Sei sulla pagina 1di 59

, .

: :

The University of Jordan Authorization Form

I Wafa' Saleh Jebril Shehadeh, authorize the University of Jordan to supply copies of my Thesis to libraries or establishments or individuals on request.

Signature: Date:

INTRODUCTION
Gelatin is a heterogeneous mixture of watersoluble proteins of high average molecular weight. It is obtained by hydrolysis of collagen by boiling skin, ligaments, tendons, etc. gelatin swells when water is added and dissolves in hot water to form a solution that sets to a gel on cooling. It is used in photographic emulsions, pharmaceutical, adhesives, in jellies and other foodstuff.
( http://www.singaporescienceCenterScienceNetPhysicalSciencesGeneralChemistry.htm )

Gelatin is one of the largely produced chemicals and used in pharmaceutical industry especially for medicine capsules. Its presence in wastewater results in considerable harmful effects to the environment and water resources due to its proteinacious nature which results in high BOD and COD levels in the discharged wastewater thus, depleting all the available oxygen (Ayyoub, 1999). Methods and techniques suitable for the treatment of wastewater can roughly be classified in the following three categories: 1. Mechanical / physical methods, e.g. sedimentation, filtration, membrane processes (ultrafiltration, reverse osmosis), and drying. 2. Physical / chemical methods, e.g. vaporization, absorption, chemical precipitation, incineration, and adsorption. 3. Biological methods, such as an anaerobic digestion, aerobic treatment. (Lettinga et al., 1999). Anaerobic digestion is a biological process in which anaerobic and facultative bacteria convert 40-60% of the organic solids in sludge to carbon dioxide and methane gases. The organic matter that remains is chemically stable and practically odorless and contains 90 to 95% moisture (Ray et al., 1992).

3 A wide variety of engineered systems have been specifically developed for anaerobic digestion of different waste streams. To make practical use of the lagoon technology and shorten the digestion time requirements, methods were devised to improve the contact between the organic matter and the bacterial biomass. This type of system now widely used for agricultural and some industrial wastes also can include coverings for gas collection Up-flow anaerobic sludge blanket (UASB) reactor, hybrid up-flow anaerobic sludge blanket reactor (HUASB), anaerobic up-flow fluidized bed reactor, expanded granular sludge bed (EGSB) reactor, are a number of anaerobice systems that are used in the whole world for wastewater treatment purposes (David, 1996). Gelatin has great tendency to foam and separate upon aeration. This is due to its proteinacious nature, it is also an easily biodegraded organic material when applied to an activated sludge process. Since aeration and mixing can remove gelatin from its solution physically, it is quite reasonable to consider the flotation process before the activated sludge process. Dispersed air flotation is much easier, quicker, and very efficient with the advantage of no needing additives or chemicals (Ayyoub, 1999). Lack of water, which is now a problem, makes treatment of industrial wastewater a necessity if treated efficiently to meet the required specifications. The treated wastewater may be beneficial in agricultural and other industrial uses. The parameters, organic loading rate (OLR) that is the mass of influent organic material per unit of reactor volume, up-flow velocity that is the influent flow rate per cross sectional area of the reactor and hydraulic retention time (HRT) that is the volume of the reactor per the influent flow rate, were studied to know their effect on the COD removal percentage. The objective of this work is to examine the feasibility of treating gelatin wastewater by anaerobic digestion using UASB reactor and to study the effect of OLR, up-flow velocity and HRT on the UASB performance system.

LITERATURE REVIEW 1. Gelatin


In common with all proteins, gelatin is made up of amino acids joined together by peptide linkages to form polymer chains. The primary structure of gelatin can be illustrated as shown in Figure 1.

Amino acid R N2N-CH-CO2H Polypeptide R R -CH-CO-] NH-CH-CO ]n -NH-CH-CONHFigure 1: The primary structure of gelatin The nature of the side-chain group (R) will vary for different amino acids. Gelatin is composed of 18 different amino acids and its composition is remarkably consistent, irrespective of its physical properties or whether it is derived from hide/skin or bone collagen or manufactured by an acid or alkali process. Amino acids compositions may be expressed in various ways, but commonly are reported as grams of amino acids per 100 grams of protein or as residues per 1000 residues ( Corda Gelatin Ltd. Gel fax). These values for gelatin are given in Table 1. R

5 Table 1. Gelatin values for different amino acids (Corda Gelatin Ltd. Gel fax) Amino Acid Alanine Glycine Valine Leucine Isoleucine Proline Phenylalanine Tyrosine Serine Threonine Methionine Arginine Histidine Lysine Aspartic acid Glutamic acid Hydroxyproline Hydroxylysine g amino acids per 100 g protein 8.3-9.2 20.5-21.2 2.1-2.8 2.8-3.1 1.4 -1.6 13.4 14.2 2.0 2.4 0.2 0.6 3.1 3.6 1.8 2.2 0.5 0.8 5.4 8.7 0.6 0.9 3.8 4.1 5.7 6.2 9.6 10.4 11.3 13.1 0.7 1.0 Residues per 1000 residues 105-117 325-335 20-26 22-25 11 13 124 132 12 14 14 32 37 17-20 36 30 50 46 26 29 44 48 67 73 90 105 47

In particular, gelatin has high glycine content (representing approximately one third of the total number of residues), also it has high hydroxyproline content, but it is not a complete protein source because it is deficient in tryptophan and low in methionine content. (Corda Gelatin Ltd. Gel fax).

