomass of U lactuca that must be renewed every

10 days. Under these conditions and time frame, the phosphate content of the

effluent was reduced by about 50% (Tsagkamilis et al., 2010). In Italian study

made by Lawton, he discovered that the majority of macroalgae grow

17

submerged in water and all are capable of taking up dissolved nutrients across

the entire surface area. Ulva (chlorophyta) is one genus of opportunistic green

macroalgae that owing to its folios morphology has efficient nutrient uptake and

high growth rates, enabling these organisms to proliferate fast upon fortunate

conditions (Lawton et al., 2013). A vegetative clone of Ulva lactuca was

selected by Vandermeulen and Gordin, for mass culture and nutrient uptake

experiments with fish pond wastewater. The plants could not survive on the

macronutrients provided by a weekly pulse of wastewater. A continuous supply

of fish pond wastewater was required to maintain good growth rates. An

‘uncoupling’ of growth rate and thallus nitrogen content was observed. The

plants were able to store nitrogen from a pulsed ammonium supply and allot the

nitrogen reserves to new tissue growth. Plants with slower growth rates or a

continuous supply of ammonium had higher thallus nitrogen content. Ulva

efficiently removed up to 85% of the ammonium from fish pond wastewater in

darkness or light independently of temperature fluctuations (Vandermeulen

and Gordin, 1990).

18

3. Materials and Methods

3.1 Sampling:

3.1.1 Sampling of U. lactuca algae:

Samples of floating U. lactuca were collected from Gaza Sea's rocks and beach

from April to May, 2016. They were identified washed with filtered water very

well to eliminate the concentration of salts, dried in shade then by oven at 55c°at

the ministry of agriculture laboratories, after that, they were grinded by blinder

to have a powder algae, in addition some algae remained as whole algae.

3.1.2 Sampling of secondary treated wastewater:

Secondary treated wastewater was collected from Al-Shaikh Ejleen plant (Gaza

city), Gaza strip, Palestine, by gallons and bottles.

3.2 Study duration:

The study duration continued for approximately 13 months, from April 2016

(when algae appear in the sea) to April 2017.

3.3 Tools and equipment of the study:

1- Six gallons to collect wastewater samples (16 L)

2 -Thirty buckets to filter wastewater after treatment.

3- Many bottles and sterile cups to transport wastewater.

4- Sterile gauzes for filtration.

5- Cups for planting.

6- Imported Arugula seeds (Eruca sativa variety: JU-R0111. LOT No:

R02333616EE03) was from JUMARSTM Company.

19

7- Microbiological and chemical laboratory supplies.

8- Isolated soil (peat moss) and compost.

3.4 Experimental and laboratory work:

Each liter of wastewater was treated by (1, 2, 5 g) of powder and whole algae

for 24, 48hours (Three replicas for each conc.) respectively.

3.4.1 Physicochemical analyses of treated wastewater

1- pH using pH meter checked at the laboratory (pH Testing in Wastewater

Treatment).

2-Electrical conductivity (EC) checked using conductivity meter (Munoz-

Carpena et al., 2005).

3-Nitrate NO3- were estimated using uv.vis double beam spectrophotometer

(Ultra violet Spectrophotometric Screening Method, 2018).

4-Chloride ions Cl- were estimated by Titration (Mohr’s Method).

*These tests were checked at the Ministry of Agriculture laboratories.

3.4.2 Physicochemical, COD, and Heavy metals analyses

According to the primary results, the process was repeated using 1, 2g of

powder and 1g whole algae for 12 and 24 hours (Three replicas for each conc.).

1-Physicochemical analyses (pH, EC, NO3-, Cl

-) were estimated again.

2- COD were estimated by spectrophotometer (Bullock et al., 1996),

3- Heavy metals were determined by atomic absorption spectrometry (AAS)

(Baysal et al., 2013).

21

*Both of COD and heavy metals tests were checked at the Institute of Water

and Environment Al-azhar University.

3.4.3 Microbiological analyses

Microbiological analyses (bacterial and fungal) were estimated, whereas,

Nutrient agar (for bacterial count), SS agar (for detection of salmonella

&shigella spp.), MacConkey agar (for detection of total coliform; E.colia ,

Klebsiella, Enterobacter app) , and Sabouraud dextrose agar( for detection of

fungi). All these media were prepared, and then were inoculated by the treated

samples (Hauser, 2006; Growing Bacteria in Petri Dishes, 2018).

-Finally, the treatment process was repeated by the treatment of the same

amount of wastewater by (1, 2, 5 g) of powder and whole algae for 2 and 24

hours. The treatment process was done in static and shaking conditions at 50

rpm. Isolation, enumeration and characterization of the bacteria and fungi were

done according to the standard methods mentioned above.

*these tests were estimated at microbiology laboratory, faculty of science in Al-

azhar university.

3.4.4 Arugula planting

The treated wastewater with the best results (1, 2 g powder and 1g whole, 24

hours) was used for irrigation of Arugula seeds (Eruca sativa) variety: JU-

R0111. Lot. No: R02333616EE03 bought from JUMARSTM

Company, as well

as others were irrigated by medium salinity water (5000 mS) and by filtered

water (each with 3 replicas). Thirty-six cups (each one with a plate) were filled

by 50 g of the following formulations, 6 of them were filled by 2/3 peat moss,

compost, 3 of them were irrigated by filter water, and 3 by medium salinity

water, those considered as control. Three cups were filled by peat moss,

compost and powder algae (5 g). Twelve cups were filled by peat moss and

21

different amounts of powder algae 0.25, 0.5, 1.5, 10 g, and these were irrigated

also by medium salinity water (5000 mS). The last 12 cups were filled only with

peat moss but irrigated by the different concentrations of treated wastewater.

Five seeds were sowed in each cup, irrigated each 3 days by 30 ml water for 3

weeks then they were irrigated weekly by the same amount of water. When the

seeds grew and formed leaves, the best one was chosen. Each cup was put in

small plate, and exposed daily to sunlight evenly in field from October to Marsh

in16 -18°C average temperature and 68.5% average humidity, 19°C average

temperature, and 58% average humidity in laboratory. Crop was incubated

under controlled conditions until the rose started to form. The length of roots,

area and number of leaves were calculated.

3.5 Data entry and analysis:

The Statistical Package for the Social Sciences (SPSS) used in data entry,

statistical analysis and treatments. Descriptive, frequencies, central tendency,

dispersion measurements, cross tabulation and statistical treatment tests like t-

test, ANOVA, chi-square, correlation and regression used to clarify the

relationship between the research variables.

3.6 limitations:

The worst is power shortage, which causes the delay of work and sometimes

cause corruption, as well as the lack of chemicals necessary for many of

laboratory tests and this is what the occupation imposed on us.

22

4 Results

4.1 Physicochemical analysis Results

4.1.1 Physicochemical analysis of wastewater samples treated with powder

algae for 24 & 48 hr.

4.1.1.1 Electrical conductivity (EC)

Physicochemical analysis (EC, pH, Cl- & NO3

-) results of treated wastewater by

powder algae after different durations and concentrations are presented in Table

4.1. In study groups, the mean EC(mS) was 6±0, 7.0±0.2, 7.3±0.2, 8.3±1.2,

7.9±1.5, 7.5±0.3 and 9.4±1.3 mS for secondary treated wastewater, treated

wastewater with 1 g powder algae-24 hr., treated wastewater with 2 g powder

algae-24 hr., treated wastewater with 5 g powder algae-48 hr., treated wastewater

with 1 g powder algae-48 hr, treated wastewater with 2 g powder algae-48 hr. and

treated wastewater with 5 g powder algae-24hr, respectively (Figure4.1). There

was statistically significant difference in secondary treated wastewater vs. treated

wastewater with 1 & 5 g powder algae-48 hr. and treated wastewater with 1 & 2 g

powder algae-24 hr. p ≤ 0.05.


Regarding pH among treated wastewater by powder algae after different durations

and concentrations was 7.4±0, 8.2±0.2, 7.9±0.3,7.9±0.2, 8±0.1, 8.2±0.7 and

8.1±0.1 for secondary treated wastewater, treated wastewater with 1g powder

algae-24 hr., treated wastewater with 2 g powder algae-24 hr., treated wastewater

with 5 g powder algae-48 hr., treated wastewater with 1 g powder algae-48hr,

treated wastewater with 2 g powder algae-48hr and treated wastewater with 5 g

powder algae-24 hr., respectively (Figure 4.2).The results indicated that there was

statistically significant difference in secondary treated wastewater vs. treated

wastewater with 1 g powder algae-24 hr., treated wastewater with 1 g powder

algae-48 hr., treated wastewater with 2 g powder algae-48 hr. and treated

23

wastewater with 5 g powder algae-48 hr. p ≤ 0.05. In contrast, no significant

difference for secondary treated wastewater vs. treated wastewater with 2 g

powder algae-24 hr., treated wastewater with 5 g powder algae-24 hr. p > 0.05.


