An-Najah National University 

Faculty of Engineering and Information Technology 

Chemical Engineering Department 

Graduation Project Ⅱ 

Toward Biogas Production from Olive Mill Wastewater (OMWW) and Constructing 

Anaerobic Lab Scale Bioreactor  

Prepared by:  

Farah Baghdadi 

Yasmeen Odeh 

Zain Abu Lebdeh 

 

Supervisor:  

Dr. Majd Eshtaya 

Graduation Project Submitted in Partial Fulfilment of the Requirement for the Degree of 

B. Sc in Chemical Engineering 

June, 2020



  

i 

 

Acknowledgements  

 

We would like to express our deepest gratitude to our supervisor, Dr. Majd Eshtaya, for her 

excellent guidance, caring, patience, and providing us with an excellent atmosphere for doing 

research. 

Our thanks also go to Dr. Abed Al Fattah Mallah, for his support as well as for providing us with 

the required laboratory facilities to accomplish our research experiments.  

Special thanks to Eng. Bilal Al-Shayeb for being our guide and supervisor during all the 

experiments. Also, thanks for his thoughtful comments and suggestions. 

We are also grateful for Eng. Mohammed Homidan for helping us in sample collection also for his 

valuable comments and information. 

Finally, we would like to thank our families for their support and encouragement. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



  

ii 

 

Abstract 

 

Olive oil production is a traditional industry in Palestine; about 20,754 tons of olive oil are 

produced annually in Palestine. However, Olive Mill Wastewater (OMWW) -which is the black 

liquid byproduct of the olive oil extraction process- management is a crucial environmental 

challenge facing the Mediterranean countries including Palestine. Based on previous studies, Co-

digestion of OMWW with sludge from the digester in a designed bioreactor under specific 

conditions provides the possibility to eliminate the OMWW toxicity and produce biogas at the 

same time.  

The main goal of this study is to treat the OMWW with sludge at different ratios under anaerobic 

digestion process at a mesophilic condition with an optimum temperature equal to 37C using a 

laboratory-scale bioreactor.  

The project started by constructing and build a laboratory-scale bioreactor. Depending on the 

economical and qualitative aspects 5 liters in size pressure cooker (four identical pressure cookers) 

was chosen to be used as a lab-scale bioreactor. Due to the sensitivity of the bacteria and 

sustainability, stainless steel was considered as a material of construction.  

The choice of a pressure cooker as a constructed lab-scale bioreactor was basically to perform the 

digestion process to treat the OMWW samples. Thus, the four pressure cookers were modified in 

a way to reach the desired construction of the bioreactor. The modifications were done in manner 

to transform the pressure cooker into a lab-scale bioreactor as follows;¾‶ hole with a PVC pipe 

was inserted in the led as the feed inlet, a washing nozzle (stainless steel water tap), 2 PVC pipes 

(¾‶) with 3 elbows were also inserted; lastly, a gas tube was connected with the bike tire for gas 

collection purpose. The produced biogas will be utilized as the stirring system to prevent bacteria 

scum.  

The project work was mostly involved around the construction part as explained above due to the 

current situations (the spread of Covid-19) but some tests were made to assure that the system is 

working properly and to check some parameters like leaking and stability. Another test was the 

cow manure test which was done to confirm the design stability. 



  

iii 

 

This project is a continues work for the previous work that was accomplished in the first semester 

were fresh OMWW samples were collected from Al- Kafryat pressing mill, near Tulkarem and the 

Wastewater Treatment Plant (WWTP), near Nablus. Characterization of OMWW samples were 

done based on number of physical and chemical parameters such as chemical oxygen demand 

(COD), ammonia, total nitrogen, pH and total dissolved solids (TDS). The results that obtained 

were respectively 60,000 mg/L, 280 mg/L, 630 mg/L, 7.4 and 20,746 mg/L. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



  

iv 

 

Table of Contents 

Acknowledgements .......................................................................................................................... i 

Abstract ........................................................................................................................................... ii 

List of Figures ................................................................................................................................ vi 

List of Tables ................................................................................................................................ vii 

Nomenclatures ............................................................................................................................. viii 

Chapter One .................................................................................................................................... 1 

1. Introduction ............................................................................................................................. 1 

1.1. Problem Statement ........................................................................................................ 2 

1.2. Objectives ..................................................................................................................... 3 

1.3. Challenges .................................................................................................................... 3 

1.4. Report Structure ............................................................................................................ 4 

Chapter Two.................................................................................................................................... 5 

2. Literature Review.................................................................................................................... 5 

2.1. Olive Mill Wastewater Treatment ................................................................................ 5 

2.1.1. Physical treatment ..................................................................................................... 5 

2.1.2. Forced Evaporation ................................................................................................... 5 

2.1.3. Physio-chemical treatment ........................................................................................ 5 

2.1.4 .  Electrocoagulation .................................................................................................... 6 

2.1.5. Biological treatment .................................................................................................. 6 

2.2. OMWW Characteristics ............................................................................................... 7 

1.1. Potential Studies for Biogas Production from OMWW ............................................... 9 

1.2. Anaerobic Bioreactor Design ..................................................................................... 12 

1.3. Biogas ......................................................................................................................... 19 

1.3.1. Biogas Substrate...................................................................................................... 19 

1.4. Environmental Impact of OMWW ............................................................................. 20 

1.5. Factors affecting the anaerobic digestion process through biogas scope: .................. 21 

1.6. Anaerobic Digestion Stages ........................................................................................ 24 

1.7. Olive Pressing Techniques ......................................................................................... 26 

1.7.1. Traditional Pressing Method. .................................................................................. 26 

1.7.2. Modern Method (Integral Centrifuge Systems) ...................................................... 26 

Chapter Three................................................................................................................................ 28 



  

v 

 

3. Methodology and Analytical Methods ....................................................................... 28 

3.1. Analytical Methods..................................................................................................... 28 

3.1.1. Measurement of Chemical Parameters ................................................................... 28 

3.1.2. Measurement of Physical Parameters ..................................................................... 29 

Chapter Four ................................................................................................................................. 31 

4. Lab-Scale Bioreactor .................................................................................................. 31 

4.1. Overview .................................................................................................................... 31 

4.2. Bioreactor Material of Construction ........................................................................... 34 

4.3. Construction Implementation ..................................................................................... 36 

4.4. Schematic Diagrams for the Bioreactor Construction ................................................ 42 

4.5. Tests Methodology ..................................................................................................... 44 

Chapter Five .................................................................................................................................. 50 

5.1. Recommendations and Future work ........................................................................... 50 

a. Preparing the substrate materials ................................................................................ 50 

b. Characterization of OMWW samples ......................................................................... 50 

c. Operating the designed bioreactors ............................................................................ 50 

d. Analytical Methods..................................................................................................... 51 

e. OMWW and Sludge Ratios ........................................................................................ 51 

f. Methods for Measuring Produced Biogas Volume. ................................................... 53 

g. Challenges could be faced: ......................................................................................... 54 

Chapter Six.................................................................................................................................... 55 

6. Conclusion ............................................................................................................................ 55 

References ..................................................................................................................................... 56 

Appendices .................................................................................................................................... 60 

 

 

 

 

 

 

 



  

vi 

 

List of Figures 

 

Figure 2.4. 1: Schematic diagram of continuous-stirred tank reactor  (Khanal et al., 2017) ........ 12 

Figure 2.4. 2:  Schematic diagram of a typical covered anaerobic lagoon (Khanal et al., 2017). 13 

Figure 2.4. 3 :Schematic diagram of anaerobic contact reactor  (Khanal et al., 2017). ................ 14 

Figure 2.4. 4: Schematic diagram of a UASB reactor  (Khanal et al., 2017). .............................. 15 

Figure 2.4. 5: Schematics of anaerobic baffled reactor  (Khanal et al., 2017). ............................ 16 

Figure 2.4. 6:Schematics of anaerobic sequencing batch reactor (Khanal et al., 2017). .............. 17 

Figure 2.4. 7: Schematic diagram of an anaerobic filter (A) upflow anaerobic filter and (B) 

downflow anaerobic filter  (Khanal et al., 2017). ......................................................................... 18 

 

Figure 2.8. 1: Anaerobic digestion stages  (Manchala et al., 2017) .............................................. 25 

 

Figure 2.9.2. 1: Schematic flowchart of industrial olive oil extraction techniques; traditional, 

three-phase and two-phase centrifuge process (ElMekawy et al., 2014). ..................................... 27 

  

Figure 4.3.1. 1: Routes to solve leaking........................................................................................ 40 

Figure 4.3.1. 2: Gasket maker with the original rubber. ............................................................... 41 

Figure 4.3.1. 3: Drilled holes with rubbers. .................................................................................. 41 

Figure 4.3.1. 2: Gasket maker with the original rubber. ............................................................... 41 

Figure 4.3.1. 3: Drilled holes with rubbers………………………………………………………41  

Figure 4.5.1. 1: Gas leaking test ................................................................................................... 45 

 

1. The cow manure samples were mixed with a ratio of (2 L fresh: 1 L old) and diluted with 

tap water as shown in Figure 4.5.2. 1. .......................................................................................... 46 

2. The feed was injected in the reactor through the feed inlet using a plastic funnel as shown in 

Figure 4.5.2. 2. .............................................................................................................................. 46 

  

Figure 4.6. 1: Cumulative of Biogas with HRT (Baati et al, 2018) .............................................. 23 

 

Figure 5.1. 1: COD removal percentage using (70:30) ratio  (Azbar et al., 2004). ...................... 51 

Figure 5.1. 2: Total Nitrogen percentage in using (70:30) ratio  (Azbar et al., 2004). ................. 52 

Figure 5.1. 3 : Polyphenols removal percentage in (70:30) ratio  (Azbar et al., 2004). ............... 53 

Figure 5.1. 4: Anaerobic digestion system including the biogas measurement (Abushattal et al., 

2017) ............................................................................................................................................. 54 

 

Figure A- 1: Hl 83214 Multi-parameter Bench photometer for wastewater treatment application.