6 2. Treatment of wastewater containing gelatin Removal of gelatin from industrial wastewater was investigated under laboratory conditions, using two techniques. First is the dispersed air flotation in batch and continuous modes. The second is a biological treatment process applying the batch activated sludge technique. For the continuous flow flotation, the effect of both residence time and air flow rate on effluent quality were studied while for the batch flotation, the study was concerned with the effect of initial concentration of gelatin, air flow rate, temperature and pH. The removal efficiency in continuous flow flotation was up to 80% at the optimum residence time and air flow rate of 45 minutes and 60 L/min, respectively, at pH of the solution 7.5-8.5. Batch experiments had the same efficiency as the continuous flow process. Unlike temperature, pH showed great effect on the process. Best removal of gelatin was obtained at low pH (4-6). The second treatment process was applied following the flotation process. Gelatin was easily biodegraded using activated sludge at a temperature of 30C and pH 7.5-8.5. The removal efficiency was 80% in four days. During this period, concentration dropped from 80 to 15 mg/L (Ayyoub, 1999). Yu and Fang (2003) investigated the influence of temperature and pH on the acidification of synthetic gelatin based wastewater. Experiments showed that temperature and pH had a different influence on the acidification of the gelatin rich wastewater. Gelatin degradation efficiency substantially increased with pH, from 60.0% at pH 4.0 to 97.5% at pH 7.0. The degree of acidification increased from 32.0% at pH 4.0 to 71.6% at pH 6.5, but dropped to 66.8% when pH increased to 7.0. The optimum pH for the overall acidogenic activity was found to be 6.0. Operation at pH of 4.0-5.0 favored the production of propionate, hydrogen, whereas the operation at pH 6.0-7.0 encouraged the production of acetate, butyrate, and i-butyrate. Temperature affected the

7 acidogenesis of gelatin according to the arrhenius equation with low activation energy of 1.83 Kcal/mol. Abu-Zurayk (2004) studied the effect of two operating conditions, up-flow velocity and OLR, on the COD removal percentage using HUASB reactor. The study showed that the COD removal percentage increased linearly from 75% to 83% as the up-flow velocity was increased from 0.1 m/hr to 1.45 m/hr at OLR of 17 kg COD/m3.day. However increasing the OLR from 17 kg COD/ m3.day to 23 kg COD/ m3.day at upflow velocity of 1.45 m/hr reduced the COD removal percentage from 83% to 50%. The methane content of the biogas is also increased from 58% to 64% as the OLR increased from 17 kg COD/ m3.day to 23 kg COD/ m3.day. 3. Anaerobic wastewater treatment For industrial wastewater, the objective of the biological treatment is to remove or reduce the concentration of organic and inorganic compounds. Anaerobic fermentation is used primarily for the treatment of waste sludge and high-strength organic wastes (Metcalf and Eddy, 2003). 3.1 Advantages of Anaerobic fermentation processes over aerobic i Less energy is required (Metcalf and Eddy, 2003). ii Fewer nutrients are required (Metcalf and Eddy, 2003). iii Smaller reactor volume is required ( Metcalf and Eddy, 2003). The loading rates ranges of 3.2 to 32 kg COD/m.day, compared to 0.5 to 3.2 kg COD/ m.day for aerobic processes (Metcalf and Eddy, 2003). iv Anaerobic digestion produces lower amounts of sludge. The net amount of cells produced per metric ton of COD destroyed is 20-150 kg, as compared to 400-600 kg for aerobic digestion (www.united-tech.com/wd-anaerobicdigestion.html).

8 v Anaerobic digestion produce a useful gas, CH4. This gas contains about 90% of the energy, and can be burned on site to provide heat for digesters or to generate electricity (www.united-tech.com/wd-anaerobicdigestion.html). 3.2 Mechanism of anaerobic digestion Anaerobic digestion occurs in four distinct steps that can be illustrated as following: i Hydrolysis: Anaerobic bacteria break down complex organic molecules (proteins, cellulose, lignin, and lipids) into soluble monomer molecules such as amino acids, glucose, fatty acids, and glycerol. The monomers are directly available to the next group of bacteria. Hydrolysis of complex molecules is catalyzed by extracellular enzymes such as proteases. ii Acidogenesis: Acidogenic (i.e. ,acid-forming) bacteria play a role in this step. It converts sugars, amino acids, and fatty acids to organic acids (e.g., acetic, propionic, formic, lactic, butyric acids), alcohol and ketones (e.g., ethanol, methanol, glycerol, acetone), acetate, CO2 , and H2 . iii Acetogenesis (intermediary acid-production): Acetognic bacteria convert fatty acids (e.g., propionic acid, butyric acid) and alcohols into acetate, hydrogen, and carbon dioxide, which are used by the methanogens. iv Methanogenesis: Anaerobic digestion of organic matter in the environment releases 500-800 million tones of methane per year into the atmosphere and this represents 0.5% of the organic matter derived from photosynthesis. Methanogenic microorganisms grow slowly in wastewater and their generation time ranges from 2 days at 35C to as high as 50 days at 10C. About two thirds of methane is derived from acetate conversion by methanogens. The other third is the result of carbon dioxide reduction by hydrogen. So, the final product in this step is CH4, CO2, traces of other gases e.g. H2S, H2 and new cell-matter. (www.united-tech.com/wd-anaerobicdigestion .html).

9 A simplified schematic of the overall mechanism of anaerobic digestion, which involves a multitude of microbial species, is

Insoluble Organics

liquefaction by exteracellular hydrolytic enzymes

Bacterial cells Soluble acid producing organics Other products bacteria Volatile acids + CO2 + H2 gasification by methane producing bacteria

CH4 + CO2 + traces of H2S, H2 + bacterial cells (Bailey and Ollis, 1986).