Cleary, Chloride ions (Cl- ) levels among treated wastewater by powder algae after

different durations and concentrations were 1322.1±0, 1548.7±223.9,

1433.3±121.1, 1628.1±2.7, 1864.2±510.4, 1705.2±227.4 and 2018.4±382 mg/l for

secondary treated wastewater, treated wastewater with 1 g powder algae-24 hr.,

treated wastewater with 2 g powder algae-24 hr., treated wastewater with 5 g

powder algae-48hr, treated wastewater with 1 g powder algae-48 hr., treated

wastewater with 2 g powder algae-48hr. and treated wastewater with 5 g powder

algae-24 hr., respectively (Figure4.3). The difference between secondary treated

wastewater vs. treated wastewater with 5 g powder algae-48 hr. showed

statistically significant p ≤ 0.05. In comparison, no significant difference for

secondary treated wastewater vs. treated wastewater with 5 g powder algae-24 hr.

p > 0.05.

4.1.1.4 Nitrate (NO3)

Also, nitrate ions (NO3-) levels among treated wastewater by powder algae after

different durations and concentrations were 131.0±0, 89.8±47.9, 109.8±18.1,

148.8±1.6, 157.9±3.0, 154.0±20.8 and 182.0±11.0 for secondary treated

wastewater, treated wastewater with 1, 2 & 5 g powder algae-24 hr. and treated

wastewater with 1, 2 & 5 g powder algae-48 hr. (Figure4.4). The results showed

statistically significant difference in Secondary treated wastewater vs. treated

wastewater with 1 & 2 g powder algae-24 hr., treated wastewater with 5 g powder

algae-48 hr. p ≤ 0.05.

24

Table 4.1: Physicochemical analysis of wastewater samples treated with powder

algae for 24 & 48 hr.

Parameters EC (mS)

pH

Cl- (mg/l)

NO3- (mg/l)

Stww

6±0 7.4±0 1322.1±0 131.0±0

T1- 24hr

7.0±0.2 8.2±0.2 1548.7±223.9 89.8±47.9

T2-24 hr

7.3±0.2 7.9±0.3 1433.3±121.1 109.8±18.1

T5-24 hr

8.3±1.2 7.9±0.2 1628.1±2.7 148.8±1.6

T1 -48 hr

7.9±1.5 8±0.1 1864.2±510.4 157.9±3.0

T2-48 hr

7.5±0.3 8.2±0.7 1705.2±227.4 154.0±20.8

T5-48 hr

9.4±1.3 8.1±0.1 2018.4±382 182.0±11.0

stww: secondary treated wastewater g: grams of powder hr.: hour. EC: Electrical

conductivity T1:1g powder algae, T2:2g powder algae, T5:5g powder algae *P-

value significant at P ≤ 0.05.

25

Figure 4.1: the mean EC of treated wastewater by powder algae

26

Figure 4.2: The mean pH of treated wastewater by powder algae

Figure 4.3: The mean Cl- of treated wastewater by powder algae

27

Figure 4.4: The mean NO3- of treated wastewater by powder algae

4.2 Physicochemical analysis of wastewater samples treated by whole algae

for 42 hr.

4.2.1 Electrical conductivity (EC)

As shown in table 4.2 the mean values of chemical analysis results (EC, pH, Cl-

, NO3-) of treated wastewater by whole algae after 24 hr. of different

concentrations. In study groups, the mean of EC was 6±0, 6.5±0.2, 7.1±0.2,

10.7±0.8 for secondary treated wastewater, treated wastewater with 1g whole

algae-24 hr., treated wastewater with 2 g whole algae-24 hr., treated wastewater

with 3 g whole algae-24 hr., respectively (Figure 4.5). The results showed

statistically significant difference between secondary treated wastewater vs.

treated wastewater with 2 g whole algae-24 hr., treated wastewater with 3 g

whalgae-24 hr. p ≤ 0.05, no significant difference for secondary treated

wastewater vs. treated wastewater with 1 g whole algae-24 hr. p > 0.05.

28

4.2.2 Power of hydrogen (pH)

There was statistically significantly between studied groups for pH. The average

of pH was 7.4±0, 8.4±1.5, 7.5±0.1, 7±0.1 for secondary treated wastewater,


whole algae-24 hr. and treated wastewater with 3 g whole algae-24 hr.,

respectively (Figure 4.6). However, there were no significant difference in pH

values between secondary treated wastewater and treated wastewater with 1, 2, 3

g whole algae-24 hr. p > 0.05.

4.2.3 Chloride (Cl-)

Likewise, Cl- levels among treated wastewater by whole algae after 24 hr. of

different concentrations were 1322.1±0, 1289.3±240., 1361.7±4.2, 1746.8±65.2

mg/l for secondary treated wastewater, treated wastewater with 1 g whole algae-

24 hr., treated wastewater with 2 g powder algae-24 hr., treated wastewater with

3 g whole algae-24 hr., respectively (Figure 4.7). The results showed statistically

significant difference between secondary treated wastewater and treated

wastewater with 3 g whole algae-24 hr. p ≤ 0.05. In contrast, no significant

difference for secondary treated wastewater vs. treated wastewater with 1 & 2 g

powder algae-24 hr. p > 0.05.

4.2.4 Nitrate NO3

The levels of NO3- among treated wastewater by whole algae after 24 hours of

different concentrations were 131±0, 78.7±4.5, 63.7±0.6, 143.7±29.6 mg/dl for

secondary treated wastewater, treated wastewater with 1 g powder algae-24 hr.,


powder algae-24 hr., respectively (Figure 4.8). The results indicated Statistical

significant difference between secondary treated wastewater vs. treated

wastewater with 1, 2 g powder algae-24 hr. p ≤ 0.05.

29

Conversely, no significant difference for secondary treated wastewater vs.

treated wastewater with 3 g whole algae -24 hr. p > 0.05.

Table 4.2: Physicochemical analysis of wastewater samples treated with whole

algae for 24

Parameters EC (mS)

pH

Cl- (mg/l)

NO3-(mg/l)

Stww

6±0

7.4±0

1322.1±0

131±0

T1- 24hr

6.5±0.2

8.4±1.5

1289.3±240.1

78.7±4.5

T2-24 hr

7.1±0.2

7.5±0.1

1361.7±4.2

63.7±0.6

T3-24hr

10.7±0.8

7±0.1

1746.8±65.2

143.7±29.6

T1:1gram of powder, T2: 2gram of powder, T3: 3g of whole algae, hr: hour,

stww: secondary treated wastewater *P- value significant at P ≤ 0.05

31

Figure 4.5: The mean EC of treated wastewater by whole algae

Figure 4.6: The mean pH of treated wastewater by whole algae

31

Figure 4.7: The mean Cl- of treated wastewater by whole algae

Figure 4.8: The mean NO3- of treated wastewater by of whole algae

32

4.3 Physicochemical and heavy metals analysis of wastewater samples

treated by whole and powder algae 24 hr.

4.3.1 Physicochemical analysis of wastewater samples treated by whole and

powder algae 24 hr.

4.3.1.1 Electrical conductivity EC

Physicochemical analysis (EC, pH, Cl-, COD and Nitrate) results of treated

wastewater after different durations and concentrations of powder and whole

algae pointed on Table 4.3. The mean of EC levels (mS) were 7.8±0 for

secondary treated wastewater, 6.58±0 for blank-12 hr., 9.81±0 for blank -24hr ;

where "blank" is un treated secondary wastewater, 5.7±0.7 for treated

wastewater with 1g powder algae-12 hr., 6.6±2.4 for treated wastewater with 2 g

powder algae-12 hr., 5.8±0.2 for treated wastewater with 1 g whole algae-12 hr.,

7.2±2.2 for treated wastewater with 1g powder algae-24hr, 5.9±1.5 for treated

wastewater with 2 g powder algae-24hr and 7.7±1.5 for treated wastewater with

1g whole algae-24 hr. (Figure 4.9). The results showed that there was

statistically significant difference between blank -24hr and treated wastewater

with 1 & 2 g powder algae-12 hr., treated wastewater with 1g whole algae-12

hr., treated wastewater with 1g powder algae-24hr, treated wastewater with 2 g

powder algae-24 hr. p ≤ 0.05.

4.3.1.2 Power of hydrogen (PH)

Clearly, the mean of pH levels was 7.5±0 for secondary treated wastewater,

7.9±0 for blank-12hr, 8±0 for blank -24hr, 7.7±0.1 for treated wastewater with

1g powder algae-12hr, 7.5±0.15 for treated wastewater with 2 g powder algae-

12hr, 7.8±0.1 for treated wastewater with 1 g whole algae-12 hr., 7.8±0.06 for

treated wastewater with 1 g powder algae-24hr, 7.6±0.2 for treated wastewater

with 2 g powder algae-24hr and 7.9±0.1 for treated wastewater with 1 g whole

33

algae-24 hr. (Figure4.10). Post hoc test shows statistically significant difference

in secondary treated wastewater vs. treated wastewater with 1 g powder algae-

12 hr., treated wastewater with 1 g whole algae-12 hr., treated wastewater with 1

g powder algae-24 hr., treated wastewater with 1 g whole algae-24 hr. p ≤ 0.05.