....................................................................................................................................................... 67 

Figure A- 2: Hl 839800 COD Reactor. ......................................................................................... 68 

Figure A- 3: Hl93764-0, Nessler Reagent. ................................................................................... 68 

Figure A- 4: Ammonia test vial. ................................................................................................... 69 

file:///C:/Users/Farah/Desktop/Final%20Draft/Graduation-Project-2-Final12.docx%23_Toc43813481
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Figure A- 5: COD test vial after digestion. ................................................................................... 69 

Figure A- 6: KUBOTA 5100 centrifuge. ...................................................................................... 70 

Figure A- 7: Vacuum flask. .......................................................................................................... 70 

Figure A- 8: Whatman filter paper................................................................................................ 71 

Figure A- 9: General properties of Whatman filter paper. ............................................................ 71 

 

 

 

List of Tables 

Table 2.5. 1: Composition of Biogas  (Kossmann and Pönitz, 2011) ........................................... 19 

 

Table 4.2. 1: Material of construction type and advantages, disadvantages. ................................ 35 

Table 4.3. 1: Bioreactor construction specifications ..................................................................... 37 

 

Table A- 1: COD required reagent. ............................................................................................... 60 

Table A- 2: Ammonia required reagents. ..................................................................................... 62 

Table A- 3: Total nitrogen required reagent. ................................................................................ 64 

Table A- 4: Number of oil presses and methods of waste disposal of olive cake and waste water.

....................................................................................................................................................... 72 

 

 

 

 

 

 

 

 

 

 

 

 



  

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Nomenclatures 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BOD Biochemical Oxygen Demand 

CH4 Methane 

CO2 Carbon Dioxide 

COD Chemical Oxygen Demand 

CSTR Continuous Stirred Tank 

HRT Hydraulic Retention Time 

LCM Liquid Cow Manure 

OLR Organic Load Rate 

OMWW Olive Mill Wastewater 

SRT Solid Retention Time 

TCOD Total Chemical Oxygen Demand 

TDS Total Dissolved Solid 

TP Total Phenolic 

TPOMWW Two Phase Olive Mill Wastewater 

TPOMWW Two Phase Olive Mill Wastewater 

TS Total Solid 

VFAs Volatile Fatty Acids 

TSS Total Suspended Solid 

WWTP Wastewater Treatment Plant  



  

1 

 

Chapter One 

1. Introduction  

 

Wastewater is defined as a complex matrix that contains several concentrations such as solids (total 

solids 350–1200 mg/l), dissolved and particulate matter (chemical oxygen demand (COD) 

250– 1000 mg/l), microorganisms (up to 109 number/ml), nutrients and heavy metals and micro-

pollutants  (Warwick et al., 2013). Wastewater is classified mainly into four groups; domestic 

wastewater, industrial wastewater, storm-water, and infiltrations. These four groups can be present 

in a combined system or separate sewage systems  (Henze et al., 2008).  

According to the studies, industrial wastewater is considered as one of the most pollutants elements 

for the water environment. Especially during the last decades, the discharge of industrial 

wastewater into rivers, lakes and coastal areas has significantly increased. Therefore, a specific 

treatment system is truly needed. Developing a treatment system is depended on the type of 

industrial wastewater as there are many types of industrial wastewater, each type requires special 

treatment technique  (Hanchang, 2009). 

The anaerobic digestion process is one of the most common treatments that are widely used to 

treat different industrial effluents. To maintain the anaerobic process, an appropriate bioreactor 

design is required. The selection of a specific bioreactor design includes a series of choices ranging 

from basic microbiology and biochemistry to process engineering and marketing which must 

contribute in a symmetric way to make a proper determination of the basic type of bioreactor 

system and its correct dimensions to provide an optimal environment for the biological system, 

operating at minimal cost in order to maximize the benefits and the productivity of the 

process (Asenjo, 1994).  

Typically, the design of biological reactors involves determining operating conditions, sizing the 

reactor, controlling temperature and sterility, determining the means of feed introduction and 

product removal, designing for mixing and mass transfer and controlling operating variables, such 

as pH and oxygen concentrations (McDuffie, 2013). The importance of selecting the suitable 

construction of bioreactor is highly needed  to achieve the desired results such as the amount of 

the biogas compositions and the optimum ratios to produce biogas  (Mandenius, 2016) 



  

2 

 

The anaerobic digestion treatment with other types of waste (e.g. cow manure, chicken manure, 

and sludge from the wastewater treatment plant), in other terms anaerobic co-digestion treatment 

is the presuming solution for the reduction of the OMWW Chemical Oxygen Demand (COD), and 

generating the methane gas  (Battista et al., 2013).  

 

1.1. Problem Statement  

 

In the current Palestinian situation, the enormous produced amounts of OMWW composes a real 

threat to the environment. The annual OMWW production in the Mediterranean countries is 

estimated to amounts ranging from 7 to over 30 million m3. In the West Bank, the annual 

production is up to 120,000 m3 besides thousands of tons of solid olive residues. This huge amount 

of OMWW has no pre-treatment system before discharging it directly into Wadi or sewage 

networks  (Nazzal, 2017).  

The OMWW has a very high organic load, as the load is 100-150 times greater than the organic 

load of domestic wastewater (1080 mg/L)  (Al-Khatib et al., 2009). The process of discharging the 

OMWW directly into Wadi or the public sewage networks has a huge effect on the efficiency of 

the Wastewater Treatment Plant (WWTP) and the activity of microorganisms and the effluent 

quality.  

The most problem of OMWW is the high organic content, acidity pH and high phenols content 

which is toxic to many microorganisms  (Nazzal, 2017). 

Despite the importance of the olive oil production industry for the Palestinian economy, the 

problem of produced OMWW is becoming a real and serious environmental problem. Thus, it’s 

required to solve the problem and apply a reasonable and practical treatment technique. The 

proposed treatment in the project is an anaerobic co-digestion process taking into consideration 

the design of an anaerobic bioreactor to perform the target process. 



  

3 

 

 

1.2. Objectives 

  

The main objectives of this project are:  

1. Construct a laboratory-scale bioreactor with the required specifications (such as the size, 

the material of construction and temperature controlling).  

2. To produce biogas from the anaerobic digestion of OMWW locally named as “Zibar” 

mixed with sludge (from digester) obtained from the domestic wastewater treatment plant. 

3. To measure the volume of produced biogas and determine the methane (CH4) content.  

4. To examine the optimum ratio between OMWW and the sludge for maximum biogas 

production. 

 

1.3. Challenges  

There are many challenges that were encountered during the graduation project’s journey, 

here are some of these challenges:  

• The spread of pandemic Covid-19, which threatened the entire world and because 

of it, part of the graduation project was suspended and thus the possibility to 

complete the planned experimental work.  

• Time delay through the construction of lab-scale bioreactors due to the facilities 

to accomplish the desired design.  

1.4  Constraints and Engineering Standards  

Constraints: 

• Operating the digestion process under mesophilic conditions at 37 ℃ (Stable, less energy and wide 

use in practice). Olive Mill Wastewater samples are only available seasonally (September -

November), so that it was mandatory to store the samples in glass bottles inside the 

refrigerator at 4 ℃. 

• Transfer the sludge for wastewater treatment plant to the laboratory immediately 

 



  

4 

 

Standards:  

1. Chemical Oxygen Demand Test (COD), Ammonia Test, Total Nitrogen Test and Total 

Dissolved Solid Test must be done according to the standards. 

2. The recommended bioreactor working is  3.5 L (70% of 5 L)  (Kurt Eyer, 2020). 

3. Retention time should be about (15–30) days to treat waste under mesophilic conditions. 

 

1.4. Report Structure  

 

The report consists of six chapters, the first one includes the introduction in general, also the 

problem statement, the objectives and the challenges of this project. The second chapter 

includes the literature review part that contains eleven sections: OMWW treatment, OMWW 

characteristics, potential studies for biogas production, olive pressing techniques (traditional 

and modern methods), olive oil production in Palestine, the environmental impact of OMWW, 

biogas from OMWW, anaerobic digestion stages, also factors affecting anaerobic digestion 

process through biogas and anaerobic digestion stages. Chapter three is about methodology 

and analytical methods. The fourth and fifth chapters contain lab-scale bioreactor construction 

and recommendations respectively. Finally, chapter six is about the conclusion



  

 5  

 

Chapter Two 

2. Literature Review 

 

2.1. Olive Mill Wastewater Treatment  

 

  There are several processes that could be used to treat the OMWW which includes the physical 

method, the physio-chemical methods (flocculation, filtration, incineration, open evaporating 

ponds, and coagulation), and biological treatment methods that reduce the organic load of 

OMWW. 

2.1.1.  Physical treatment 

    Physical processes are applied to separate the various phases (solids, liquids, and gases) of 

OMWW. However, physical processes are not able to achieve acceptable limits for toxicity and 

organic load alone when used in OMWW treatment. physical processes include: dilution, 

sedimentation, filtration, centrifugation, evaporation filtration, and dissolved-air 

flotation  (Abushattal et al., 2017). 

2.1.2. Forced Evaporation 

    For two phase oil extraction process, the appropriate management and treatment option for 

OMWW in is the “natural evaporation method”. It can be considered a low-cost solution to 

incorporation of wastewater pollutants into dried sludge. However, due to the long evaporation 

periods this technique is suitable only for low to medium wastewater flows. This alternative 

treatment is characterized by the generation of dried sludge that can be disposed of or reused as 

fertilizer  (Shaheen and Abdel Karim, 2007). 