4. Types of microorganisms The microorganisms in an anaerobic digester are highly specialized bacteria. The two groups of bacteria which can live in the digester are the facultative bacteria and the

10 obligate anaerobic bacteria. The acid formers are made up predominantly of facultative bacteria, with a few strict anaerobes. Various species of Pseudomonas, Flavobcterium, Alcaligens, Echerichia, and Aerobacter contribute to the acid production. The methane formers are small specialized group of bacteria that are obligate anaerobes. Those bacteria which have been isolated belong to the general Methanobacterium, Methnosarcina, and Methanococcus (McKinney, 1962). Table 2 lists some methanogenic bacteria, substrtes and products. Table 2. Methanogenic bacteria (Price and Cheremisinoff, 1981) Bacterium Methanobacterium formicum M. mobilis M. propionicum M. ruminantium M. sohngenii M. suboxydans Methanococcus mzei M. vannielii Methanosarcina barkeri Substrates H2 + CO2 Products CH4a

H2 + CO2 CH4 Propionate CO2 + Acetatea H2 + CO2 CH4 Acetate +Butyrate CH4 + CO2 Caproate + Butyrate Propionate + Acetatea Acetate +Butyrate CH4 + CO2 H2 + CO2 CH4 H2 + CO2 CH4 Methanol CH4 Acetate CH4 + CO2 M. methanica Acetate +Butyrate CH4 + CO2 a: Acetate or propionate converted to CH4 in two-step process.

5. Factors controlling anaerobic digestion i Temperature: Anaerobic digestion is carried out in the mesophilic range from 25C 40C with the optimum at approximately 35C. Thermophilic digestion operates at temperature ranges of 50C- 65C (www.united-tech.com/wd-anaerobic digestion.html) ii Retention Time: Hydraulic retention time HRT depends on wastewater characteristics and environmental conditions and it must be long enough to allow metabolism by anaerobic bacteria in digesters. (www.united-tech.com/wd-anaerobic digestion.html)

11 iii pH: Most methanogenic bacteria function in a pH range between 6.7 and 7.4, but optimally at pH (7.0-7.2) .The process my fail if the pH is close to 6.0. Acidogenic bacteria produce organic acids, which tend to lower the pH of the bioreactor. Acidity is more inhibitory to methanogens than of acidogenic bacteria. An increase in volatile acid levels thus serves as an early indicator of system upset. iv Toxicants: A wide range of toxicants is responsible for the occasional failure of anaerobic digesters. Inhibition of methanogenesis is generally indicated by reduced methane prodution and increased concentration of volatile acids. (www.unitedtech.com/wd-anaerobic digestion.html). v Organic Loading Rate: Removal efficiencies of 90 to 95 percent for COD have been achieved at COD loadings ranging from 12 to 20 kg COD/m3. day on a variety of wastes at 30 to 35C with UASB reactors. (Metcalf and Eddy, 2003) vi Up-Flow Velocity: For weaker wastewater the allowable velocity and reactor height will determine the UASB reactor volume, and for stronger wastewater it will be determined by the volumetric COD loading. (Metcalf and Eddy, 2003). Mohan et al. (2001) studied the anaerobic treatment of the pharmaceutical using the Anaerobic Suspended Film Contact Reactor (ASCR). In their investigation, the effluent from a bulk drug industry, which utilizes several organic chemicals, have been taken to assess their applicability for anaerobic treatment. The characteristics of the wastewater used by Mohan et al., (2001) are shown in Table 3. Table 3. Characteristics of the pharmaceutical wastewater, at pH of 6.65-7.40

12

Parameter
Total alkalinity TDS VS SS COD BOD5 Chlorides Sulphates Phosphates

Concentration (mg/L)
1800-2100 15500-16700 6400-6600 9150-9240 23700-24500 11500-12100 3900-4100 400-650 <98

The basis of ASCR reactor design that was used in their investigation is to ensure effective contact with the anaerobic biomass in suspended form with the organic load to achieve high organic removal. To achieve this the inlet feed was introduced through the

13 bottom of the reactor. This enables the organic substrate to have good contact with the suspended film of the active biomass to ensure effective degradation. The organic loading rates were varied from 0.25 Kg COD/m3.day to 2.5 Kg COD/m3.day and the COD reduction was found to be in the range of 60-80%. Long term operation of an anaerobic suspended film contact reactor carried out with 1.25 Kg COD/m3.day was found to be optimum. During the degradation process, the biogas generated was monitored and the methane content was found to be 60-70%. Azbar et al. (2001) studied the roles of substrate complexity (molecular size of the substrate) and process configuration to know its effect on the performance of anaerobic process. Five reactor configurations were operated: batch fed single-stage continuous stirred tank reactor (CSTR), continuously fed single-stage CSTR, two-phase CSTR, two- stage CSTR, and single-stage UASB. The feed concentration was 20.000 mg/L as COD for the five substrates (glucose, propionate, butyrate, ethanol, and lactate) plus a mixed waste (60% carbohydrate, 34% protein, and 6% Lipids). The SRT and HTR in the CSTR reactors were 20 days, while HRT in the UASB was 2 days. All reactors were operated for at least 60 days (equal to 3SRT). The results of the study indicate that the substrate complexity was less significant under two- phase, two-stage UASB reactor configurations. Twophase CSTR, two-stage CSTR, and single-stage UASB configurations yielded the lowest effluent chemical oxygen demands (130-550, 60-700, and 50-250 mg/L, respectively). The highest effluent chemical oxygen demands were detected when feeding glucose, ethanol, propionate and lactate to continuously fed single-stage CSTRs (10,400,9900, and 4700 mg/L COD, respectively) and to Batch-fed single-stage CSTRs (11,200,2500, and 2700 mg/L COD, respectively). 6. UASB reactor