The mean of Cl-(mg/l) levels were 1780.3±0 for secondary treated wastewater,

1901±0 for blank-12 hr., 2036.8±0 for blank -24 hr., 1820.6±75.9 for treated

wastewater with 1g powder algae-12hr., 1825.6±117.8 for treated wastewater

with 2 g powder algae-12 hr. , 1820.6±17.4 for treated wastewater with 1g

whole algae-12 hr. , 1820.6±115.2 for treated wastewater with 1 g powder

algae-24 hr., 1755.2±34.8 for treated wastewater with 2 g powder algae-24 hr.

and 1875.9±98.2 for treated wastewater with 1 g whole algae-24 hr. (Figure

4.11). The results showed that there is a statistically significant difference in

secondary treated wastewater vs. treated wastewater with 1 g powder algae-

12hr, treated wastewater with 2 g powder algae-12hr p ≤ 0.05, blank -24 hr. vs.

treated wastewater with 1 & 2 g powder algae-12 hr., treated wastewater with 1

g whole algae-12 & 24 hr., treated wastewater with 1 & 2 g powder algae-24hr,

treated wastewater with 2 g powder algae-24 hr. p ≤ 0.05.

4.3.1.4 Chemical oxygen demand (COD)

Obviously, the mean of COD (mg/l) levels were 608±0 for secondary treated

wastewater, 480±0 for blank-12 hr., 508±0 for blank -24 hr., 435±35 for treated

wastewater with 1g powder algae-12 hr., 450±9.2 for treated wastewater with 2

g powder algae-12 hr., 400±50 for treated wastewater with 1 g whole algae-12

hr., 450±8.9 for treated wastewater with 1 g powder algae-24 hr., 481±6.9 for

treated wastewater with 2 g powder algae-24 hr. and 430±10 for treated

wastewater with 1g whole algae-24 hr. (Figure 4.12). Post hoc test shows


34

wastewater with 1 & 2 g powder algae-12 hr., treated wastewater with 1g whole

algae-12 hr., treated wastewater with 1 & 2 g powder algae-24 hr., treated

wastewater with 1 g whole algae-24 hr. P ≤ 0.05, blank-12hr vs. treated

wastewater with 1 g powder algae-12 hr., treated wastewater with 1g whole

algae-12 hr. p ≤ 0.05, blank-24 hr. vs. treated wastewater with 1g powder algae-

24 hr., treated wastewater with 1 g whole algae-24 hr., p ≤ 0.05.

Conversely, no significant difference for blank-12 hr. vs. blank -24 hr., treated

wastewater with 2 g powder algae-12 hr., treated wastewater with 1 & 2 g


4.3.1.5 Nitrate (NO3)

As can be expected, the mean of nitrate (mg/L) levels were 155.0±0 for

secondary treated wastewater, 76.1±0 for blank-12 hr., 77.6±0 for blank -24hr ,

77.8±11.3 for treated wastewater with 1g powder algae-12 hr., 76.1±12.4 for

treated wastewater with 2 g powder algae-12 hr., 72.4±9.4 for treated

wastewater with 1g whole algae-12hr., 66.7±13.5 for treated wastewater with 1

g powder algae-24hr, 113.9±38.6 for treated wastewater with 2 g powder algae-

24hr and 72.7±1.3 for treated wastewater with 1 g whole algae-24 hr. (Figure

4.13). Post hoc test shows statistically significant difference in secondary treated

wastewater vs. blank-12 hr., blank -24 hr., treated wastewater with 1 & 2 g

powder algae-12hr, treated wastewater with 1g whole algae-12 & 24 hr., treated

wastewater with 1 & 2g powder algae-24 hr., p ≤ 0.05.

On the contrary, no significant difference for blank-12 hr. vs. treated wastewater

with 1 & 2 g powder algae-12 hr., treated wastewater with 1g whole algae-12 &

24 hr., treated wastewater with 1 g powder algae-24 hr. p > 0.05.

35

Table 4.3: Physicochemical analyses of wastewater samples treated with whole

& powder algae for 12 & 24 hr.

Parameters

EC

(mS)

pH

Cl- (mg/l)

COD

(mg/l) NO3

-(mg/l)

1Stww 7.8±0

7.5±0

1780.3±0

608±0

155.0±0

blank-12 hr. 6.58±0

7.9±0

1901±0

480±0

76.1±0

blank -24hr 9.81±0

8±0

2036.8±0

508±0

77.6±0

T1-12 hr. 5.7±0.7

7.7±0.1

1820.6±75.9

435±35

77.8±11.3

T2-12 hr. 6.6±2.4

7.5±0.15

1825.6±117.8

450±9.2

76.1±12.4

T1(w)-12hr 5.8±0.2

7.8±0.1

1820.6±17.4

400±50

72.4±9.4

T1-24 hr. 7.2±2.2

7.8±0.06

1820.6±115.2

450±8.9

66.7±13.5

T2-24 hr. 5.9±1.5

7.6±0.2

1755.2±34.8

481±6.9

113.9±38.6

T1(w)-24hr

7.7±1.5

7.9±0.1

1875.9±98.2

430±10

72.7±1.3

stww: secondary treated wastewater; g: grams hr.: hour; EC: Electrical

conductivity T1:1g powder algae T2:2g powder algae T5:5g powder algae *P-


36

Figure 4.9: The mean EC of treated wastewater by whole & powder algae

Figure 4.10: The mean pH of treated wastewater by whole & powder algae

37

Figure 4.11: The mean Cl- of treated wastewater by whole & powder algae

Figure 4.12: The mean COD of treated wastewater by whole & powder algae

38

Figure 4.13: The mean Nitrate of treated wastewater by whole & powder algae

4.3.2 Heavy metals analysis results of treated wastewater after different

durations and concentrations of powder and whole algae

4.3.2.1 Lead (pb)

Table 4.4 showed some heavy metals (Pb, Fe, Zn, Mn & Sr) analysis results of

treated wastewater after different durations and concentrations of powder and

whole algae. The mean levels of Pb were 0.147±0, 0.134±0.004, 0.127±0.015,

0.111±0.014, 0.137±0.021, 0.106±0.012, 0.097±0.007 mg/l for secondary

treated wastewater, treated wastewater with 1 g powder algae-12 hr., treated

wastewater with 1 g whole algae-12 hr., treated wastewater with 2 g powder


with 1g powder algae-24 hr., treated wastewater with 2 g powder algae-24 hr.,

respectively (Figure 4.14). The results of lead analysis showed that there was a

statistically significant difference between secondary treated wastewater and

treated wastewater with 2 g powder algae-12 hr., treated wastewater with 1 & 2

g powder algae-24 hr. p ≤ 0.05. In contrast, no significant difference for

39

secondary treated wastewater vs. treated wastewater with 1 g powder algae-12

hr., treated wastewater with 1 g whole algae-12 hr., treated wastewater with 1 g

whole algae-24 hr. p > 0.05.

4.3.2.2 Ferrous (Fe)

The mean levels of Fe were 0.083±0, 0.065±0.036, 0.081±0.042, 0.001±0.001,

0.099±0.015, 0.041±0.038, 0.0001±0.0001 mg/l for secondary treated

wastewater, treated wastewater with 1 g powder algae-12 hr., treated wastewater

with 1 g whole algae-12 hr., treated wastewater with 2 g powder algae-12 hr.,

treated wastewater with 1g whole algae-24 hr., treated wastewater with 1g

powder algae-24 hr., treated wastewater with 2 g powder algae-24 hr.,

respectively (Figure 4.15). The results showed statistically significant difference


12 hr., treated wastewater with 2 g powder algae-24 hr. p ≤ 0.05. Conversely, no

significant difference for secondary treated wastewater vs. treated wastewater

with 1 g powder algae-12 hr., treated wastewater with 1 g whole algae-12 hr.,

treated wastewater with 1 g whole algae-24 hr., treated wastewater with 1g


4.3.2.3 Zinc (Zn)

The mean levels of Zn were 0.051±0, 0.024±0.025, 0..042±0.032, 0.002±0.004,

0.029±0.011, 0.003±0.005, 0.0002±0.0001 mg/l for secondary treated

wastewater, treated wastewater with 1 g powder algae-12 hr., treated wastewater

with 1 g whole algae-12 hr., treated wastewater with 2 g powder algae-12 hr.,


powder algae-24 hr., treated wastewater with 2 g powder algae-24 hr.,

respectively (Figure 4.16). The results showed statistically significant difference


41

12 hr., treated wastewater with 1 g whole algae-24 hr., treated wastewater with 1

& 2 g powder algae-24 hr. p ≤ 0.05.

4.3.2.4 Manganese (Mn)

The mean levels of Mn were 0.392±0, 0.051±0.025, 0.065±0.016, 0.024±0.03,

0.071±0.018, 0.02±0.005, 0.016±0.025 mg/L for secondary treated wastewater,


whole algae-12 hr., treated wastewater with 2 g powder algae-12 hr., treated

wastewater with 1g whole algae-24 hr., treated wastewater with 1 g powder

algae-24 hr., treated wastewater with 2 g powder algae-24 hr., respectively

(Figure 4.17). Results of Mn determination for the different samples showed


wastewater with 1 & 2 g powder algae-12 hr., treated wastewater with 1g whole


with 1 & 2 g powder algae-24 hr. p ≤ 0.05. On the contrary, no significant

difference for treated wastewater with 1 g powder algae-12 hr. vs. treated

wastewater with 1 g whole algae-12 hr., treated wastewater with 2 g powder


with 1 g powder algae-24 hr. p > 0.05.