 

2.1.3.  Physio-chemical treatment 

     This method is mainly used to remove organic matter from the liquid phase by adding 

chemicals. Many techniques used in this method such as, neutralization technique, flocculation 

technique, precipitation, adsorption, chemical oxidation, and Ion exchange. However, it has many 

disadvantages such as; sludge-disposal problems, low efficiency, and high cost  (Abushattal et al., 

2017). 



  

 6  

 

2.1.4. Electrocoagulation 

   Electrocoagulation is a process that consists of creating a floc of metallic hydroxides within the 

effluent to be cleaned, by electro dissolution of soluble anodes. Compared with traditional 

flocculation and coagulation, electrocoagulation has, in theory, the advantage of removing the 

smallest colloidal particles; the smallest charged particles have a greater probability of being 

coagulated because of the electric field that sets them in motion. It is used to reduce the phenolic 

content of OMWW effluent.  Electrocoagulation is an effective pre-treatment technique which 

aims to reduce the organic load of OMWW prior to an upcoming biological treatment which 

resulted in a higher volume of biogas compared to crude or diluted OMWW  (Sounni et al., 2018). 

 

2.1.5. Biological treatment 

   Biological treatment is the most appropriate and modern cheapest way to treat OMWW. The 

biodegradable chemical species present in OMWW can be degraded by this method using 

microorganisms that break down these species with environmental consideration. Biological 

therapy can be achieved by using aerobic, anaerobic treatment  (Abushattal et al., 2017). 

Detoxification processes run by fungi is more effective than that run by bacteria that applied to 

degrade the phenolic compound. The removal rate of fungi for COD as 40 – 88 %, for phenols as 

60 – 100 %. 

2.1.5.1. Aerobic processes 

     Aerobic processes are applied as a pre-treatment to reduce COD, TSS, and phenolic compounds 

which enhancing the anaerobic treatment. The aerobic digestion conditions include the availability 

of oxygen and nutrient where the aerobic microorganism thrives in these conditions.   However, 

the aerobic treatment is not highly recommended due to the number of reasons: a. It is an expensive 

treatment. b. Needs dilution. c. High residence time is necessary. d. Requires a pH adjustment. 

2.1.5.2. Anaerobic process 

    The anaerobic treatment is a cost-effective alternative, when it is compared to the aerobic 

treatment, especially for high organic industrial wastewater. Anaerobic wastewater treatment 

processes for effluent treatment of olive presses were tested in experimental measures  (Shaheen 

and Abdel Karim, 2007). 



  

 7  

 

Anaerobic treatments include the degradation of organic matter and the production of biogas such 

via a microorganism in the condition of absence of oxygen. Many parameters affect the anaerobic 

digestion process which is Solids Retention Time (SRT), Temperature, pH and Alkalinity.  

Anaerobic treatment is widely used especially for the production of biogases which has big 

capability to recover energy, saving energy, and reduce operational cost  (Abushattal et al., 2017). 

2.2. OMWW Characteristics  

 

Serious environmental problems arise when OMWW is discharged into the environment without 

applying any treatment or controls because OMWW contains a high amount of chemical species 

that resist degradation such as phenolic compound and high COD content. 

In general, OMWW contains water with 83 wt %, 15 wt % organic compounds and the rest 

inorganic compounds as illustrated in Figure 2.9.2 

 

Figure 2.9.2.: The main composition of OMWW 

 

Also, it is characterized by the high concentration of cations and anions, COD, BOD, Polyphenolic 

compounds, Fat and Nutrient. 

Water

83%

Organic

15%
Inorganic

2%

Water

Organic

Inorganic



  

 8  

 

Usually, OMWW can be described in dark violet to black colour, and its organic compound load 

is 100-150 times higher than the organic load of domestic wastewater, which is the highest 

percentage of all ingredients and has a specific oil odor  (Yosef and Abdallah, 2016). 

OMWW pH between 4.9 and 5.1, high electrical conductivity, high concentration of total 

suspends solid (TSS) and total dissolved solids (TDS), COD (up to 247 g/L) and high polyphenol 

concentration (up to 21 g/L). 

Table 2 below shows the general characteristic of OMWW  (Yosef and Abdallah, 2016). 

Table 2.5.1 1: General Characteristic of OMWW from (2004 – 2016) 

Parameter Unit Yosef (2016) 
Aladham 

(2012) 

Khtib et al., 

(2009) 

Basheer et al., 

(2004) 

BOD5 mg/L 33532 11375 45624.67 27500 

COD mg/L 246652 137525 98999.67 163500 

TS mg/L 75328 67478   

TSS mg/L 62117 52014 16963.6 86840 

TDS mg/L 13211 15464 35212.67  

Total polyphenols mg/L 21000 4592 3149.33 6800 

pH  5.1 4.9 4.99 5 
 

All of the above parameters must be taken into consideration in the design of a well-integrated 

treatment process of OMWW.



  

 9  

 

 

1.1. Potential Studies for Biogas Production from OMWW 

 

Several studies were inducted all over the world to study the anaerobic treatability of OMWW. A 

group of these studies from different regions are illustrated below. 

A study in Turkey was inducted to evaluate the anaerobic digestion process of a group of OMWW 

samples with a collected digester sludge from Ankara wastewater treatment plant. The substrate 

was injected in 125 mL serum bottles for 44 days, the gas production rate was monitored and 

measured using a gas displacement device. Additionally, the CH4 content was measured using a 

20 g/L KOH solution in other serum bottles. It was found that the CH4 content of the produced 

biogas from olive-oil mill wastewater was 77 ± 6%. Error! Reference source not found. 

illustrates the cumulative gas production for the OMWW samples  (Demirer et al., 2000).  

 

 

 

 

 

 

 

 

 

 

 

(Demirer et al., 2000).

Figure 2.3. 1:Cumulative gas production for different OMWW samples  



  

 10  

 

According to a different study in Vegas Altas Spain, it was confirmed to perform an aerobic 

pretreatment for the collected OMWW samples. The aerobic pretreatment was followed up by the 

anaerobic digestion process. The anaerobic digestion process was incited and activated via 

injecting the sludge inoculum to the OMWW samples. The experiments were performed for a 

period of time that ensured that reliable results would be obtained. The results for the anaerobic 

digestion process (methane yield, pH, reduction of total chemical oxygen demand) are illustrated 

in Figure 2.3. 2. A reduction in polyphenols concentration of 78% (5 days aeration time), achieves 

a methane production of 0.39 m3/kg removed TCOD and 65.17% TCOD degradation  (González-

González and Cuadros, 2015). 



  

 11  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.3. 2: Results of anaerobic digestion of OMWW: no pretreatment stage and 5- and 

7-days aeration process. (a)Methane yield, (b) pH, (c) reduction of Total Chemical 

Oxygen Demand  (González-González and Cuadros, 2015). 



  

 12  

 

1.2. Anaerobic Bioreactor Design 

 

The biochemical reactor is defined as a system that provides a biologically active environment, 

where the microorganisms and enzymes are immobilized, and biochemical reactions are 

performed (Somiya, 2013). 

 

Generally, the anaerobic bioreactor design is dependent on the experimental basis. Each 

anaerobic system has its requirement and specifications based on several conditions and factors. 

There are different types of bioreactors are represented below in details   (Khanal et al., 2017): 

 

1. Continuous-Stirred Tank Reactor (CSTR): 

The CSTR is the most commonly used reactor configuration employed for anaerobic treatment 

of industrial effluent containing medium to high suspended solids with total solids (TS) content 

of 0.5% or higher. The contents in the reactor are maintained under completely-mixed 

conditions by mixing continuously or intermittently using the mechanical mixture, biogas 

sparging, or liquid recirculation. A schematic diagram for the CSTR is illustrated in Figure 

2.4.1.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.4. 1: Schematic diagram of continuous-stirred tank 

reactor  (Khanal et al., 2017) 



  

 13  

 

2. Covered Anaerobic Lagoon (CAL): 

The CAL is an inexpensive option for treating the industrial effluents with TS content of 

0.5- 3%. The CALs are designed as earthen pits constructed with impermeable liners (e.g., 

clay) at the bottom and sides. A typical CAL uses neither mechanical mixing nor external 

heating. It is operated at ambient conditions; thus, the treatment efficiency is tied to the 

geographical location and climate. Figure 2.4.2 shows the typical CAL. 

 

 

 

3. Anaerobic Contact Reactor (ACR) 

The ACR is mainly used for treating the industrial wastewater with high suspended solids (e.g., 

wastewater from a meat packing plant). It mainly includes a CSTR and a downstream settling tank. 

The settled microbial biomass is recycled back to the reactor, due to the ACR configuration it 

maintains a high biomass concentration. Also, the produced biogas (CH4 and CO2) are removed 

from the liquid phase via degassifier to prevent biomass floating. Figure 2.4.3 below shows the 

ACR design.  

Figure 2.4. 2:  Schematic diagram of a typical covered anaerobic lagoon (Khanal et 

al., 2017). 



  

 14  

 

 

 

4. Upflow Anaerobic Sludge Blanket Reactor (UASB):  

The UASB reactor is a suspended growth reactor mainly utilized for the very high 

concentration of microbial biomass (e.g. food processing, agro-based industries, 

and other carbohydrate-rich industries) by promoting granulation. The anaerobic 

granules are 1-3 mm in diameter and dense enough to settle down in the reactor. 

The substrate is uniformly distributed at the bottom of the reactor, where the 

anaerobic granules come in contact with the organic matter and degrade it. UASB 

design based on two approaches, the first one the maximum allowable volumetric 

OLR to obtain the desired organic removal efficiency. And the other approach 

based on the specific sludge activity or the specific substrate utilization rate.  

 

 

 

 

 

Figure 2.4. 3 :Schematic diagram of anaerobic contact reactor  (Khanal et al., 2017). 