14 The UASB reactor is the most widely and successfully used high-rate anaerobic systems for several types of wastewaters (Lettinga, 1996). The great success of the UASB reactor can be attributed to its capability to retain a high concentration of active suspended biomass with simple and low cost means. Moreover, the formation of granular sludge improves the maximum loading rate of the UASB system. Figure 2 shows the UASB reactor concept. The wastewater feed passes upwards through an anaerobic sludge bed where the microorganisms in the sludge come in contact with the wastewater-substrate. The sludge bed is composed of microorganisms that naturally form granules that have a high sedimentation velocity and thus resist wash-out from the system. The resulting anaerobic degradation process typically is responsible for the production of gas (biogas containing CH4 and CO2 ). The upward motion of released gas bubbles causes hydraulic turbulence that provides reactor mixing without any mechanical parts. At the top of the reactor, water phase is separated from sludge solids and gas in a (gas-liquid-solids separator). The three -phase-separator is commonly a gas cap with a settler situated above it. Below the opening of the gas cap, baffled are used to deflect gas to the gas-cap opening. (www.uasb.org/discover/agsb.htm)

15

Figure 2: The up flow anaerobic sludge blanket (UASB) concept

16 Liu et al. (1991) found that the granular sludge formed in the UASB reactor when treating protein - containing wastewater for the sludge loading rate was above 0.67 Kg/ (Kg VSS.d) and pH of effluent was in the range of 7.2-7.5, the propionate in the effluent was below 300 mg/L. Microbiological analyses demonstrated that the sludge has granulation when a variety of bacteria (fermentative bacteria, propionate degraders, butyrate degraders, acetoclastic methanogens and fomate/H-2 + CO-2 methanogens) had got a proper number and in appropriate proportion . After granular sludge matured, its components were relatively stable. The activity of granular sludge was much higher as a result of increasing bacteria in the granular sludge compared with the seed sludge. Sandberg and Ahring (1992) investigated the influence of high pH on anaerobic degradation of fish process waste-water with a high total ammonia concentration in UASB reactor. More than 99% of VFA in the process waste-water were degraded up to pH 7.9. Above pH 7.9 only the conversion of acetate was slightly decreased. At pH 8.3 serious disintegration of the granules occurred leading to process failure. Increasing the pH changed the physical characteristics of the granules leading to decreased density, size, and volatile solids content. After 4 months of acclimatization to high pH in the reactor, the specific methanogenic activity of the granular biomass was the same from pH 7.1 to 8.5. At pH 8.3 and 8.5, acclimatization had improved the specific activity by 25 and 50%, respectively. However, the acclimatized biomass generally showed a decreased activity (60%) at all pH values tested below the acclimatization pH. Note that this type of organic material is suspended in the wastewater and not dissolved. Jayanth and Ramanujam (1995) investigated the treatment of spent wash (distillery effluent) as substrate using a laboratory scale UASB reactor. They studied the gas composition before and after recirculation, the volatile acids content and the solids variations along the height before and after biogas recirculation. The recirculation of

17 biogas was done on 44th day. Their study revealed that the start-up of UASB reactor can be enhanced through biogas recirculation at low organic loading rates, they found that the process efficiency and methane content increases substantially after recirculation of biogas. Almost not much change was noticed in the solids profile before and after recirculation. The volatile fatty acids (VFA) reduced considerably indicating a response favorable for methanogens. After 8 weeks of start-up they observed by using scanning Electron Micrographs active dark brown granules of about 1-1.5 mm size of long multicellular filaments of rod shaped organisms, presumably Methanothrix soehngenii. Torkian et al. (1997) studied the performance of UASB system treating slaughterhouse wastewater. They found that granulation formed after 4 months of operation. The granules were dark brownish with a diameter of 1-4 mm and a settling velocity of 20 m/hr. Up-flow velocity was gradually increased to 0.8-1 m/hr, HRT of 2.5 hr and volatile suspended solids (VSS) reached 25 g/L once granules were formed. It was possible to increase the loading up to 14 kg COD/m3.day at 29C with total COD removal efficiencies of 85-90%. At these conditions 250-350 L gas (75% methane) was generated for each kg COD removed. UASB reactor, HUASB reactor and anaerobic baffled reactor (ABR), which belong to the group of high-rate anaerobic reactors with a sludge bed (Hutnan et al. 1999). They compared between these three reactors. They conclude the following: i The lowest biomass wash-out (means that biomass escape from the reactor) observed from the ABR. Intensive biomass wash-out from UASB, while significant biomass losses from the HUASB were noticed. ii Negligible gradients of pH and VFA concentration were observed along the HUASB and the UASB. Exactly the contrary was the case of the ABR, where the differences along the reactor were notable.

18 iii A faster biomass granulation was observed in the ABR than in the other two reactors. Singh (2000) examined the feasibility of treating municipal wastewater by UASB system under low temperature conditions. He found that UASB reactor could be successfully started up at 20C. Start-up Lasted for about 60 days with a COD removal efficiency ranging from 80 to 85% at the end of this period. The performance of reactor was found to be optimum at HRT of 6 hours at all operating temperatures. It was found that the performance of UASB reactor was good and stable up to a low-temperature of 11C, however the performance of UASB reactor was not very stable at 6C, even though 30 to 50% of COD removal could be achieved. Tagawa et al. (2002) investigated the performance of a multi-staged UASB (MSUASB) reactor by feeding with a food processing wastewater containing high strength of lipid and protein. Temperature inside the reactor was operated at 55C (Thermophilic condition). The experiment continued for a period of 600 days. The results showed that the soluble COD removal was 90% (based on the effluent filtered COD), while the overall COD removal was 60-70% (based on the effluent COD- total). Also, they found that high concentration of Mg and Ca ions present in raw wastewater caused a severe scum and/or insolubilized substance formation within the UASB sludge bed, and this hindered the contact efficiency between substrate, and sludge. Because of this a deterioration of sludge methanogenic activity took place. Martinez et al (2002) investigated the kinetics of anaerobic treatment of slaughterhouse wastewater in batch and up-flow anaerobic sludge blanket UASB reactors. The characteristics of slaughterhouse wastewater used in this study are given in Table 4. In batch digesters, methane and nitrogen production stopped after 30-40, 20-30h, respectively, and in UASB reactors it was terminated after 30-40 days.