4.3.2.5 Strontium (Sr)

The mean levels of Sr were 6.9±0, 5.7±1.2, 5.7±1.0, 3.0±0.1, 6.1±0.2, 4.28±0.2,

2.7±0.2 mg/l for secondary treated wastewater, treated wastewater with 1 g

powder algae-12 hr., treated wastewater with 1 g whole algae-12 hr., treated

wastewater with 2 g powder algae-12 hr., treated wastewater with 1g whole

algae-24 hr., treated wastewater with 1 g powder algae-24 hr. treated wastewater

with 2 g powder algae-24hr, respectively (Figure 4.18). There was a statistically

significant difference in secondary treated wastewater vs. treated wastewater

with 1 & 2 g powder algae-12 hr., treated wastewater with 1 g whole algae-12

41

hr., treated wastewater with 1 & 2 g powder algae-24 hr., treated wastewater

with 2 g powder algae-24 hr. p ≤ 0.05. Conversely, no significant difference for

secondary treated wastewater vs. treated wastewater with 1 g whole algae-24 hr.

p > 0.05.

Table 4.4 heavy metals analyses of wastewater samples treated with whole &

powder algae for 12 & 24 hr.

samples Pb (mg/l) Fe (mg/l) Zn (mg/l) Mn (mg/l) Sr (mg/l)

stww

0.147±0

0.083±0

0.051±0

0.392±0

6.9±0

T1-12hr

0.134±0.004

0.065±0.036

0.024±0.025

0.051±0.025

5.6±1.2

T1(w)12hr

0.097±0.007

0.081±0.042

0.042±0.032

0.065±0.016

5.7±1.1

T2-12hr

0.111±0.014

0.001±0.001

0.002±0.004

0.024±0.03

3.0±0.1

T1(w) 24hr

0.137±0.021

0.099±0.015

0.029±0.011

0.071±0.018

6.1±0.2

T1 24hr

0.106±0.012

0.041±0.038

0.003±0.005

0.02±0.005

4.3±0.2

T2-24hr

0.127±0.015

0.0001±0.0001

0.0002±0.0001

0.016±0.025

2.7±0.2

Stww: secondary treated wastewater, T1: 1gram of powder, T2:2gram of

powder, T1 (w): 1g of whole algae, hr.: hour *P- value significant at P ≤ 0.05.

42

Figure 4.14: The mean Pb of treated wastewater by whole & powder algae

Figure 4.15: The mean Fe of treated wastewater by whole & powder algae

43

Figure 4.16: The mean Zn of treated wastewater by whole & powder algae

Figure 4.17: The mean Mn of treated wastewater by whole & powder algae

44

Figure 4.18: The mean Sr of treated wastewater by whole & powder algae

4.4 Microbiological results

4.4.1 Bacterial count of treated wastewater samples after 12 and 24 hours

Bacterial count of treated wastewater after 12 and 24 hours was demonstrated in

Table 4.5. The mean levels (103) of bacterial count during 12 hr. were

2970000±0, 50 ±0 (106), 190±13.2, 119±6.6, 206.7±15.3 for secondary treated

wastewater, Blank (un treated secondary wastewater) , (T1) treated wastewater

with 1 g powder algae, T1 (w) treated wastewater with 1 g whole algae, (T2)

treated wastewater with 2 g whole algae, respectively. The results of bacterial

count indicated that there was a statistically significant difference among

different groups, secondary treated wastewater vs. Blank, treated wastewater

with 1 & 2 g whole algae p ≤ 0.05, Blank vs. treated wastewater with 1 g

powder algae, treated wastewater with 1 g whole algae, treated wastewater with

2 g powder algae p ≤ 0.05.

45

Obviously, the mean levels (103) of bacterial count after 24 hr. were 2970000±0,

50±0 (106), 86±1.0, 32±2, 36±1.0 for secondary treated wastewater, blank,

treated wastewater with 1 g powder algae, treated wastewater with 1g whole

algae, treated wastewater with 2 g powder algae, respectively. The Post hoc test

showed statistically significant difference in secondary treated wastewater vs.

blank, treated wastewater with 1 g powder algae, treated wastewater with 1g

whole algae, treated wastewater with 2 g powder algae p ≤ 0.05, blank vs.

treated wastewater with 1 g powder algae, treated wastewater with 1 g whole

algae, treated wastewater with 2 g powder algae p ≤ 0.05.

Table 4.5: Bacterial count of treated wastewater after 12 and 24 hours

Samples

bacterial count* (103)

12hr 24hr

Stww 2970000±0**

2970000±0**

blank * 106 50.0

50.0

T1 190±13.2

86±1.0

T1(w) 119±6.6

32±2

T2 206.7±15.3

36±1.0

T1: 1gram of powder T2:2 gram of powder T1(w): 1 g whole algae hr: hour

stww: secondary treated wastewater *: Too numerous to count *P- value

significant at P ≤ 0.05.

46

4.4.2 Detection of Salmonella & Shigella spp. and fungi treated in


-Table 4.6 showed detection of Salmonella & Shigella spp. and, fungi in treated

wastewater after 12 & 24 hours. Heavy growth of Salmonella & Shigella spp.

after 12hr among all studied groups (3 (100%) blank, treated wastewater with 1g

whole algae, treated wastewater with 1 & 2 g powder algae with no statistically

significant between different studied groups (x2 =12, P=1.000). On the other

hand, heavy growth of Salmonella & Shigella spp. after 12hr among studied

groups (3(100%) blank, treated wastewater with 1 & 2 g powder algae-24 hr.)

and light growth (3(100%) in treated wastewater with 1g whole algae with

statistically significant between studied groups (x2 = 24, P= 0.000). The results

showed statistically significant difference in blank vs. treated wastewater with

1g powder algae 24 hr. p ≤ 0.05, no significant difference for blank vs. Treated

wastewater with 1g whole algae, treated wastewater with 2 g powder algae p >

0.05.

-Heavy growth of fungi in treated wastewater after 12 hr. among all studied

groups (3 (100%) blank, treated wastewater with 1 & 2 g powder algae, and few

growth (3(100%) for treated wastewater with 1 g whole algae with statistically

significant between studied groups (x2=12, P=0.004).

The results indicated that there was a statistically significant difference blank vs.

Treated wastewater with 1 g whole algae p ≤ 0.05. Conversely, there is no

significant difference for blank vs treated wastewater with 1 & 2 g whole algae

p > 0.05.

47

Table 4.6: Detection of Salmonella & Shigella spp. and fungi in treated


Sample

Salmonella & Shigella spp. Fungi

12 hr.

24 hr.

12 hr.

24 hr.

heavy

growth

(full)

No

growth

heavy

growth

light

growth

heavy

growth

few

growth

heavy

growth

medium

growth

few

growth

Blank +ve -ve +ve -ve +ve -ve +ve -ve -ve

T1(w) +ve -ve -ve +ve -ve +ve -ve -ve +ve

T1 +ve -ve +ve -ve +ve -ve +ve -ve -ve

T2 +ve -ve +ve -ve +ve -ve -ve +ve -ve

T1 (w):1 g of whole algae; T1:1 g of powder algae; T2:2 g hr: hour

*P- value significant at P ≤ 0.05.

48

Figure 4.19: detection of Salmonella & Shigella spp.

.

Figure 4.20: detection of fungi after 12h treatment

Figure 4.21: detection of fungi after 24h treatment

49

4.4.3 Effect of shaking conditions on bacterial count after 2, 24 hours’

-Bacterial count (X104) after 2 & 24 hr. treatment under static and shaking

conditions are showed in Table 4.7.

- The mean levels of bacterial counts after 2 hours treatment under shaking

conditions were 297000±0 for secondary treated wastewater, 50±0 for blank (un

treated secondary wastewater) (X105), 24±4 for treated wastewater with 1g

whole algae, 8000±500 for treated wastewater with 2 g whole algae, 300±20 for

treated wastewater with 4g whole algae, 120±10 for treated wastewater with 5 g

whole algae, 75333.3±1527.5 for treated wastewater with 1g powder algae,

5600±400 for treated wastewater with 2 g powder algae, 14333.3±152.8 for

treated wastewater with 4 g powder algae, 120000±10000 for treated wastewater

with 5 g powder algae, respectively. The results showed statistically significant

difference in secondary treated wastewater vs. blank and all the treated samples

p ≤ 0.05.

-The mean levels of bacterial counts (X104) after 2hr treatment under static

conditions were 297000±0 for secondary treated wastewater, 50±0 for Blank

(X105), 800±50 for treated wastewater with 1g whole algae, 62.3±2.1 for treated

wastewater with 2 g whole algae, 0.3±0.1 for treated wastewater with 4 g whole

algae, 42.3±2.5 for treated wastewater with 5 g whole algae, 72.3±2.5 for treated

wastewater with 1 g whole algae, 1773.3±20.8 for treated wastewater with 2 g

powder algae, 1886.7±15.3 for treated wastewater with 4 g powder algae,

95000±5000 for treated wastewater with 5 g powder algae, respectively. The

results showed statistically significant difference in secondary treated

wastewater vs. blank and all treated samples p ≤ 0.05, Blank vs. treated

wastewater with 5 g whole algae p ≤ 0.05.