  

 15  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5. Anaerobic Baffled Reactor (ABR) 

The ABR consist of a group of baffles that are arranged in a certain way to force the 

wastewater to flow over and under the baffles. Microbial biomass accumulates between 

the baffles forming granular biomass with time. It has the advantage of promoting 

contact between the wastewater and the sludge blanket. However, it’s not suitable to 

treat the industrial effluents with high suspended solid content. Figure 2.4.5 shows the 

ABR design.  

 

 

 

 

 

 

 

Figure 2.4. 4: Schematic diagram of a UASB reactor  (Khanal et al., 

2017).  



  

 16  

 

 

 

 

 

6. Anaerobic Sequencing Batch Reactor (ASBR) 

The ASBR offers the ability to treat high-strength industrial effluents with medium 

solid content TS (0.5-4%).  Since the ASBR is operating based on a sequence of cycles. 

The substrate is fed into the reactor during the “feed” cycle. The biomass is degraded 

in the react cycle, mechanical mixture or the produced biogas could be used as an 

agitation system. After a sufficient period of time (settle phase) all the microbial 

biomass will be completely degraded and the degree of the anaerobic treatment is 

achieved. Figure 2.4.6 illustrates the schematic diagram for ASBR. 

 

 

 

 

 

 

 

 

Figure 2.4. 5: Schematics of anaerobic baffled reactor  (Khanal et al., 2017). 



  

 17  

 

 

 

7. Anaerobic Filter (AF): 

The (AF) is a packed-bed attached-growth reactor primarily developed to treat highly 

soluble wastewater. The two common types of AR are the upflow and the downflow 

aerobic filter reactors. A large amount of microbial biomass is retained in an anaerobic 

filter. Thus, extremely long SRT can be achieved irrespective of HRT. The excess 

microbial biomass may require a periodic removal from the bottom of the reactor to 

prevent the filter blocking. Figure 2.4.7 demonstrates the schematic diagram of the AF 

reactor.  

  

 

 

 

 

 

 

 

Figure 2.4. 6:Schematics of anaerobic sequencing batch reactor (Khanal et al., 2017). 



  

 18  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.4. 7: Schematic diagram of an anaerobic filter (A) upflow anaerobic filter and 

(B) downflow anaerobic filter  (Khanal et al., 2017). 



  

 19  

 

1.3. Biogas  

 

Biogas originates from bacteria in the process of biodegradation of organic material under 

anaerobic (without air) conditions. The natural generation of biogas is an important part of the 

biogeochemical carbon cycle. Methanogens (methane-producing bacteria) are the last link in a 

chain of microorganisms, which degrade organic material and return the decomposition products 

to the environment. In this process biogas is generated, a source of renewable energy  (Kossmann 

and Pönitz, 2011) 

Biogas is a mixture of gases that is composed chiefly of composition as shown in Table 2.5.1 

below. 

Table 2.5. 1: Composition of Biogas  (Kossmann and Pönitz, 2011) 

Composition of Biogas Vol % 

Methane (CH4) 40 - 70  

Carbon dioxide (CO2) 30 – 60 

Other gases 5-1 

Hydrogen (H2) 0 – 1 

Hydrogen Sulfide (H2S) 0 – 3 

 

 

1.3.1. Biogas Substrate 

 

Biogas substrate, the production of biogas has started from biomass energy sources. Biomass has 

several different forms such as food crops (e.g. corn or sugar cane, sugar beets, canola), cellulosic 

plant material (e.g. switch grass or miscanthus), complex biomass (e.g. animal waste) and plants 

(e.g. Jatropha, soybeans, or sunflowers)  (Rittmann, 2008). The major obstacle against using food 

crops as the energy source that the competition with food or feed production as long as all food 

crops require to be grown on a high-grade arable land.  (Mussgnug et al., 2010).  

 

Combination of different biomass forms also considered a reliable source for biogas production. 

A considerable combination is pretreated OMWW with chicken manure (a rich source of nitrogen) 

and sludge  (Sounni et al., 2018). 

 



  

 20  

 

1.4. Environmental Impact of OMWW 

 

OMWW is a problematic waste that has become a major cause of health and environmental 

concerns. OMWW is frequently dumped, untreated, either in soil or into water sources causing:  

1. Phytotoxicity,  

2. The proliferation of insects, 

3. Increasing salinity, 

4. Reducing the permeability of the soil, 

5. Decreasing the degree of aeration 

6. Colouring of natural waters, 

7. Changing plant growth and, 

8. Pungent odors 

 

According to different studies, the environmental impact of OMWW was investigated. It has long 

term effects on soil and crops as it contains phenolic compounds (Antimicrobial effects), fats, and 

salts. Therefore, it should not be used directly in agricultural areas. OMWW contains different 

concentrations of ionic species (K+1, Na+1 and HCO3) that cause soil salinity if it is directly applied 

in irrigation without pre-treatment. Improper discharge of OMWW into domestic wastewater 

basins disrupts biological activities due to a large load of toxic organic compounds. Aerobic 

digestion in an open system causes strong odor and problems of surface and groundwater  (Al-

Khatib et al., 2009). 

 

Therefore, a precautionary measure should be taken before discharging non-dry water into the 

environment (soil and water) and disposing of untreated water directly without treatment is 

unacceptable. 

 

The number of oil presses and methods of waste disposal of Zibar in Palestine in 2018 was shown 

in Table A- 5.  

 



  

 21  

 

1.5. Factors affecting the anaerobic digestion process through biogas scope:  

  

1. Temperature 

Temperature is considered as the most crucial parameter that directly affects the anaerobic 

process through two scopes; the first one is affecting the enzymes and co-enzymes. The 

other scope, it affects the methane yield and digestate (effluent) quality. 

 

Generally, anaerobic digestion bacteria are thermophilic (55-70℃) and mesophilic bacteria 

(25-50℃). It is observed that higher growth rates, higher metabolic rates, and higher 

productivity are all obtained via thermophilic digestion. However, it is more sensitive to 

environmental changes. In contrast, mesophilic digestion exhibits better process stability 

and richer in bacteria although lower methane yields and poor biodegradability  (Mao et 

al., 2015, Zhang et al., 2014). 

 

2. pH 

The pH value has a dominant role in both methanogenesis and acidogenesis processes since 

both methanogenic and acidogenic bacteria have optimal pH operating levels. The 

methanogenesis is most efficient at pH (6.5–8.2) and the optimal pH is 7.0. On the other 

hand, the optimum pH level for acidogenesis at (5.5-6.5). Otherwise, any change in pH 

levels will reduce the growth rate and bacteria activity  (Mao et al., 2015).   

 

3. Carbon to nitrogen (C/N) ratio 

Carbon to nitrogen ratio is a sensitive parameter as it affects the process stability and gas 

production rate  (Sievers and Brune, 1978). Low C/N ratio indicates a low protein 

solubilization rate and this led to lower total ammonia nitrogen rates in other words 

inhibition of ammonia. Whereas, a high C/N ratio means insufficient nitrogen to maintain 

cell biomass. Therefore, fast nitrogen degradation and lower gas production. The optimal 

C/N ratio is (20-30) and 25 is mostly used  (Mao et al., 2015). 

 

 

 



  

 22  

 

4. Organic load rate (OLR) 

OLR represents the number of volatile solids fed into a digester per day under continuous 

feeding. Volatile solids represent the amount of solids that can be digested in the digestion 

process. The OLR depends on the waste type that is fed to the digester, as the types of 

waste determine the biochemical activity that will occur in the digester is directly 

associated with gas production, studies showed increasing OLR increases the production 

although extremely high OLR values cause bacterial inhibition  (Mao et al., 2015, Babaee 

and Shayegan, (2011)). 

 

5. Retention time 

Retention time is defined as the time required to complete the degradation process. There are 

mainly two types of retention time: solid retention time (SRT) and hydraulic retention time (HRT). 

The retention time is related to the microbial growth rate and depends on the process temperature, 

OLR and substrate composition. An average retention time of (15–30) days is required to treat 

waste under mesophilic conditions  (Mao et al., 2015). Thus, the retention time has a major effect 

on the production of bigas and digestion process in general. The Hydraulic Retention Time (HRT) 

has a critical effect on the production of methane, the longer HRT more likely, the substrate will 

be broken down and stabilized also, have proper interactions with bacteria within the digester. 

Hence, a longer HRT leads to increase the methane production. Although, some studies looked at 

the HRT, many set it as a constant ranging from 1 to 20 days. A few studies maintained the 

digestion until the production of methane fell or levelled off  (Nelson, 2010).  

              Based on the previous studies, the higher amount of biogas will produce with longer HRT since 

the microorganisms have been accorded much more time to actively degrade materials. An 

increase in HRT might provide sufficient time for methanogens to mineralize the organic matter 

to methane and carbon dioxide. For example, after 12 h, volumes of 0.11, 0.16, 0.21 and 0.24 L/L 

leachate were achieved for HRT 12, HRT 24, HRT 36 and HRT 48, respectively  (Baati et al., 

2018) as shown in Figure 4.6.1 



  

 23  

 

 

Figure 4.6. 1: Cumulative of Biogas with HRT (Baati et al., 2018) 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



  

 24  

 

1.6. Anaerobic Digestion Stages  

 

Anaerobic digestion is a complex process that is capable to degrade any organic waste 

through a bacterial population mixture in the absence of oxygen. This degradation process 

produces a valuable gas mixture (mainly methane CH4 and CO2) under specific 

environmental conditions that are needed for bacteria growth. In respect of bacteria’s 

enzymes, organic waste undergoes four main reactions as shown in Figure 2.8. 1  (Lastella 

et al., 2002, Manchala et al., 2017): 

 

1. Hydrolysis 

Organic macromolecules, i.e. carbohydrates and fats, are converted to monomers in a 

de-polymerization process by enzymes. Produced monomers undergo degrading 

reactions and are converted to acetic acid, long-chain fatty acids and CO2.  