19 Based on this study, the reactions of anaerobic treatment of slaughterhouse wastewater agree with the first order kinetic model. The yield coefficient, Yp was 343 and 349 mL CH4 per g of chemical oxygen demand at standard temperature and pressure conditions for batch reactors and UASB reactor, respectively. Table 4. Characteristics of the slaughterhouse wastewater

Characteristics
COD (g/L) pH Sulphate (g/L) Phosphate (g/L) VSS (g/L) TSS (g/L) VFA (acetic acid) (g/L) Alkalinity (CaCO3) (g/L) Grease and oils (g/L) Nitrogen (Kjeldal) (g/L) Nitrates (mg/L)

Influent to UASB reactor


12.82 7.5 0.97 0.41 26.50 58.20 0.88 0.53 0.25 0.531 0.96

Effluent from UASB reactor


1.43 8.19 0.24 0.25 3.55 5.66 0.325 0.58 0.12 0.150 0.21

20

EXPERIMENTAL PROCEDURES 1. Apparatus


The digester used for treatment to remove gelatin is UASB reactor. A schematic diagram for the UASB reactor used is shown in Figure 3. The reactor is 200 cm long Plexiglas column. The internal diameter is 10 cm and thickness of the column is 1cm. Eight sampling ports are distributed equally along the column. On the top of the reactor a three-phase-separator settler is installed to separate the biogas, liquid and solid. The height of this part is 60 cm. Its cross sectional area is 30x30 cm2 and its thickness is 1cm, this part is also made of Plexiglas. The biogas is guided to a cone that has a diameter of 20 cm by means of four baffles. These baffles are present on each side of the settler. Biogas is led out from the cone to the gas bag to collect the produced biogas. The treated wastewater effluent spills into weir that is present in the upper side of the settler and it is discharged with sewage. The wastewater feed influent is fed from the bottom of the Plexiglas column The temperature was kept at 34 + 2C. Circulating water bath is used to keep temperature constant inside the reactor. The pH was kept at 7.0 + 0.2 inside the reactor, and the pH for the feed ranged from 7.0 to 7.3. Total volume of the reactor was 74 L and it represented the volume of the column plus the volume of the settler. The effective volume of the reactor was 15.7 L and it represented the volume of the column. Feed is pumped using a peristaltic pump, with model no. 7553-85 with speed of 1-100 rpm. Head model is 701721 and pat no. 3358609. Pump tube is Master flex 96400-17. From Cole-parmer Instrument Co.

21

22 2. Synthetic Wastewater Preparation A Test conducted to know what is the concentration of gelatin wastewater that was brought regularly from the Arab center for Pharmaceutical and Chemical Industries Company. Analysis showed that 2.7 g/L of gelatin was equivalent to 10 g COD/L. The COD of the wastewater of the Arab Center for Pharmaceutical and Chemicals Industries was between 8000 and 10000 mg/L. A synthetic wastewater has been prepared almost the same concentration of the gelatin wastewater of the plant. The experimental work continued for 6 months in three stages. In the first stage 80 g of gelatin was needed and dissolved in 30 L of tap water daily. In the second Stage 125 g of gelatin was needed and dissolved in 47 L of tap water daily. In the third stage 250 g of gelatin was needed and dissolved in 94 L of tap water daily. The operational parameters are listed in Table 5. Table 5. Operational parameters for the experimental work. Stage Time (Month) Concentration CODin (mg/L) Up flow velocity (m/hr) Q (m3/day) (OLR) on the base of effective volume (kg COD/m3.day) (HRT) on the base of effective volume (hr) (OLR) on the base of total volume (kg COD/m3.day (HRT) on the base of total volume (hr) 1 2.5 (8000-10000) 0.16 0.03 (15.4-19.2) 12.6 (3.3-4.1) 59.2 2 2 (8000-10000) 0.25 0.047 (23.9-29.9) 8.0 (5.1-6.4) 37.78 3 1.5 (8000-10000) 0.5 0.094 (47.9-59.9) 4.0 (10.0-12.7) 18.9

23 The properties of gelatin as a raw material of the Arab Center for Pharmaceutical and Chemical Industries Co. that was used is illustrated in appendix C. 2.1 Tests conducted on Synthetic Wastewater 1. Chemical oxygen demand test: The COD test is used to measure the oxidized organic matter chemically using dichromate as a strong oxidant in an acid solution. (Metcalf & Eddy, 2003). In This test an excess of potassium dichromate is used and the sample is refluxed in strongly acid solution. After digestion, the remaining unreduced Potassium dichromate is titrated with ferrous ammonium sulfate to determine the amount of K2Cr2O7 consumed and the oxidizable organic matter is calculated in terms of oxygen equivalent. COD test was conducted as outlined according to the Standard Methods For Examination of Water And Wastewater (Eaton et al., 1995). The test was conducted once and sometimes twice weekly. The procedure is shown in appendix A1. 2. Solids Total and volatile solids tests: The procedure is shown in appendix A2. 3. Volatile fatty acids test It is a group of volatile fatty acids that are composed of three main volatile fatty acids, these are acetic, propionic and butyric acids, on which the over all rate of methane production is dependent on. These acids are measured by using Varian Gas Chromatograph. Sampling applied by direct injection of the sample, the used detector is Flame Ionization Detector (FID) and the used column is porapak QS, 2m length that is made of stainless steel. Oven is operated at 200 C , the injector is operated at 250 C and the detector is operated at 300C.