Clearly, paired t-test illustrated bacterial count were higher statistically

significant after 2 hr. treatment under shaking conditions than static conditions

51

at the same time for treated wastewater with (2 g, 4 g) whole algae p ≤ 0.05. On

the other hand, no statistically significant after 2hr treatment under shaking

conditions than static conditions at same time for Stww p > 0.05, Blank p >

0.05, and treated wastewater with 5 g whole algae p > 0.05.

-While it shows bacterial count (X 104) after 24 hr. treatment under static and

shaking conditions.

-The mean levels of bacterial counts after 24 hr. treatment under shaking

conditions were 297000±0 for stww, 50.0±0 for blank 105, 2500±100 for treated

wastewater with 1g whole algae, 30000±1000 for treated wastewater with 2 g

whole algae, 1380±40 for treated wastewater with 4 g whole algae, 29000±1000

for treated wastewater with 5 g whole algae, 6600±200 for treated wastewater

with 1 g powder algae, 20000±700 for treated wastewater with 2 g powder

algae, 29000±1000 for treated wastewater with 4 g powder algae, 18000±1000

for treated wastewater with 5 g powder algae, respectively. The results indicated

that there was a significant difference in stww vs. blank, treated wastewater with

1, 2, 4, 5 g whole algae and treated wastewater with 1, 2, 4, 5 g powder algae p

≤ 0.05, blank vs. treated wastewater with 1, 2, 4, 5 g powder algae p ≤ 0.05.

-The mean levels of bacterial counts (X104) after 24hr treatment under static

conditions were 297000±0 for stww, 50.0±0 for Blank (X105), 3.2±0.2 for

treated wastewater with 1 g whole algae, 120±10 for treated wastewater with 2 g

whole algae, 7600±100 for treated wastewater with 4 g whole algae,

100000±5000 for treated wastewater with 5 g whole algae, 820±10 for treated

wastewater with 1 g powder algae, 11700±300 for treated wastewater with 2 g

powder algae, 30000±1000 for treated wastewater with 4 g powder algae, 0±0

for treated wastewater with 5 g powder algae, for, respectively. The results

showed statistically significant difference in stww vs. blank, treated wastewater

with 1, 2, 4 & 5 g whole algae, and treated wastewater with 1, 2, 4 & 5 g powder

51

algae p ≤ 0.05, blank vs. treated wastewater with 4 g whole algae, treated

wastewater with 5 g whole algae, treated wastewater with 2 g powder algae,

treated wastewater with 4g powder algae p ≤ 0.05.

Clearly, paired t-test illustrates that bacterial count were higher statistically

significant after 24 hr. treatment under shaking conditions than static conditions

at same time for treated wastewater with 1 g whole & powder algae P ≤ 0.05,

treated wastewater with 2 g whole & powder algae P ≤ 0.05, and treated

wastewater with 5 g powder algae P ≤ 0.05, and lower statistically significant

for treated wastewater with 4g whole algae P ≤ 0.05 and treated wastewater with

5 g whole algae P ≤ 0.05. In comparison, no statistically significant for stww P >

0.05, blank *105 P > 0.05, and treated wastewater with 4 g whole algae P > 0.05.

Table 4.7: Bacterial count after 2 & 24 hr. treatment under static and shaking

conditions

Samples

Bacterial count* (104)

after 2hr

treatment under

shaking

conditions

after 2hr

treatment

under static

conditions

after 24hr

treatment under

shaking

conditions

after 24hr

treatment under

static

conditions

Stww 297000±0 297000±0 297000±0 297000±0

blank *105 50.0±0 50.0±0 50.0±0 50.0±0

w (1g) 24±4 800±50 2500±100 3.2±0.2

w ( 2g) 8000±500 62.3±2.1 30000±1000 120±10

w (4g) 300±20 0.3±0.1 1380±40 7600±100

w (5g) 120±10 42.3±2.5 29000±1000 100000±5000

p (1g) 75333.3±1527.5 72.3±2.5 6600±200 820±10

p (2g) 5600±400 1773.3±20.8 20000±700 11700±300

p (4g) 14333.3±152.8 1886.7±15.3 29000±1000 30000±1000

p (5g) 120000±10000 95000±5000 18000±1000 50.0 *105±0

Stww: secondary treated waste wastewater; g: grams p: powder algae; w: whole

algae *P- value significant at P ≤ 0.05.

52



Table 4.8 showed Fungi, Salmonella & Shigella spp., and Coliform after 2 hr.

treatment by powder algae under shaking (sh) ,and static ( un-sh) conditions.

-In fungi, blank( which is un treated secondary wastewater) showed moderate

growth after 2 hr. (3 (100%) and no growth (3 (100%) for treated wastewater

with 1g powder algae sh, treated wastewater with 2 g powder algae sh, treated

wastewater with 4 g powder algae sh, treated wastewater with 5 g powder algae

sh, treated wastewater with 1 g powder algae un-sh, treated wastewater with 2 g

powder algae un-sh, treated wastewater with 4 g powder algae un-sh, treated

wastewater with 5 g powder algae un-sh. The results showed statistically

significant difference in blank vs. treated wastewater with 1, 2, 4 & 5 g powder

algae sh, treated wastewater with 1, 2, 4 & 5 g powder algae un-sh p ≤ 0.05.

Heavy growth of Salmonella & Shigella spp. in blank after 2 hr. among all

studied groups (3 (100%) for blank, treated wastewater with 1 g powder algae

sh, treated wastewater with 2 g powder algae sh, treated wastewater with 5 g

powder algae sh, treated wastewater with 1 g powder algae un-sh, treated

wastewater with 2 g powder algae un-sh and treated wastewater with 4g powder

algae un-sh and no growth (3 (100%) for treated wastewater with 4g powder

algae sh, and treated wastewater with 5 g powder algae un-sh. The results

showed statistically significant difference in blank vs. treated wastewater with 4

g whole algae sh, treated wastewater with 5 g whole algae un-sh p ≤ 0.05. In

contrast, no significant difference for blank vs. treated wastewater with 1 g

powder algae sh, treated wastewater with 2 g powder algae sh, treated


un-sh, treated wastewater with 2 g powder algae un-sh, treated wastewater with

4g powder algae un-sh p > 0.05.

53

-Heavy growth of coliform (pink colonies) shown in blank (3 (100%).On the

other hand, there was heavy growth of (pale colonies) in treated wastewater with

4 g powder algae and treated wastewater with 5 g powder algae (3 (100%), and

treated wastewater with 2 g powder algae sh. Light growth of (pale colonies)

shown in treated wastewater with 1g powder algae un-sh, and treated

wastewater with 5 g powder algae un-sh (3 (100%), while Light-moderate

growth of (pale colonies) shown in treated wastewater with 4 g powder algae

un-sh (3(100%). Moderate growth of (pale colonies) shown in treated

wastewater with 4 g powder algae and treated wastewater with 2 g powder algae

un-sh (3 (100%). The results indicated statistically significant difference in

blank vs. treated wastewater with 1, 2, 4 & 5 g powder algae sh and treated

wastewater with 1, 2, 4 & 5 g powder algae un-sh p ≤ 0.05.

54

Table 4.8: Detection of Fungi, Salmonella & Shigella spp., and Coliform after

2 hr. treatment by powder algae under Shaking and static conditions

Sample

Fungi

Salmonella &

Shigella

Coliform

moderate

growth

no

growth

heavy

growth

light

growth

heavy

growth

(pink

colonies)

heavy

growth

(pale

colonies)

light

growth

(pale

colonies)

light

moderate

growth

(pale

colonies)

moderate

growth

(pale

colonies)

Blank +ve -ve +ve -ve +ve -ve -ve -ve -ve

p (1g) sh -ve +ve +ve -ve -ve -ve +ve -ve -ve

p (2g) sh -ve +ve +ve -ve -ve -ve -ve -ve +ve

p (4g) sh -ve +ve -ve +ve -ve +ve -ve -ve -ve

p (5g) sh -ve +ve +ve -ve -ve +ve -ve -ve -ve

p (1g)

un-sh

-ve +ve +ve

-ve -ve -ve +ve -ve -ve

p (2g)

un-sh

-ve +ve +ve

-ve -ve -ve -ve -ve +ve

p (4g)

un-sh

-ve +ve +ve

-ve -ve -ve -ve +ve -ve

p (5g)

un-sh

-ve +ve

-ve +ve -ve -ve +ve -ve -ve

un-sh: static Sh: shaken p: powder of algae g: grams hr: hou

*P- value significant at P ≤ 0.05

55

Figure 4.22: detection of fungi after 2hr treatment


Figure 4.24: detection of coliform after 2hr treatment

56


occurrence in the 2 hr. whole algae treatment process

Salmonella & Shigella spp., Coliform & Fungi after 2 hr. treatment by whole

algae under Shaking and static conditions pointed on Table 4.9. Heavy growth

of Salmonella & Shigella spp., were in blank, treated wastewater with 1 g whole

algae sh, treated wastewater with 2 g whole algae sh, treated wastewater with 4g

whole algae sh, treated wastewater with 5 g whole algae sh, treated wastewater

with 1 g whole algae un-sh, treated wastewater with 4 g whole algae un-sh and

treated wastewater with 5 g whole algae un-sh (3(100%). Conversely, light

growth of Salmonella & Shigella spp., were in treated wastewater with 2 g

whole algae un-sh. The results showed statistically significant difference in

blank vs. treated wastewater with 2 g whole algae un-sh p ≤ 0.05. In

comparison, no significant difference in blank vs. treated wastewater with 1, 2, 4

& 5 whole algae sh, treated wastewater with 1, 4 &5 g whole algae un-sh p >

0.05.