 

2. Acid Formation (Acidogenesis)  

It is generally defined as an anaerobic acid producing microbial process without an 

additional electron acceptor or donor. In this process, the long chain of fatty acids 

serves as both the electron donors and acceptors. The main products of the hydrolysis 

process are converted volatile fatty acids (VFAs) (e.g., acetate, propionate, butyrate, 

etc.), hydrogen and CO2.  

 

3. Acetic Acid Formation (Acetogenesis) 

Acetogens bacteria are responsible for producing enzymes that are used for conversion 

reactions of long-chain fatty acids to acetic acid, molecular hydrogen and CO2. In other 

words, it’s a further fermentation of VFAs to acetate, CO2 and hydrogen, which are the 

precursors of methane formation.  

 

4. Production of Methane (Methanogenesis) 

Acetic Acid is degraded with methane production using methanogenic bacteria. This 

bacterium is highly sensitive for O2 concentration and pH value (7-8 for a well-

balanced system). 

 



  

 25  

 

 

 

 

 

 

Figure 2.8. 1: Anaerobic digestion stages  (Manchala et al., 2017) 



  

 26  

 

1.7. Olive Pressing Techniques  

 

1.7.1. Traditional Pressing Method. 

 

The traditional pressing method, Pressure method (Stone mill). The oldest method for pressing 

olives to extract oil. The extraction of oil in this method is done by spreading the olive paste after 

crushing and kneading olives on filter mats stacked together and pressurized. This process doesn’t 

require the addition of water to the olive paste unless it is difficult to separate the oily phase from 

other phases. If so, addition small quantities of water (3-5 L/100 kg of olives) during crunching 

and kneading occurs. The pressing mills are operated manually so that require more labor. 

However, the oil is extracted is high-quality oil with high polyphenol content  (Di Giovacchino et 

al., (1994), Khdair et al., 2015). 

 

1.7.2. Modern Method (Integral Centrifuge Systems) 

 

a. Three-phase centrifuge decanters. By the 1970’s, three-phase method was introduced. 

It’s based on the industrial centrifuge for phase separation as shown in Figure 1. As a first 

step, olives are crashed to get a fine paste, then paste is pumped to a horizontal centrifuge, 

in this stage addition of warm water occurs to dilute the olive paste before it goes to the 

vertical centrifuge decanter and then components are separated to three streams; olive oil, 

aqueous waste (black water) and a wet solid (paste)  (Angerosa and di Giovacchino, 1996). 

Three-phase decanters method is less bulky than the traditional method, requires a smaller 

number of labors so that the construction and working cost are much lower but it requires 

a lot of electric energy. However, the obtained oil yields in this method are lower than that 

from the traditional one and the paste is almost twice higher of moisture percentage. For 

the quality of produced oil, it is slightly less acidic, richer in coloring pigments and poorer 

in natural antioxidants that directly affect the oil flavor  (Ranalli and Martinelli, 1995). 

 

b. Two-phase centrifuge decanters were introduced by 1990. This pressing method is based 

on horizontal centrifuge so that needs a minimum amount of water to separate the olive oil 

from two-phase olive mill wastewater (TPOMWW)  (ElMekawy et al., (2014)). As long 



  

 27  

 

as, the minimum amount of water is used the energy consumption is reduced. The quantity 

of processed olives is enhanced. Furthermore, the produced oil is higher in quality since it 

is richer in natural antioxidants  (Ranalli and Angerosa, 1996). However, the produced oil 

requires a multi-stage process treatment to remove high acidity and organoleptic defects 

since the produced TPOMWW is considered as complex aqueous residue, this residue is 

highly concentrated of organic matter and toxic compounds such as polyphenols, 

polyalcohol's and volatile fatty acids  (Borja et al., (2006)).  

 

Figure 2.9.2.1 shows three olive oil extraction techniques. 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.9.2. 1: Schematic flowchart of industrial olive oil extraction techniques; traditional, 

three-phase and two-phase centrifuge process (ElMekawy et al., 2014). 

 

 

 

 

 

 

 



  

 28  

 

Chapter Three 

3.  Methodology and Analytical Methods 

 

3.1. Analytical Methods 

 

3.1.1. Measurement of Chemical Parameters  

 

a. Chemical Oxygen Demand (COD) 

COD was determined based on the Standard Methods. Details of the method are 

shown in Appendix A. 

 

COD is the amount of consumed oxygen to chemically oxidize organic waste. It is a 

measure of water and wastewater quality and used to monitor wastewater treatment 

plant efficiency  (Merk Mili Pore, 2019). 

 

Two samples of fresh and stored OMWW were tested. Before the test is performed the 

samples were diluted 5 times (1:5) and 10 times (1:10) for stored and fresh samples 

respectively. Then the samples were digested at 150℃ using HI 839800 COD 

REACTOR, HANNA instruments for two hours. The test is held based on the EPA 

(Environmental Protection Agency) approved method 410.4 for COD medium range 

(0-1500) mg/L determination that uses mercuric sulfate (HgSO4) as the oxidizing 

reagent. Then allowed to cool to room temperature. Spectrophotometer (HANNA) 

Colorimeter was used to read COD value.  

 

b. Ammonia  

Ammonia is a critical nutrient in biological wastewater treatment. It’s a requirement 

for bacteria to make proteins, including enzymes needed to break down food. It’s a 

reflective measure for nutrient deficiencies  (Plaintest Water Analysis Technologies, 

2019).  

 

A fresh OMWW sample was filtered for two times using cotton filter paper, Whatman 

(41 grade, 90 mm diameter), to obtain a clear sample before performing the test. The 



  

 29  

 

sample was diluted 5 times and the test performed according to the ASTM (American 

Society for Testing and Materials) Manual of Water and Environmental Technology, 

D1426-92, Nessler method. Four drops of the Nessler reagent were added to the filtrate 

sample to measure the concentration. The ammonia concentration was determined 

using HI 82314 Multiparameter Bench Photometer for Wastewater Treatment 

Application, HANNA. Details of method procedure in Appendix B. 

 

c. Total Nitrogen 

Total nitrogen is an essential nutrient in biological wastewater treatment. It reflects the 

nutrient efficiency  (Enviroenmental Protection Agency, 2013).  

 

A fresh OMWW sample was diluted by a factor (1:5) and digested at 150℃ using HI 

839800 COD REACTOR, HANNA instruments for two hours, the test is performed 

based on chromotropic acid method Sodium metabisulfite was added to the sample to 

remove potential interferences. The concentration of total nitrogen was determined 

using HI 82314 Multiparameter Bench Photometer for Wastewater Treatment 

Application, HANNA. Details of method procedure in Appendix C. 

 

3.1.2. Measurement of Physical Parameters  

 

a. pH Value 

The pH value of the solution was measured at ambient temperature using pH-

Meter (Jenway 3310). The obtained pH value of OMWW samples was 7.4. 

 

b. Total Dissolved Solids (TDS)  

TDS is the measure of dissolved nutrients in the water, it reflects the water 

quality as it an indicator for the presence of contaminates  (Water Research 

Center, 2014). 

 

Three representative samples were used to conduct the test. Only one sample 

was filtered using glass microfiber filter paper, Whatman (934-AH, 47 mm 



  

 30  

 

diameter) with vacuum filtration and the other two samples were centrifuged 

(KUBOTA 5100) at 3500 rpm for 15 min. All samples were placed in the oven 

at 180 ℃ for 15 min. After the samples were cooled down, they were weighed  

to perform the calculations. TDS was determined based on the standard 

method   (HANNA). Details are shown in Appendix D. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



  

 31  

 

Chapter Four 

 

4. Lab-Scale Bioreactor  

 

4.1. Overview  

 

The main function of the bioreactor is providing a controlled environment to manage optimal 

growth and/or product formation in particular cell system employed.  

The criteria for constructing a bioreactor didn’t consist of a group of steps to follow, nor does it 

have a single correct solution to the problems that were faced. The consideration in the construction 

is many and varied, however, the accepted construction was based upon the available approaches 

and economic consideration. 

At the earlier stages, it was intended to use the constructed bioreactor by the mechanical 

engineering department, which was a pilot-scale (8 litres) steel bioreactor (continuous stirred tank 

reactor (CSTR)) as shown in Figure 4.1.1 and Figure 4.1.2 respectively. 

 

 

 

Figure 4.1. 1: Reactor tank with motor  (Fares Khrashi, 2019) 

 



  

 32  

 

 

Figure 4.1. 2: Bioreactor/digester schematic diagram  (Fares Khrashi, 2019). 

 

However, when the constructed bioreactor was examined number of defects were noticed as 

follows: 

1.  The digester cover was made of steel instead of stainless steel (as mentioned in the design 

report) which was completely covered with rust.  

2. The digester cover was insulated with a piece of rubber which was completely raptured as 

shown in Figure 4.1.4.  

3. Impeller flow pattern was chosen to be radial flow which is characterized with high shear 

and turbulence, however, for the anaerobic digestion (liquid materials) the axial flow 

should be used as it known for lower shear (Caframo Lab Solution, 2019).  

From an economical point of view, the maintenance of the previous defects would cost 

both money and time. Moreover, the expectations for maintenance success is low. So, it 

was decided to construct a sustainable lab-scaled bioreactor to perform the experimental 

part 



  

 33  

 

 

 

Figure 4.1. 3: Bioreactor made of Steel and covered with rust and corrosion. 

 

 

Figure 4.1. 4: Rubber for the cover was raptured 

 

 

 



  

 34  

 

 

Figure 4.1. 5: Radial flow Impeller 

 

New construction has been studied based on the available approaches and the economic aspects. 

the bioreactor construction steps all fully clarified in later sections.   