24

3. Tests conducted on the gelatin-wastewater of the Arab Center for Pharmaceutical and Chemical Industries Co. 1. Chemical oxygen demand test This test is conducted in the same manner of COD test that is applied on the synthetic wastewater and the procedure of this test is shown in appendix A1. 2. Anaerobic biodegradability test The anaerobic biodegradability of the wastewater indicates the fraction of organic pollutants in the sewage that can be converted into methane under anaerobic conditions. This can be determined experimentally by measuring the amount of produced methane from a certain amount of wastewater with organic pollutants. The methane produced was measured by a liquid displacement test that is explained in appendix A3. The test made duplicates using two serum bottles that are prepared with 0.5L of a composite wastewater. The procedure is illustrated in appendix A3. 4. Tests conducted on sludge The used sludge was brought from a UASB reactor at khirbil Assamra that receive domestic wastewater from Amman, Ruseifa, Zarqa and Hashimiya. 1. Methanogenic activity test The methanogenic activity is the amount of methane produced expressed in gram COD per gram of sludge VS per day, so this test is used to measure the activity of the methane-producing biomass present in the sludge per day (g COD/g VSS.day). The methanogenic activity is performed using a composite sludge sample. Test conducted within 2 weeks. Temperature was kept at 33C and pH was kept between (6.3-7.8) .The procedure of the activity test is shown in appendix A4.

25 2. Solids Total solids: It is applied in the same manner in the case of total solid test that was applied for synthetic wastewater, but the used volume of sludge is 60-70mL. Volatile solids test: It is applied in the same manner in the case of volatile solids lest that was applied for synthetic wastewater, but the ignition time was 4 hours. 5. Tests conducted on biogas The biogas composed of CH4, CO2, H2S and N2 that was entrapped in the wastewater. 1. To estimate the biogas production rate: A wet Gas Meter (Schlumberger Nr 390811998) with Pmax of 100 mbar is used to measure the volume of gas whenever the gas bag that was used became full of gas. 2. To estimate the composition of the biogas a PYE-UNICAM Gas Chromatograph Instrument was used. The used column is made of stainless steel, its length is 1.5m and its diameter is 0.25 inch, its packed with porapak Q. The temperature of oven was kept at 70 C, the used detector that is thermal conductivity detector was kept at 200 C and the injector was kept at 100 C. Sampling was done using a syringe.

26

RSULTS AND DISCUSSION


In this Chapter the results are presented and discussed. 1. Characteristics of gelatin-wastewater that was collected from the Arab Center or Pharmaceutical and Chemical Industries CO. i Chemical oxygen demand. The COD for the plant was measured, it was between (8000-10000) mg/L. ii Results of the anaerobic biodegradability test This test continued for a month. Duplicates samples tested. The first sample was prepared with nutrients, the second sample was prepared without nutrients. In addition to the blank sample that was prepared in the same manner of the other samples but tap water was added instead of wastewater. The purpose of performing this test is to know whether the wastewater of the plant is biodegradable or not. The results are summarized in Table 6. Table 6. CH4 produced in the Biodegradability Test. (Joint experiment with Rand AbuZurayk). No. of sample Sample # 1 With nutrient Sample # 2 without nutrients Blank CH4 produced (mL) 70 338 327

As seen above it is noticed that the CH4 produced in case of sample # 1 that has nutrients is less than the CH4 produced in case of sample # 2 that doesnt have nutrients. This is maybe because the gelatin used as a raw material has enough concentration of nutrients and adding more nutrients caused inhibition to methanogens that are responsible for CH4 production and prevent CH4 production.

27 The amount of COD that is digested or degraded was calculated using the following equation: CH 4,t=t CH4blank,t=t COD total degraded = COD tot,t=0 100%

The total COD degraded was 76.2%. Calculation of COD (total degraded) is shown in Appendix B2. 2. Characteristics of Khirbit Assmra sludge 1. Methanogenic activity results of the used sludge is illustrated in Table 7. Table 7. Activity test for Khirbit Assamra sludge Time (day) 0 2 3 3 3 2 Acaccumulative NaOH Time (day) (ml) 0 2 5 8 11 13 0 5 3 4 2 2 Acaccumulative COD (g) ml NaOH 0 5 8 12 14 16 0 0.0114 0.0182 0.0272 0.032 0.037 gCOD/g VSS 0 0.00912 0.01456 0.02176 0.0256 0.035

The activity is calculated by plotting the accumulative methane production expressed as COD versus time. The activity is equal to the slope of the line as shown in Figure 4. So, activity is the steepest slope of the line that is 0.0026 g COD/g VSS/day. (Abu-Zurayk, 2004), reported two values of methanogenic activity in treating gelatine-wastewater using HUASB reactor, the first value was 0.0026 g COD/g VSS/day at OLR of 17 kg COD/m3.day and at up-flow velocity of 0.1 m/hr and the second value was 0.12 g COD/g VSS/day at OLR of 17 kg COD/m3.day and at up-flow velocity of 0.1 m/hr. The

28 second value was estimated at the end of the operational applied stage but the first value was estimated at the beginning of the same operational stage.

29

30 2. Results of the TS and VS for the sludge that was brought from Khirbit Aassmra were: TS and VS was 40.2 g/L and 26.1 g/L, respectively. 3. After about 4.5 months it is observed granules that had black color, they were about 1-1.5 mm size. 3. Characteristics of the synthetic wastewater 1. Solids (Total and Volatile Solids) These two tests were conducted to the effluent of the treated synthetic wastewater, they were conducted about 3 times. The readings of TS and VS that are shown in Table 8 were taken by the end of each stage. Most the TS represents the sludge that was washed out from the reactor so the main source of the TS was the sludge, because the measurements of the COD fractions were the same as total COD, so the synthetic gelatin-wastewater was completely dissolved. Results are shown in Table 8. Table 8. TS and VS Results No. of stage Up-flow velocity m/hr OLR kg COD/m3 day TS g/L VS g/L 1 0.16 (15.4-19.2) 0.70 0.40 2 0.25 (23.9-29.9) 0.74 0.45 3 0.5 (47.9-59-9) 0.65 0.34