Heavy growth of coliform under shaking and static conditions were in each of

blank, treated wastewater with 1g whole algae un-sh, treated wastewater with 2

g whole algae un-sh & treated wastewater with 4 g whole algae un-sh(3(100%),

while light growth of (pale colonies) (3 (100%) in each of treated wastewater

with 2 g whole algae sh, treated wastewater with 4 g whole algae sh & treated

wastewater with 5 g whole algae sh, light growth of (pale colonies) (3 (100%) in

treated wastewater with 1 g whole algae sh and heavy-moderate growth of (pale

colonies) (3(100%) in treated wastewater with 5 g whole algae un-sh. The chi

square test shows statically significant differences among studied groups (x2=81,

P=0.000). The results showed statistically significant difference in blank vs.

treated wastewater with 12and4&g whole algae sh, treated wastewater with 5 g

whole algae un-sh p ≤ 0.05. In contrast, no significant difference for blank vs.

57

treated wastewater with 5 g whole algae sh, treated wastewater with 1, 2 & 4 g

whole algae un-sh p > 0.05.

Light growth of fungi under static and shaking conditions (3 (100%) were in

blank and no growth in each of treated wastewater with 1 g whole algae sh,

treated wastewater with 2 g whole algae sh, treated wastewater with 4 g whole


whole algae un-sh, treated wastewater with 2 g whole algae un-sh, treated

wastewater with 4 g whole algae un-sh and treated wastewater with 5 g whole

algae un-sh. The results showed statistically significant difference in blank vs.

treated wastewater with 1, 2, 4 & 5 g whole algae sh, treated wastewater with 1,

2, 4 & 5 g whole algae un-sh p ≤ 0.05.

Table 4.9: Detection of Salmonella & Shigella spp., Coliform & Fungi after

2hr treatment by whole algae under Shaking and static conditions

samples

Salmonella &

Shigella

Coliform

Fungi

heavy

growth

light

growth

heavy

growth

(pink

colonies)

heavy

growth

(pale

colonies)

light

growth

(pale

colonies)

heavy -

moderate

growth

(pale

colonies)

light

growth

no

growth

Blank +ve -ve +ve -ve -ve -ve +ve -ve

w (1g) sh +ve -ve -ve -ve +ve -ve -ve +ve

W (2g) sh +ve -ve -ve +ve -ve -ve -ve +ve



w (1g)un-sh +ve -ve +ve -ve -ve -ve -ve +ve

w (2g)un-sh -ve +ve +ve -ve -ve -ve -ve +ve

w (4g)un-sh +ve -ve +ve -ve -ve -ve -ve +ve

w (5g)un-sh +ve -ve -ve -ve -ve +ve -ve +ve

un -sh: static; Sh: shaken g: grams; w: whole algae; hr: hour. *P- value

significant at P ≤ 0.05.

58



Table 4.10 showed the growth of Fungi, Salmonella & Shigella spp., and

Coliform after 24 hr treatment by powder algae under Shaking and static

conditions. Few growth of fungi were in blank (3 (100%)), and no growth (3

(100%)) in treated wastewater with 1g whole algae sh, treated wastewater with 2

g whole algae sh, treated wastewater with 4 g whole algae sh, treated wastewater

with 5 g whole algae sh, treated wastewater with 1 g whole algae un-sh, treated

wastewater with 2 g whole algae un-sh, treated wastewater with 4 g whole algae

un-sh and treated wastewater with 5 g whole algae un-sh. The results showed

statistically significant difference in Blank vs. treated wastewater with 1, 2, 4 &

5 g powder algae sh and treated wastewater with 1, 2, 4 & 5 g powder algae un-

sh p ≤ 0.05.

Heavy growth of Salmonella & Shigella spp. (3 (100%)) were in each of blank,

treated wastewater with 1 g powder algae sh, treated wastewater with 2 g

powder algae sh, treated wastewater with 4 g powder algae sh, treated


un-sh, treated wastewater with 2 g powder algae un-sh & treated wastewater

with 5 g powder algae un-sh and treated wastewater with 4 g powder algae un-

sh, while light growth (3 (100%)) was in treated wastewater with 4g powder

algae un-sh. The results indicated that there is a statistically significant

difference in blank vs. treated wastewater with 4 g powder algae un-sh p ≤ 0.05.

On the contrary, no significant difference for Blank vs. treated wastewater with

1 g powder algae sh, treated wastewater with 2 g powder algae sh, treated

wastewater with 4g powder algae sh, treated wastewater with 5 g powder algae

sh, treated wastewater with 1 g powder algae un-sh, treated wastewater with 5 g

powder algae un-sh p > 0.05.

59

Heavy growth of Coliform (pink colonies) (3 (100%)) were in blank, while

heavy growth of (pale colonies) were in treated wastewater with 1g powder

algae sh, treated wastewater with 2 g powder algae sh, treated wastewater with 4

g powder algae sh, light growth of (pale colonies) (3(100%)) was in treated

wastewater with 5 g whole algae sh, light growth (pale colonies) (3(100%)) was

in treated wastewater with 1 g powder algae un-sh, light-moderate growth (pale

colonies) (3(100%)) were in treated wastewater with 2 g powder algae un-sh &

treated wastewater with 4 g powder algae un-sh, and moderate growth (pale

colonies) (3(100%)) was in treated wastewater with 5 g whole algae un-sh. The

results showed statistically significant difference in blank vs. treated wastewater

with 1 g powder algae sh, treated wastewater with 2 g powder algae sh, treated


sh, treated wastewater with 1g powder algae un-sh p ≤ 0.05, treated wastewater

with 1g powder algae sh vs. 1g un-sh, treated wastewater with 2g powder algae

un-sh, treated wastewater with 4g powder algae un-sh, treated wastewater with 5

g powder algae un-sh p ≤ 0.05, treated wastewater with 2 g powder algae sh vs.

treated wastewater with 1g powder algae un-sh, treated wastewater with 2 g

powder algae un-sh, treated wastewater with 4 g powder algae un-sh, treated

wastewater with 5 g powder algae un-sh p ≤ 0.05, treated wastewater with 4g

powder algae sh vs. treated wastewater with 1g whole algae un-sh, treated

wastewater with 2 g powder algae un-sh, treated wastewater with 4 g powder

algae un-sh, treated wastewater with 5 g powder algae un-sh p ≤ 0.05, treated

wastewater with 5 g powder algae sh vs. treated wastewater with 1g powder

algae un-sh, treated wastewater with 2 g powder algae un-sh, treated wastewater

with 4g powder algae un-sh, treated wastewater with 5g powder algae un-sh p ≤

0.05, treated wastewater with 1 g powder algae un-sh vs. treated wastewater

with 2 g powder algae un-sh, treated wastewater with 4g powder algae un-sh,

treated wastewater with 5 g powder algae un-sh p ≤ 0.05

61

Table 4.10: Detection of Fungi, Salmonella & Shigella spp., and Coliform after

24hr. treatment by powder algae under Shaking and static conditions

sample

Fungi

Salmonella &

Shigella spp.

Coliform

few

growth

no

growth

heavy

growth

light

growth

heavy

growth

-(pale

colony)

heavy

growth

-(pink

colony

light-

growth

-(pale

colony

Light

moderate

growth

(pale

colony)

moderate

growth

(pale

colony)

blank +ve -ve +ve -ve -ve +ve -ve -ve -ve

p (1g)

sh

-ve +ve +ve -ve +ve -ve -ve -ve -ve

p (2g)

sh


p (4g)

sh


p (5g)

sh


p(1g)

un-sh

-ve +ve +ve -ve -ve -ve +ve -ve -ve

p(2g)

un-sh

-ve +ve +ve -ve -ve -ve -ve +ve -ve

p(4g)

un-sh

-ve +ve -ve +ve -ve -ve -ve +ve -ve

p(5g)

un-sh

-ve +ve +ve -ve -ve -ve -ve -ve +ve

un -sh: static; Sh: shaken p: powder algae ; g: grams hr: hour


61


after 24 hr. of treatment with whole algae

As shown in Table 4.11, heavy growth of Salmonella & Shigella spp. were in




wastewater with 4 g whole algae un-sh, treated wastewater with 5 g whole algae

un-sh, while light growth (few colonies) was in blank and moderate growth was

in treated wastewater with 4 g whole algae sh. The results showed statistically

significant difference in blank vs. treated wastewater with 1 g whole algae sh,

treated wastewater with 4 g whole algae sh p ≤ 0.05. Conversely, no significant

difference for blank vs. treated wastewater with 2 g whole algae sh, treated

wastewater with 5 g whole algae sh, treated wastewater with 1 g whole algae un-

sh, treated wastewater with 2 g whole algae un-sh, treated wastewater with 4 g

whole algae un-sh, treated wastewater with 5 g whole algae un-sh p > 0.05.