 

4.2.  Bioreactor Material of Construction  

As mentioned before the anaerobic digestion process is a sensitive biological process. Due to the 

sensitivity of the bacteria that is responsible for breaking down the biodigester complex feed. For 

building up this liable bacterial society a long time is required (from 3-4 weeks)  (Meegoda et 

al., 2018). Thus, to manage a reliable and successful anaerobic digestion process with optimum 

biogas production, it’s essential to select the proper type of construction material of bioreactor. 

Two scenarios were considered and Table (4.2.1) illustrates the reasons for choosing stainless 

steel over plastic. 

 

 



  

 35  

 

Table 4.2. 1: Material of construction type and advantages, disadvantages. 

Material of 

construction 

Advantages Disadvantages Reference 

 

Stainless Steel 

(selected)  

• Excellent corrosion 

resistance. 

• Long service 

(sustainable and 

durable) 

• No need for 

protective coatings. 

• Low maintenance. 

 

 

 

 

• High cost. 
 

 

 

 (Institute, 

2016) 

Plastic • Low cost. 

• Easily portable. 

 

• Short life span. 

• Plastic causes 

reduction in the 

digestion. 

• Plastics are 

nonbiodegradable. 

 

 (Rajendran 

et al., 2012, 

Muthuswamy 

and 

Nemerow, 

1990) 

 

The challenges of the material of construction: 

•Deciding whether to use plastic or stainless steel, it was decided (based on the researches) to use 

stainless steel since it is more suitable and sustainable for long term use even though plastic is 

cheaper and easier to work with.  

•Searching the local market to find good stainless steel “pressure cooker” with a suitable price and 

shape as it is available in the local markets with reasonable price. 

 

 



  

 36  

 

4.3. Construction Implementation 

 

Further, overcoming the obstacles that were experienced in the selection of the material of 

construction. The bioreactor construction criteria were executed through studying different 

scenarios, each scenario has its requirement and specifications. In the first scenario, the 

construction of bioreactor could be accomplished by a specified design company with minimum 

time. However, from an economical point of view, this option would cost much. Another scenario 

was suggested to repair all malfunctions in the old construction and reuse it. Although, this 

scenario would cost both time and money and the possibility of failure is high.  

The promising scenario involving all the economical and qualitative aspects is to use a modified 

stainless steel “pressure cooker” (5 litres), with dimensions 38.1 x 20.3 x 30.5 cm and  

4.14 kg as the weight with silver color (Amazon, 2017). It is worth mentioning that using a pressure 

cooker as a bioreactor is a new unique option in this aspect. On the other hand, the available 

pressure cookers in the Palestinian local stores (Nablus) have some problems that are listed below:  

1. The pressure cooker has a solid body (requires a lathe to create change). 

2. The high cost of each pressure cooker (project execution requires four pressure cookers). 

To cope with all pervious problems and obtain the desired construction certain modifications are 

needed. A deal for pressure cooker price was done to be within the project’s budget. Also, some 

modifications were done in a lathe workshop (Al-Kurdi workshop/ Nablus city). Each modification 

was tested to make sure the progress was going properly. All modifications that were done to 

convert the “pressure cooker” to the required bioreactor construction for performing the digestion 

of olive mill wastewater and sludge are illustrated in Table 4.3.1 below.  

 

 

 

 

 

 



  

 37  

 

Table 4.3. 1: Bioreactor construction specifications 

Bioreactor Construction 

Requirement 

Specifications The Used Materials 

Feed inlet 
A hole ¾‶ in the pressure 

cooker lid.  

¾‶ of PVC pipe was inserted 

in the hole to deliver the feed.  

Effluent (outlet) 

A hole ¾‶ is made in the 

bottom of pressure cooker 

body (2 cm above) 

2 PVC pipes (¾‶) with 3 

elbows for conjunction.  

Gas collection system 

A hole ½‶ in the pressure 

cooker lid.  

Gas tube (double-check) 

connected with bike tire (26‶ 

diameter) to store the gas 

inside. 

Washing nozzle 

A hole ¾‶ in the bottom 

of the pressure cooker 

body.  

¾‶ Stainless steel tank tap. 

Agitation system  The same hole for the gas 

collection system. 

Using a valve to block moving 

the produced biogas and keep 

it inside the reactor.  

 

The studied scenarios are summed below in Figure 4.3.1. 

 

 

 

 

 

 



  

 38  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4.3. 1: Scenarios Summary 

 

 

Studided Scenarios 

A company for design
Repair the old 
construction

Pressure cooker 

OR 

D
is

ad
v
an

ta
g
es

  

Requires money 

and time 

W
h
y
 t

o
 s

el
ec

t?
  

OR 

D
is

ad
v
an

ta
g
es

  

Very high cost 

• The first and new 

unique option in the 

bioreactor construction 

aspect. 

• Reasonable cost. 

• Provide the required 

specification for 

bioreactor (anaerobic 

system). 

• Available in the local 

stores. 

• Could be modified  

 



  

 39  

 

 

4.3.1. Modifications Implementation. 

 

Many routes were applied to prevent leaking, first coating the bioreactor’s cover with 

silicone adhesive and rubber (waiting 24 hours to dry and stick together) then applying the 

test, leaking was noticed. Another type of silicone adhesive (super 7, stronger than the 

previous one) was suggested as an alternative approach to solving the problem and the leak 

test was re-conducted, but this suggestion also failed. After many trials of using different 

kinds of adhesives, it was intended to clean and remove these adhesives, and use gasket 

maker (strong adhesive, outstanding oil resistance, excellent torque retention and suitable 

for high temperatures) to cover the leaking spots under the original rubber then applying 

the test, and no leak was noticed as shown in Figure 4.3.1. 2. The gasket maker was a good 

solution to the first part of the problem (leaking in different spots in the cover of pressure 

cooker). However, for the leakage around the drilled holes in both pressure cooker body 

and cover, the holes were covered with gasket maker fortified with rubbers (both sides) to 

ensure that there is no leak as illustrated in Figure 4.3.1. 3. Since the implemented material 

(as mentioned in Table 4.3.1) which is PVC was a different material than stainless steel 

material. As a conclusion, no adhesive can stand up with stainless steel material (based on 

the previous trials and tests), but in our project, the best solution was to use gasket maker 

fortified with rubbers to overcome this problem. Figure 4.3.1. 1summaries all the routes. 

 

 

 

 

 

 

 

 

 



  

 40  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4.3.1. 1: Routes to solve leaking. 

 

 

 

 

Routes to solving leaking 

Silicone adhesive  

Testing 

Super 7 (silicone) 

No 

Testing 

No 

Gasket Testing 

No 

Gasket with 

rubber 
Testing Finish 

Search for 

other solutions 

No 

Yes 

Yes 

Yes 

Yes 



  

 41  

 

 

 

 

 

 

 

 

 

 

 

Figure 4.3.1. 2: Gasket maker with the original rubber. 

 

 

 

 

 

 

 

 

 

 

Figure 4.3.1. 3: Drilled holes with rubbers. 

 



  

 42  

 

4.3.2. Mixing Options (Stirring System) 

 

Mixing in an anaerobic digester keeps the solids in suspension and homogenizes the incoming feed 

with the active microbial community of the digester content. The following options are all valid 

as a mixing system in the bioreactor: 

1. Usage of an old pump by inserting it to the system. 

2. Stirring by the produced biogas using a valve. 

3. Inserting a mixer with certain dimensions to fit the bioreactor dimensions.  

 

4.4.Schematic Diagrams for the Bioreactor Construction  

 

The following scheme Figure 4.4. 1 illustrates the constructed bioreactor dimensions in cm and 

the bioreactor working volume. Typically, the working volume is 70-80% of the total bioreactor 

volume  (Kurt Eyer, 2020). As the pressure cooker volume is 5 L, the working volume is 3.5 L 

(70% of 5 L). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4.4. 1: The constructed bioreactor dimensions and working volume. 



  

 43  

 

 

Whereas, the Figures below  (Figure 4.4.2-Figure 4.4.6) show the bioreactor constructions 

stages:  

 

 

Figure 4.4. 2: A solid pressure cooker 

 

 

Figure 4.4. 3: Effluent (Outlet hole. 

 

 



  

 44  

 

Figure 4.4. 4: Washing nozzle (stainless steel water tap) 

 

 

Figure 4.4. 5: Final schematic for bioreactor construction 

 

 

 

 

 

 

 

 

 

Figure 4.4. 6: Feed inlet and Agitation system holes 

 

 

4.5.Tests Methodology 

 

4.5.1. Testing bioreactor for gas and fluid leakage 

 

First, a leak test must be performed to make sure that there is no gases and fluids escape from the 

bioreactor. A leak test method is a quality control step to assure device integrity and solidity, it is 



  

 45  

 

considered as a non-destructive test with no impact on the environment or operators. Several leak 

testing methods are available, but the most commonly used methods are bubble test, pressure and 

vacuum decay, tracer gas detectors (halogen, helium and hydrogen) and acoustic leak 

detection  (Bergoglio and Mari, 2012). In our case, the bubble test method was selected.  

Bubble Test Method: 

This method is cheap and easy to apply, it allows detection of leaks up to 10-5 Pa.m³/s and is 

suitable for very large systems. Pressurizing technique was used in this experiment. The cover of 

the bioreactor was moistened with a foam-forming soap solution, then the pressure was applied to 

ensure the visibility of tiny leaks, it was observed that the cover was leaking and bubbles were 

formed as shown in Figure 4.4.1. 1. To observe if there were any leaks in the body, fluid leaking 

test was done by filling the bioreactor with water (liquid leak was noticed). Figure 4.3.1. 4 above 

illustrates all the work. 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4.5.1. 1: Gas leaking test 

 

 

 



  

 46  

 

 

4.5.2. Cow Manure Test  

 

It was intended to conduct a test to check the constructed reactor and confirm construction stability. 