As seen from the Table 8, it is noticed that increasing the OLR from 15.4-19.2 kg COD/m3.day to 47.9-59.9 kg COD/m3.day, TS and VS decreased because it is noticed in the third stage a decrease in the production rate of the gas when the OLR increased to 47.9-59.9 kg COD/m3.day, although the up-flow velocity was increased and this is supposed to enhance the sludge particles to rise up. This can be explained by the fact that the gas production decreased in the third stage due to the reduction in the organic

31 matter digestion which reduces the sludge wash out from the reactor, this result agrees with the results of Abu-Zurayk, (2004). 2. Chemical oxygen demand The results for COD of the synthetic wastewater for all stages are illustrated in Tables 9, 10, 11. Table 9. COD Results for first stage (up-flow velocity) is 0.16 m/hr and (OLR)eff.v. is between (15.4-19.2)Kg COD/m3.day,and (OLR)Tot.V. is between (3.3-4.1) kg COD/m3.day Time (day) 0 15 29 41 50 60 75 COD in (mg/L) 10000 9180 9384 8568 8976 8287 COD out (mg/L) 8500 5967 4692 3170 1975 1823 Max % removal COD Tot 15 35 50 63 78 78

Table 10. COD Results for second Stage (Up-flow velocity) is 0.25 m//hr and (OLR)eff.v. is between (23.9-29.9) Kg COD/m3.day, (OLR)Tot.v. is between (5.1-6.4) Kg COD.m3.day.

32 Time (day) 0 10 20 30 40 50 60 COD in (mg/L) 9268 8459 8594 8633 9014 9847 COD out (mg/L) 6488 4230 3094 2331 1442 1477 Max % removal COD Tot 30 50 64 73 85 85

Table 11. COD Results for the third stage (up-flow velocity) is 0.5 m/hr and (OLR)eff.v. is between (47.9-59.9) Kg COD/m3.day, (OLR)Tot.v. is between (10-12.7) kgCOD/m3.day . Time (day) 0 5 10 20 30 38 45 COD in (mg/L) 9456 8347 9662 9043 8920 8624 COD out (mg/L) 7565 6260 6667 5695 4817 4657 Max % removal COD Tot 20 25 31 37 46 46

Figures 5, 6, 7 show the relationship between COD removal % vs. time in the all stages.

33

34

35

Organic loading rate effect Organic loading rate is an important parameter affecting the microbial ecology and characteristics of the UASB system. In first stage when OLR is between 15.4-19.2 Kg COD/m3.day, the removal efficiency was 78% by the end of this stage as illustrated in

36 Table 9, the relationship between time and COD removal percentage is represented in Figure 5. In the second stage when OLR is between 23.9-29.9 Kg COD/m3.day, the removal efficiency increased to 85% by the end of this stage as illustrated in Table 10, the relationship between time and COD removal percentage is represented in Figure 6. In the third stage when OLR is between 47.9-59.9 Kg COD /m3 day, the removal efficiency decreased to 46% as and illustrated in Table 11 and as seen in Figure 7. In the third stage the pH measured and it was 6.87 and this drop to the pH was due to the increase of the VFA concentration that were measured in this stage and this increase might associated with inhibition that affected the methanogens (Hobson and Shaw, 1976). So, an inhibition might occur because the OLR was increased suddenly and may be it caused a shock to the microorganisms. But this is not an absolute conclusion because complete steady state was not reached so if more time was given after increasing the OLR, the COD removal percentage could be better. Also In this stage the HRT was reduced to 4 hrs so, there was no enough contact time between the organic matter and the microorganisms. This result almost agrees with Abu-Zurayk, (2004) result in treating gelatine-rich wastewater using hybrid up-flow anaerobic sludge blanket HUASB reactor who mentioned that increasing the OLR from 17 Kg COD /m3.day to 23 Kg COD /m3.day at up-flow velocity of 1.45 m/hr reduced the COD removal percentage from 83% to 50% when the HRT decreased from 11 hrs to 8 hrs. Also this result agrees with (Torkian and Hashemian, 2003) who reported that increasing the OLR to above 30 Kg COD /m3.day, caused a drastically decreased to below 70% of removal and this affect negatively on the performance of UASB reactor treating slaughterhouse effluent.

37 Higher OLR values of up to 45 kg COD/m3.day have been reported only for hybrid reactors using a combination of UASB reactor and a bentonite packing as a biomass support (Borja et al., 1995). So, in this work the optimum OLR was 23.9 29.9 Kg COD/m3.day for the highly COD removal percentage. Calculations of COD removal percentage are illustrated in appendix B1. Table 12. Up-Flow velocity and HRT and their effect on COD removal % in the three stages Stage No. Up-flow velocity (m/hr) First Second Third 0.16 0.25 0.5 HRT (hr) 12.6 8 4 Max % removal COD Tot. 78 85 46

38

39

Up- flow velocity effect and hydraulic retention time effect The up-flow velocity should be selected such that sufficient contact time is available between wastewater and microbial population (HRT in the 6-8 hr range) (Torkian et al.1997).