Heavy growth of Coliform (pink colonies) after 24 hr treatment by whole algae

under Shaking and static conditions (3 (100%) was in blank and moderate

growth (pale colonies) (3 (100%) in treated wastewater with 1 g whole algae sh,




wastewater with 4 g whole algae un-sh and treated wastewater with 5 g whole

algae un-sh. The results showed that there was a statistically significant

difference in blank vs. treated wastewater with 1 g whole algae sh, treated

wastewater with 2 g whole algae sh, treated wastewater with 4 g whole algae sh,


algae un-sh, treated wastewater with 2 g whole algae un-sh, treated wastewater

with 4 g whole algae un-sh, treated wastewater with 5 g whole algae un-sh p ≤

62

0.05. moderate growth of Fungi after 24 hr. treatment by whole algae under

shaking and static conditions (3(100%) were in blank and light growth (few

colonies) (3(100%) in treated wastewater with 1 g whole algae sh, treated

wastewater with 2 g whole algae sh, treated wastewater with 4g whole algae sh,

treated wastewater with 5 g whole algae sh, treated wastewater with 1g whole

algae un-sh, treated wastewater with 2 g whole algae un-sh, treated wastewater

with 4 g whole algae un-sh and treated wastewater with 5 g whole algae un-sh.

The results indicated that there was a statistically significant difference in Blank

vs. treated wastewater with 1 g whole algae sh, treated wastewater with 2 g

whole algae sh, treated wastewater with 4 g whole algae sh, treated wastewater

with 5 g whole algae sh, treated wastewater with 1 g whole algae un-sh, treated

wastewater with 2 g whole algae un-sh, treated wastewater with 4g whole algae

un-sh, treated wastewater with 5 g whole algae un-sh p ≤ 0.05. In comparison,

no significant difference for blank treated wastewater with 1 g whole algae sh

vs. treated wastewater with 2 g whole algae sh p > 0.05

63

Table 4.11: Detection of Salmonella & Shigella spp., Coliform & Fungi, after

24 hr. treatment with whole algae under shaking and static conditions

Samples

Salmonella & Shigella

Coliform

Fungi

heavy

growth

light

growth

(few

colonies)

moderate

growth

heavy

growth

(pink

colonies)

moderate

growth

(pale

colonies)

moderate

growth

light

growth

(few

colonies)

Blank +ve -ve -ve +ve -ve +ve -ve

w (1g)

sh

-ve +ve -ve -ve +ve -ve +ve

w (2g)

sh

+ve -ve -ve -ve +ve -ve +ve

w (4g)

sh

-ve -ve +ve -ve +ve -ve +ve

w (5g)

sh


w (1g)

un-sh


w (2g)

un-sh


w (4g)

un-sh


w (5g)

un-sh


un -sh: static Sh: shaken g: grams w: whole algae hr: hour


64

Figure 4.25: detection of coliform after 24hr treatment

Figure 4.26: detection of fungi after 24hr treatment


65

4.5 Arugula results

4.5a Area leaves average, leaves number and root length of Arugula


-Table 4.12a illustrates area leaves average (cm2), leaves number and root length

(cm) for arugula planted in the field, in small cups with the same size and under

the same conditions, but with different irrigation sources and fertilizers. Area

leaves average (cm2) among studied groups were 20±5.3 for treated wastewater

with 1g powder algae, 19.9±3.7 for wastewater treated with 2g powder algae,

15.7±1 for treated wastewater with 1g whole algae, 15.4±1.1 for filtered (peat

moss and compost samples were irrigated with filtered water) , 10±1.4 for P

(0.25g), 11.8±1.3 for P (1.0 g), 11.2±0.5 for alternately (plants irrigated on a day

with treated wastewater and a day with medium salinity "5000mS"), 8±1.0 for

medium saline water, 13.9±1 for P (0.5 g) respectively.

The results showed that there was a statistically significant difference in treated

wastewater with 1g powder algae vs. treated wastewater with 1g whole algae,

Filtered, p(0.25g), p (1.0 g), alternately, medium Salinity (peat moss and

compost samples were irrigated by water with medium saline"5000mS"), p (0.5

g) p ≤ 0.05.

-Leaves number means among studied groups were 20.3±2.3 for treated

wastewater with 1g powder algae, 21.7±2.1 for wastewater treated with 2g

powder algae, 23±3 for treated wastewater with 1g whole algae, 30.3±1.5 for

filtered, 14.7±1.5 for P (0.25g), 15.3±0.6 for P (1.0 g), 18±1.0 for alternately,

18±1.0 for medium salinity, 18±1.0 for P (0.5 g) respectively. The results

showed statistically significant difference in treated wastewater with 1g powder

algae vs. filtered, p (0.25g), p (1.0g) p ≤ 0.05. In comparison, no significant

difference for treated wastewater with 1g powder algae vs. P (0.5 g) p > 0.05.

66

-Root length (cm) among studied groups were 2.3±0.4 for treated wastewater

with 1g powder algae, 2.5±0.3 for wastewater treated with 2g powder algae,

2.7±0.2 for treated wastewater with 1g whole algae, 4±0.9 for filtered, 2.9±0.3

for p (0.25g), 2.6±0.3 for p (1.0 g), 2.9±0.4 for alternately, 1.0±0.1 for medium

saline, 2.0±0.4 for P (0.5 g) respectively. The results indicated that there was a

statistically significant difference in treated wastewater with 1g powder algae vs.

filtered, saline water p ≤ 0.05.

Table 4.12a: Area leaves average, leaves number and root length for arugula


Sample

Area leaves average

(cm2)

Leaves no.

Root length (cm)

T1 20±5.3

20.3±2.3

2.3±0.4

T2 19.9±3.7

21.7±2.1

2.5±0.3

T1(w) 15.7±1

23±3

2.7±0.2

Filtered 15.4±1.1

30.3±1.5

4±0.9

P (0.25g) 10±1.4

14.7±1.5

2.9±0.3

P (1.0 g) 11.8±1.3

15.3±0.6

2.6±0.3

P (0.5 g) 13.9±1 18±1.0 2.0±0.4

Medium salinity 8±1.0

18±1.0

1.0±0.1

Alternately 11.2±0.5 18±1.0 2.9±0.4 TI: wastewater treated with 1g powder algae; T2: wastewater treated with 2g powder algae; T1

(w): wastewater treated with 1g whole algae treated waste Filtered: peat moss and compost

samples were irrigated with filtered water. Alternately: plants irrigated on a day with treated

wastewater and a day with medium salinity "5000mS"). 0.25, 0.5, 1g p: Fertility concentrations

of powder algae; these samples were irrigated with medium saline water; Medium salinity: peat

moss and compost samples were irrigated by water with medium saline water. g: grams; *P-


67

Figure 4.28(a): Arugula in the field

Figure 4.28(b): Arugula in the field

68

4.5b: Area leaves average, leaves number and root length for Arugula


-Area leaves average (cm2), leaves number and root length (cm) for Arugula

planted in the laboratory in small cups with the same size and under the same

conditions, with different irrigation sources and fertilizers as illustrated in Table

4.12b the average of area leaves (cm2) among studied groups were 7.6±1.7,

14.9±2.5, 11.2±7.5, 5.0±1.0, 4±1.0 and 3±1.0 for wastewater treated with 1g

powder algae, wastewater treated with 2g powder algae, wastewater treated with

1g whole algae, p (0.25g), p (0.5g) and medium salinity (peat moss and compost

samples were irrigated by water with medium saline water), respectively. The

results showed statistically significant difference in wastewater treated with 1g

powder algae vs. wastewater treated with 2g powder algae p ≤ 0.05, wastewater

treated with 2g powder algae vs. P (0.25g), P (0.5g), medium salinity p ≤ 0.05,

Treated wastewater with 1g whole algae vs. P (0.25g), P (0.5g), and medium

saline water p ≤ 0.05.

-The average of leaves number was 13.3±2.5, 14.9±2.5, 11.2±7.5, 5.0±1.0,

4±1.0 and 3±1.0 for wastewater treated with 1g powder algae, wastewater

treated with 2g powder algae, treated wastewater with 1g whole algae, p (0.25g),

p (0.5g) and medium salinity, respectively. The results showed statistically

significant difference in wastewater treated with 1g powder algae vs. P(0.25g),

P(0.5g), medium salinity p ≤ 0.05, wastewater treated with 2g powder algae vs.

P(0.25g), P(0.5g), medium salinity p ≤ 0.05, treated wastewater with 1g whole

algae vs. P(0.25g), P(0.5g), medium salinity p ≤ 0.05, P(0.25g) vs. medium

salinity p ≤ 0.05. In comparison, no significant difference for wastewater treated

with 1g powder algae vs. wastewater treated with 2g powder algae, treated

wastewater with 1g whole algae p > 0.05.

69

-The average of root length (cm) were 4.6±1.2, 6.6±1.1, 7.9±1.6, 4.0±1.0,

3.7±0.9 and 2.0±1.0 for wastewater treated with 1g powder algae, wastewater

treated with 2g powder algae, treated wastewater with 1g whole algae, p(0.25g),

p(0.5g) and medium salinity, respectively. The results indicated statistically

significant difference in wastewater treated with 1g powder algae vs. medium

salinity p ≤ 0.05.

Table 4.12b: Area leaves average, leaves number and root length for arugula


Sample

Area leaves

average (cm2)

Leaves no.