The cow manure was chosen as the feeding material (before injecting the olive mill wastewater 

and sludge that will be studied in this project.) 

The cow manure samples were collected from Al-Afouri farms (Zawata, near Nablus). The 

samples were consisted of fresh and old cow manure and stored in glass bottles. 

The methodology for performing the test as following: 

1. The cow manure samples were mixed with a ratio of (2 L fresh: 1 L old) and diluted with 

tap water as shown in Figure 4.5.2. 1. 

2. The feed was injected in the reactor through the feed inlet using a plastic funnel as shown 

in Figure 4.5.2. 2. 

3. The reactor was incubated in the incubator at temperature (35-40)℃ as shown in Figure 

4.5.2. 3. 

4. The incubator was placed near the gas detector (to observe the gas leakage). 

5. A thermometer was injected inside the incubator to check the temperature.  

6. The produced gas from the digestion is collected in the bike tire as illustrated in Figure 

4.5.2. 4. 

7. The pH of the system was checked continuously (ensuring that the system has remained 

neutral 7) as shown in Figure 4.5.2. 5. 

8. Sodium acetate (1.2 M) was added as feeding material for the bacteria. 

9. The test was held for 4 days to ensure system stability. 

 

 

 

 



  

 47  

 

 

 

 

 

 

 

’  

 

Figure 4.5.2. 1: Mixing fresh and old cow manure and diluted by tap water. 

 

 

   

 

 

 

 

 

 

 

 



  

 48  

 

Figure 4.5.2. 2: Injecting the mixture to the reactor using a plastic funnel. 

 

 

Figure 4.5.2. 3: Injecting the reactor inside the incubator. 

 

     

 

 

 

 

 

 

Figure 4.5.2. 4: Produced gas is collected in the bike tire. 

 

 

 

 



  

 49  

 

 

Figure 4.5.2. 5: Testing the pH for the sample from the cow manure mixture. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



  

 50  

 

 

Chapter Five 

5.1. Recommendations and Future work  

 

For future work, it is proposed to preview the project's plan deeply in detail from an experimental 

point of view. Our role in the project is situated between operating the bioreactor and examine the 

quantity and quality of the produced biogas with other evolutionary techniques. All the sections 

below define the project plan. 

a. Preparing the substrate materials  

OMWW samples were collected from two different sources through the olive collecting season 

(October 2019). The majority of samples were collected from the wastewater treatment plant 

located near Nablus city. The samples were stored using glass bottles in the refrigerator at -4 ℃. 

However, the OMWW lacks efficient bacteria to initiate and activate the anaerobic digestion 

process.  It was decided to use the sludge as the inoculum to activate the bio digestion process, the 

sludge (as Eng. Mohammed, the wastewater treatment Operator recommends) will be collected 

from the digester (a rich active bacterial society). After the collection, the sludge should be 

transported to the laboratory and injected immediately to the reactor. 

b. Characterization of OMWW samples  

The collected samples must be characterized again to determine their biological oxygen demand 

(BOD), chemical oxygen demand (COD), pH and total nitrogen before performing the experiments 

(the samples were characterized immediately after the collection). 

c. Operating the designed bioreactors 

As it is previously mentioned in the design chapter, four bioreactors were constructed with the 

same specifications. One of the bioreactors will be operated as a control (reference) bioreactor, 

thus it will be filled only with OMWW samples without addition of sludge. The three remained 

bioreactors will be filled with OMWW and sludge which are mixed with different ratios. Then the 

bioreactors will be incubated in the incubators to keep the temperature at 37 ℃ (mesophilic range) 

All bioreactors will be completely closed for nearly 40 days. 

 



  

 51  

 

d. Analytical Methods  

To quantify the anaerobic digestion process and the gas productivity, many of measurements are 

utilized, including pH, alkalinity, Chemical Oxygen Demand (COD), polyphenols concentration 

and Total Solids (TS). All tests will be conducted according to the standard methods (HANNA). 

The samples will be taken from the bioreactor sampler tap to monitor the previously mentioned 

parameters.  

e. OMWW and Sludge Ratios  

As already stated, the bioreactors substrate will consist of a mixture of OMWW and sludge at 

different ratios. The intended OMWW/Sludge ratios as follows (70:30, 80:20, 90:10). Referring 

to the previous studies, the presumed ratio has achieved the highest gas production levels and the 

highest COD removals is (70:30) of OMMW to sludge  (Azbar et al., 2004). Additionally, this 

ratio provides a pH level near 6.4 which is a perfect medium for the bacterial society activities and 

growth.  

As illustrated in  Figure 5.1. 1the COD removal using (70:30) ratio is nearly 69.5%. 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5.1. 2: COD removal percentage using (70:30) ratio  (Azbar et al., 

2004). 



  

 52  

 

For total Nitrogen, the highest rate of transformation was almost 81.8% in the ratio (70:30) and 

the highest percentage was among other ratios as shown in Figure 5.1. 3.   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Furthermore, the total polyphenols removal percentage was found the highest in (70:30) ratio and 

almost is 22.2% as shown in  Figure 5.1. 5 

 

 

 

 

 

 

 

 

Figure 5.1. 4: Total Nitrogen percentage in using (70:30) ratio  (Azbar et 

al., 2004). 



  

 53  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

f. Methods for Measuring Produced Biogas Volume.  

The proposed method to measure the produced biogas volume is the Liquid Displacement Method. 

As long as, the biogas will be collected using a plastic tube and the tube will be connected to the 

other liquid basin which contains the graduated cylinder (1 L) and caustic soda solution (1 M). 

Biogas is first purified from CO2 and H2S by simultaneous reaction and absorption in a bath 

containing caustic soda solution (1M). Dissolved methane gas is released from caustic soda 

solution and assembled into an inverted 1-liter cylinder for quantitative measurement. The biogas 

will be collected from each bottle separately in an inverted graduated cylinder  (Abushattal et al., 

2017).  

The liquid displacement scheme is illustrated in  Figure 5.1. 7: 

 

 

Figure 5.1. 6 : Polyphenols removal percentage in (70:30) ratio  (Azbar et al., 

2004). 

 



  

 54  

 

 .  

Figure 5.1. 8: Anaerobic digestion system including the biogas measurement (Abushattal et al., 

2017) 

 

 

g. Challenges could be faced: 

 

• The digester sludge is a sensitive bacterial medium, thus it is required to collect the sample 

and inject them directly without any time delay, despite the distance between the WWTP 

and the university lab.  

• Delay in time for the sludge samples collection due to the Nablus municipality protocols 

to have permission for samples collection.  

 

 

 

 

 

 

 

 

 



  

 55  

 

Chapter Six 

 

6. Conclusion 

 

The main objective of this study was to evaluate the capability to treat the OMWW by the 

anaerobic co-digestion process (with the sludge from digester) and produce biogas, taking into 

account the construction of a new bioreactor to perform the experimental work using it. The 

bioreactor construction part and part of experimental testing techniques were accomplished before 

the quarantine. Based on the previously accomplished work the following conclusions were drawn:  

1. The most appropriate material to construct the bioreactor is stainless steel compared to 

plastic (sustainable and corrosion resuscitated). A local modified “pressure cooker” was 

chosen. 

2. No adhesive can stand up with stainless steel material based on the performed trials, for 

the project case, the best solution was to use gasket maker fortified with rubbers. 

3. The constructed bioreactor suites well the anaerobic digestion process conditions and 

specifications as an experimental test using the cow manure was implemented for four days 

and no leakage observed from the produced gas.  

 

 

 

 

 

 

 

 

 

 

 



  

 56  

 

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Appendices  

 

Appendix A: Chemical Oxygen Demand Test 

➢ Method of testing  

Adaptation of the USEPA 410.4 approved method for the COD determination on surface waters 

and wastewaters. Oxidizable organic compounds reduce the dichromate ion (orange) to the 

chromic ion (green). The amount of remaining dichromate is determined 

➢ Required reagent  

Table A- 1: COD required reagent. 

Code Description Q. ty/test Q. ty/set 

* Reagent 1 vial 25 vials 

----- Deionized Water 0.2 mL optional 

 

*Reagent Vial identification: No letter, red cap.  

Note: Store the unused vials in their container in a cool and dark place. 

Dichromate could be replaced with mercuric sulphate (same procedure, method and 

ranges). 

Interference may be caused by:  

Chlorides (Cl-): above 20000 mg/L. Samples with higher chloride concentration should be diluted. 

➢ Measurement procedure  

 

Before starting to use the reagent kit it is recommended to read carefully all the instructions and 

the Health & Safety Data Sheet (HSDS). Pay particular attention to all warnings, cautions and 

notes. Failure to do so may result in serious injury to the operator.  

Reagent Blank Correction: This method needs a reagent blank correction. A single blank vial may 

be used more than once. The blank vial is stable for several months (room temperature). For most 

accurate measurement, run a blank for each set of measurements and always use the same lot of 

reagents for blank and samples. 



  

 61  

 

1. Choose a homogeneous sample. Samples containing settleable solids need to be homogenized with 

a blender. 

2. Preheat the Hanna Reactor C 9800 to 150 °C (302°F). For correct use of the reactor follow Reactor 

Instruction Manual. Use of the optional HI 740217 safety shield is strongly recommended. Do not 

use an oven or microwave because leaking samples can generate a corrosive and possibly explosive 

atmosphere.  

3. Remove the cap from two Reagent Vials. 

4. Add exactly 0.2 mL of sample to one vial (sample vial), and 0.2 mL of deionized water to the other 

vial (blank vial), while keeping the vials at a 45-degree angle. 

5. Replace the cap tightly and mix by inverting each vial a couple of times.  

Warning: as the vials become very hot during mixing, be careful in handling them. 

6. Insert the vials into the reactor and heat them for 2 hours at 150°C.  

7. At the end of the digestion period switch off the reactor. Wait for twenty minutes to allow the vials 

to cool to about 120°C.  

8. Invert each vial several times while still warm, then place them in the test tube rack. Warning: as 

the vials are still hot, be careful in handling them.  

9. Leave the vials in the tube rack to cool to room temperature. Do not shake or invert them anymore 

otherwise the samples may become turbid. 

 

10. Select the program number corresponding to Oxygen Demand, Chemical HR (COD) on 

the secondary LCD by pressing PROGRAM t and s.  

11. Place the blank vial into the holder and push it completely down. 

12. Press ZERO and "SIP" will blink on the display. 

13. Wait for a few seconds and the display will show “-0.0-”. Now the meter is zeroed and 

ready for measurement. 

14. Remove the blank vial. 

15. Place the sample vial into the holder and push it completely down. 

16. Press READ DIRECT and "SIP" will blink during measurement. 

17. Multiply the reading on the Liquid Crystal Display by 10 to obtain the concentration in 

mg/L of oxygen demand. 

 



  

 62  

 

 

Appendix B: Ammonia Test  

Form HANNA’S catalog, standard methods were used  (HANNA). 

Ammonia Test  

➢ Method of testing 

 

Adaptation of the ASTM Manual of Water and Environmental Technology, D1426-92, Nessler 

method. The reaction between ammonia and reagents causes a yellow tint in the sample. 

➢ Required reagent  

Table A- 2: Ammonia required reagents. 

Code Description Q.ty/test Q.ty/set 

* Reagent 1 vial 25 vial 

HI 93764-0 Nessler Reagent 4 drops 1 bottle 

 

* Reagent Vial identification: A, green cap. 

Note: Store the unused vials in their container in a cool and dark place. 

Interference may be caused by:  

• Organic compounds like: chloramines, various aliphatic and aromatic amines, glycine or 

urea above 100 ppm N (positive error), to eliminate these interferences distillation is 

required.  

• Organic compounds like: aldehydes, alcohols (e.g. ethanol) or acetone above 1 % 

(negative error), to eliminate these interferences distillation is required. 

 • Sulfide: may cause turbidity. 

 

 

 

 



  

 63  

 

➢ Measurement procedure  

 

1. Select the program number corresponding to Ammonia HR on the secondary LCD by 

pressing PROGRAM t and s. 

2. Remove the cap from a Reagent Vial. 

3. Add exactly 1.0 mL of sample to the vial, while keeping the vial at a 45-degree angle. 

4. Replace the cap and mix by inverting the vial a couples of times. This is the blank. 

5. Mark the vial with a pencil on the white band to place an orientation sign. Use this sign to 

insert the test vial always in the same position into the holder. 

6.  Place the vial into the holder and push it completely down. 

7. Press ZERO and “SIP” will blink on the display. 

8.  Wait for a few seconds and the display will show “-0.0-”. Now the meter is zeroed and 

ready for measurement. 

9. Remove the vial. 

10. Remove the cap and add 4 drops of HI 93764-0 Nessler Reagent. 

11. Replace the cap tightly and mix by inverting the vial a couples of times. This is the sample. 

12.  Place the vial into the holder and push it completely down. Ensure that the vial orientation 

respect to the holder is the same as the blank. 

13.  Press TIMER and the display will show the countdown prior to the measurement. 

Alternatively, wait for 3 minutes and 30 seconds and press READ DIRECT. In both cases 

"SIP" will blink during measurement. 

 

 

 

 

 

 

 

 

 

 



  

 64  

 

Appendix C: Total Nitrogen Test  

➢ Method of testing  

Chromotropic acid method. A Persulfate digestion converts all forms of nitrogen to nitrate. 

Then the reaction between nitrate and the reagents causes a yellow tint in the sample. 

➢ Required reagent  

Table A- 3: Total nitrogen required reagent. 

Code Description Q.ty/test Q.ty/set 

* Digestion Vial 1 vial 50 vial 

----- Deionized Water 0.5 ml 1 bottle 

----- Potassium Persulfate 1 packet 50 packets 

----- Sodium Metabisulfite 1 packet 50 packets 

HI 93767-0 Total Nitrogen Reagent 1 packet 50 packets 

** Reagent Vial 1 vial 50 vial 

 

* Digestion Vial identification: N, red cap.  

**Reagent Vial identification: N, white cap.  

Note: Store the unused vials in their container in a cool and dark place. 

 

Interference may be caused by:  

• Bromide (Br-): above 240 mg/L (positive error) . 

• Chloride (Cl-): above 3000 mg/L (positive error).  

• Chromium (Cr3+): above 0.5 mg/L. 

 

➢ Measurement procedure  

 

Before starting to use the reagent kit it is recommended to read carefully all the instructions 

and the Health & Safety Data Sheet (HSDS). Pay particular attention to all warnings, 

cautions and notes. Failure to do so may result in serious injury to the operator. Reagent 

Blank Correction: This method needs a reagent blank correction. A single blank vial may 



  

 65  

 

be used more than once; the blank vial is stable up to one week if stored in a dark place at 

room temperature. Always use the same lot of reagents for blank and samples. For most 

accurate measurement, run a blank for each set of measurements. 

 

1. Preheat the Hanna Reactor C 9800 to 105 °C (221°F). For correct use of the reactor 

follow Reactor Instruction Manual. Use of the optional HI 740217 safety shield is 

strongly recommended. Do not use an oven or microwave because leaking samples 

can generate a corrosive and possibly explosive atmosphere.  

2. Remove the cap from two Digestion Vials (red cap vials). 

3. Add the content of one packet of Potassium Persulfate for Total Nitrogen analysis 

to each vial. 

4. Add exactly 0.5 mL of sample to one vial (sample vial), and 0.5 mL of deionized 

water to the other vial (blank vial), while keeping the vials at a 45-degree angle. 

5. Replace the cap tightly and shake vigorously the vials for about 30 seconds until all 

the powder is completely dissolved. 

6. Insert the vials into the reactor and heat them for 30 minutes at 105°C. Note: to 

obtain most accurate results, it is strongly recommended to remove the vials from 

the reactor after exactly 30 minutes. 

7. At the end of the digestion place the vials carefully in the test tube rack and allow 

to cool to room temperature. Warning: as the vials are still hot, be careful in 

handling them. 

8. Select the program number corresponding to Total Nitrogen HR on the secondary 

LCD by pressing PROGRAM t and s. 

9. Remove the cap from the vials and add the content of one packet of Sodium 

Metabisulfite for Total Nitrogen analysis to each vial. Replace the cap tightly and 

shake gently the vials for 15 seconds. 

10. Wait for 3 minutes (without shaking the vials) to allow the reaction to complete. 

11. Remove the cap from the vials and add the content of one packet of HI 93767-0 

Total Nitrogen Reagent to each vial. Replace the cap tightly and shake gently the 

vials for 15 seconds. 

12. Wait for 2 minutes (without shaking the vials) to allow the reaction to complete. 



  

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13. Remove the cap from two Reagent Vials (white cap vials). 

14. Add exactly 2.0 mL of digested sample (from the digested red cap sample vial) to 

one Reagent Vial (sample vial), and 2.0 mL of digested blank (from the digested 

red cap blank vial) to the other vial (blank vial), while keeping the vials at a 45-

degree angle. 

15. Replace the cap tightly and invert the vials 10 times. Warning: as the vials become 

hot during mixing, be careful in handling them.  

16. Place the blank vial into the holder and push it completely down. 

17. Press TIMER and the display will show the countdown prior to the measurement. 

Alternatively, wait for 5 minutes and press ZERO. In both cases "SIP" will blink 

during measurement. 

18. Wait for a few seconds and the display will show “-0.0-”. Now the meter is zeroed 

and ready for measurement. 

19.   Remove the blank vial.  

20.  Place the sample vial into the holder and push it completely down. 

21. Press READ DIRECT and "SIP" will blink on the display. 

22. The instrument directly displays concentration in mg/L of total nitrogen (N) on the 

Liquid Crystal Display. The method detects all organic and inorganic forms of 

nitrogen present in the sample.  

23. To convert the reading to mg/L of ammonia (NH3), multiply by a factor of 1.22.  

24. To convert the reading to mg/L of nitrate NO3-, multiply by a factor of 4.43. 

 

 

 

 

 

 

 

 

 

 



  

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Appendix D: Total Dissolved Solid Test 

➢ Measurement procedure  

1- First, clean the crucibles (3 crucibles) and glass watches with deionized water and soap. 

2- Put the crucibles and Whatmen filter paper (41, ashless, circles 90mm Dia, Cat No 1441 

090), which is placed in the center of the glass watch in the oven at 105 ℃ for 15 minutes.  

3- Mark the crucibles and the filter papers, weigh them. 

4- Put 30 mL samples in 50 mL graduated cylinder and 50 mL centrifuge tubes. 

5- Do vacuum filtration for the sample in 50 mL graduated cylinder. 

6- Put the tubes in centrifuge, KUBOTA 5100, with 3500 rpm for 15 minutes in order to 

separate the solid and the liquid into two layers, which is easier while doing vacuum 

filtration. 

7- Do vacuum filtration for the centrifuged tubes. 

8- Put the filter papers with the precipitate in the glass watches and the filtrate in the crucibles.  

9- Put them in the ovens in order to dry. 

10- Weigh the crucibles and the filter papers, do calculations.  

 

 

Figure A- 1: Hl 83214 Multi-parameter Bench photometer for wastewater treatment application. 



  

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Figure A- 2: Hl 839800 COD Reactor. 

 

Figure A- 3: Hl93764-0, Nessler Reagent. 



  

 69  

 

 

Figure A- 4: A