Root length (cm)

T1 7.6±1.7

13.3±2.5

4.6±1.2

T2 14.9±2.5

12.3±1.2

6.6±1.1

T1 (w) 11.2±7.5

12.7±3.2

7.9±1.6

P (0.25g) 5.0±1.0

8±1

4.0±1.0

P (0.5g) 4±1.0

5±1

3.7±0.9

Medium salinity 3±1.0

3±1

2.0±1.0

T1: wastewater treated with 1g powder algae; T2: wastewater treated with 2g powder algae; T1

(w) wastewater treated with 1g whole algae, 0.25g p, 0.5g p: Fertility concentrations of powder

algae; these samples were irrigated with medium salinity, medium salinity: peat moss and

compost samples were irrigated by water with medium saline water; g: gram

*P- value significant at P ≤ 0.05.

71

Figure 4.29: Arugula in the laboratory

71

5. DISCUSSION

Data regarding wastewater treatment in Gaza strip are insufficient recently.

Previous reports from Gaza strip have mostly focused on the determination of

BOD, COD, and total suspended solids "TSS" and fecal coliform of the treated

wastewater injected to the Sea.

Here, we examined for the first time the efficiency of the sea weed Ulva lactuca in

post treatment of the secondary treated wastewater. Also, we determined the

biofertilizing capacity of Ulva lactuca.

The macroalgae have applications in the removal of nutrients from effluent waters

of sewage. Chemical analysis results of treated wastewater by U. lactuca powder

and whole algae after different durations and concentrations showed decrease in

nitrate concentration using 1gram powder or whole algae for 24 hr. In agreement

with Neori et al., who stated that U. lactuca has proven to be a good seaweed

biofilter in the treatment of fishpond effluents (Neori et al., 2003), Ulva lactuca

has a capacity for high rates of nutrient assimilation, especially ammonium (NH4+),

and grows well in eutrophic waters which qualify this species for bioremediation

purposes (Gevaert et al., 2007). Several authors have reported that different Ulva

species have been verified as successful biofilters of wastewaters (Martinez-

Aragon et al., 2002). According to these results, U. lactuca can be used in

wastewater treatment plants for removing the biologically available nitrogen to

avoid eutrophication of adjacent water bodies. Here the cycle is closed as reactive

inorganic forms of nitrogen are converted back into free nitrogen through

microbial activity and re-liberated to the atmosphere (Schmidt et al., 2003).

Rethinking this pathway, by recycling the biologically available nitrogen could

offer a more sustainable and less energy demanding resource flow, while still

satisfying the need for nitrogen fertilizer as well as for removal of nutrients from

wastewater.

72

Chemical analysis results of treated wastewater by U. lactuca powder and whole

algae also revealed that there is slight increase in pH, chloride ion and electrical

conductivity. The alkaline environment enhances the biosorption capacity of the U.

lactuca for heavy metals removal as mentioned in previous study of (Bulgariu

and Bulgariu, 2014). The increase of chloride ion concentration may be due to

salts which still present in the powder or whole algae.

The COD test is rarely used in effluent discharge control, but primarily in

assessing the strength of trade or industrial effluents (Tchobanoglous and

Schroede 1999). For the reason that the COD test is a simple chemical assay, it

is easy to point out its drawbacks and limitations. The results showed that the

average COD values for effluent decreased from 605 mg/l to an average of 400

mg/l and the COD removal efficiency is around 66% which indicates that U.

lactuca whole algae and powder could be used as biofilter for wastewater

treatment. Our results concur with those obtained by Gvns et al., (2011) in India

using algal mat of Ulva sp., Cladophora sp. and Chlorella sp. showed that the

percentage of reduction was 52.1 (COD) and 50.8 (BOD) along with changes in

dissolved oxygen (DO) and pH (Gvns et al., 2011).

It is worth mentioning here that the numeric value of the COD removal

efficiency is less than the BOD because of the non-removal of the non-

degradable fraction of the COD. The theoretical values 2:1 of COD: BOD ratio

of municipal settled sewage (Horan, 1990). In view of its simplicity and

rapidity, the COD test is the most suitable assay for the determination of the

strength of both raw and treated wastewater.

Regarding to heavy metals removal efficiency of U. lactuca, there are

significant decrease in values of Pb, Fe, Zn, Mn and Sr ions compared with the

standard , which is in agreement with several previous studies (Bulgariu and

Bulgariu, 2014; Freitas et al., 2008; Kalesh and Nair, 2005; Mudhoo et al.,

73

2012). As mentioned before, the alkaline treated marine green algae have better

biosorption characteristics than untreated waste biomass, and have potential for

serving as biosorbent for removal of heavy metals from aqueous solution (Sodea

et al., 2013). In agreement with our results, it was showed by Lupea et al., that

the marine green algae can be efficient use for the removal of heavy metal ions

from aqueous solution (Lupea et al., 2012). In addition to the bioremediation

effect, Lu revealed that Ulva has an antibacterial effect on the waste water, thus

reducing the health related problems of the waste water (Lu et al., 2008). The

treatment of the secondary treated wastewater with U. lactuca powder and

whole algae (1g/L) reduces the total bacterial count sharply as well as fungi,

Salmonella spp. and Shigella spp. The antimicrobial activity of macroalgae

(seaweeds) is well documented (Gvns et al., 2011, Lu et al., 2008; Shannon

and Abu-Ghannam, 2016). The effect of shaking on the antimicrobial

potentials of U. lactuca against bacteria and fungi was studied during this work.

The results reveals that the antimicrobial activity of U. lactuca algae increased

under shaking conditions, and that is could be due to increase of gases transfer

and increase the amount of oxygen required for algae respiration which

increases the growth of algae and may contribute to its antimicrobial activity.

The shaking conditions also increase the capacity of the algae powder in

reduction of the bacterial and fungal as well as certain types of pathogenic

bacteria studied such as Salmonella spp. and Shigella spp. The effect of shaking

on the powder efficiency in removal of microorganisms may be due to increase

the contact between the algal powder and microorganisms present in

wastewater. Both macroalgae (seaweeds) and microalgae (diatoms) contain

pharmacologically active compounds such as phlorotannins, fatty acids,

polysaccharides, peptides, and terpenes which combat bacterial invasion and

illustrate the antimicrobial potentials of U. lactuca (Shannon and Abu-

Ghannam, 2016). The results of the present study clearly enunciate the

potentials of Ulva sp. for employing in wastewater treatment. The use of

74

naturally existing seaweeds (U. lactuca) for wastewater bioremediation is an

innovative, economical and environmentally safe alternative for treating

wastewater in Gaza strip. Macroalgae powder and extract are natural organic

fertilizers for many crops. Unlike, chemical fertilize nonpolluting and non-

hazardous to humans, animals and birds (Dhargalkar, 2014). The growth

promoting effect of liquid extracts of seaweeds on vegetative growth in

agricultural crops was reported (Thirumalthangam et al., 2003). The beneficial

results from their use in crop plants like early seed germination and

establishment, improved crop performance and yield, elevated resistance to

biotic and abiotic stress and enhanced post-harvest shelf life of seeds are

documented (Hankins and Hockey, 1990; Guiry and Blunden, 1991; Booth,

1965). To study the capacity of U. lactuca powder and whole algae as fertilizer,

we examined its effect on leaves number and area as well as root length of

Arugula (salad rocket) plant. Arugula (Eruca sativa) is an annual plant belong to

Brassicaceae family grows 20–100 centimeters in height and has a very short

life cycle, is a species of Eruca native to the Mediterranean region. The results

revealed an increase in leaves number and area as well as root length of Arugula

plant fertilized with U. lactuca powder. The planted Arugula was cultivated in

field conditions (outdoor) and in laboratory conditions at fixed temperature

20°c, and fixed humidity 55. The root growth measurements (root length) of

Arugula plant revealed that optimum root length obtained in the laboratory

conditions. Meanwhile, the shoot growth (leaves number and area) increased in

the field more the laboratory conditions. The fertilizing capacity of U. lactuca

and some other seaweed could be to its high content of growth promoting

substances (Sylvia et al., 2005), such as IAA, kinetin, zeatin and gibberellins

(Zodape et al., 2009), auxins and cytokinins (Zhang and Ervin, 2004),

metabolic enhancers (Zhang and Schmidt, 1997), macro and micro elements,

amino acids, vitamins (Strik et al., 2003).

https://en.wikipedia.org/wiki/Brassicaceae

https://en.wikipedia.org/wiki/Eruca

https://en.wikipedia.org/wiki/Mediterranean

75

6. Conclusion and Recommendations

6.1 Conclusion:

Ulva lactuca as intact organism and powder form have a tangible impact

in tertiary treatment of wastewater and reuse in irrigation and the powder

algae also has a tangible impact as a biofertilizer.

6.2 Recommendations:

Macro algae should be considered as clean and sustainable energy in

different aspects such as wastewater treatment and re-use in irrigation or

discharge more safely to the sea

Use Ulva lactuca as natural fertilizer rather than chemical

Use Ulva in different industries as medicines, food, dyes and cosmetics

manufacturing

Further studies must be done on the treatment of wastewater using macro

algae

More studies must be done on the use of seaweeds as biofertilizers.

76

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