40 As seen in Table 12 and as seen in Figure 8, the maximum COD removal percentage was 85%. This removal percentage was obtained at the end of the second stage at OLR of 23.9-29.9 kg COD/m3.day and at up-flow velocity of 0.25 m/hr. This result almost agrees with (Abu-Zurayk, 2004) in treating the gelatine-wastewater using HUASB reactor who mentioned that the COD removal percentage was 75% at OLR of 17 kg COD/m3.day and at up-flow velocity of 0.1 m/hr. While in third stage when up-flow velocity increased to 0.5 m/hr, the COD removal percentage decreased to 46% and this decrease was due to the higher OLR and to the lower HRT as mentioned previously. As HRT decreased from 8 hrs in the second stage to 4 hrs in third stage, the COD removal percentage was also decreased from 85%-46%. Therefore, in this work the optimum up flow velocity was 0.25 m/hr and the optimum HRT was 8 hr. The up-flow velocity effect is represented in figure 8, the change of up-flow velocity with COD removal % is represented in a polynomial relationship as following equation: COD removal % = (-687.58 (up flow velocity)2 + 359.69 (up flow velocity) +38.052) this equation is applied just for the same conditions of this experiment (temperature of 34 + 2C and pH of 7.0 + 0.2) The HRT effect is represented in Figure 9, the change of HRT with COD removal % is represented in a polynomial relationship as following equation: COD removal % = (1.3107 HRT2 + 25.478 HRT 34.941) this equation is applied just for the same conditions of this experiment, (temperature of 34 + 2C and pH of 7.0 + 0.2). 3. Volatile Fatty Acids

41 Duplicates analyses were made for the volatile fatty acids first analysis was made at the end of the second stage and the second analysis was made at the end of third stage. The results are illustrated in Table 13. Table 13. Results of the volatile fatty acids VFA for effluent of the treated synthetic wastewater in second and third stages Operational Parameters Acetic Acid (mg/L) Propionic Acid (mg/L) Butyric Acid (mg/L) Total VFA (mg/L) Up-flow velocity = 0.25 m/hr Up-flow velocity = 0.5 m/hr OLR = (23.9-29.9) kg OLR = (47.9-59.9) kg 3 COD /m . day COD /m3. day 58.0 (67.3)% 26.9 (31.2)% 1.3 (1.5)% 86.2 125.1 (77.7)% 33.2 (20.6)% 2.75 (1.7)% 161.05

From Table 13, it is noticed an increase of the VFA concentration in the third stage than in the second stage and so, this had bad effect on the COD removal and on the production of the biogas. (Mc Kinney, 1962) reported that the high acid concentration retards further bacterial metabolism. If more time was given it is possible that the VFA reach zero and so this will increase the efficiency of the COD removal and will improve the performance of the reactor. 4. Characteristics of biogas 1. Biogas production rate There was extremely leakage for the gas produced. So, it was very difficult to measure the production rate of biogas. Almost 3-4 L/hr of gas production was measured and this value was less than expected. 2. Biogas composition The composition of the effluent was measured twice, once at the end of second stage and another measurement was at the third stage. The results are illustrated in Table 14.

42 Table 14. Results of the biogas composition. Up-flow velocity=0.25m/hr Biogas Content OLR= (23.9-29.9) kg COD/m3. day (CH4) % (CO2) % (N2) % 65.0 18.60 15.86 Up-flow velocity = 0.5 m/hr OLR = ( 47.9-59.9 ) kg COD/m3. day 49.7 13.0 30.4

As noticed from Table 14, increasing the OLR from 23.9-29.9 kg COD/m3.day to 47.959.9 kg COD/m3.day affected the results of the biogas composition and so the performance of the UASB system because OLR was increased suddenly and it might caused a shock to the acetogenesis that are responsible for the conversion of the fatty acids into acetate and so effected the function of methanogens that convert acetate into CH4 and CO2 as a result of highly OLR.

43

CONCLUSIONS AND RECOMMENDATIONS


Within the reported results of this work, the following conclusions and recommendations are obtained: 1. Anaerobic digestion using UASB is appropriate for the removal of gelatin from Industrial wastewater. So, treating the wastewater of the Arab center for pharmaceutical and Chemical Industries Co. could be feasible because the biodegradability of the gelatin wastewater of the plant was relatively high and it was 76.2%. 2. Treatment can be achieved without adding nutrients to the feed, since the gelatin as raw material has enough concentration of nutrients. 3. Increasing the OLR to 47.9-59.9 kg COD/m3.day decreased the COD removal percentge to 46%. 4. Optimum OLR was between 23.9-29.9 kg COD/m3.day and COD removal percentage was 85% in this stage. 5. Increasing the up-flow velocity from 0.16m/hr to 0.25 m/hr increased the COD removal percentage to 85%. 6. The optimum HRT for this work was 8hr and the COD removal percentage increased to 85 %. It is recommended to: i Study the effect of up-flow velocity over 0.5m/hr on the performance of the UASB system ii Improve the design for the settler to prevent leakage of biogas is recommended. iii Use the produced biogas as fuel source is recommended, since the produced CH4 gas contains about 90% of energy and can be burned on site to provide heat for digester or to generate electricity.

44

REFERENCES
Abu-Zurayk, R. (2004). Anaerobic Treatment of Gelatine-Rich Wastewater Using Hybrid Up-flow Anaerobic Sludge Blanket (HUASB) Reactor. Published Master Thesis. University of Jordan, Amman, Jordan. Ayyoub, R. (1999). Treatment of Industrial Wastewater Containing Gelatin. Published Master Thesis. University of Jordan, Amman, Jordan. Azbar, N., Ursillo, P. and Speece R. (2001). Effect of Process configuration And Substrate Complexity On the performance of Anaerobic Processes. Water Research, 35 (3), 817-829. Bailey, J., and Ollis, D. (1986). Biochemical Engineering Fundamentals, (2nd. Ed.). New York : Mc Graw- Hill. Borga, R., Banks, CJ., and Wang, Z. (1995). Performance of a hybrid anaerobic reactor, combining a sludge blanket and a filter, treating slaughterhouse wastewater. Applied Microbial Biotechnology, 43, 351-357. (Corda Gelatin Ltd. Gelfax). A series of useful facts on all aspects of gelatin quality (manual). David, R. (1996). Anaerobic Digestion for MSW and Industrial Wastewater BioCycle, 37(11),77-81. Eaton, A.D., fronson, M.A.H., Greenberg, A.E., and Clesceri, L.S. (1995). Standard methods for examination of water and wastewater. 19th. Washington: Public health for the examination of water and wastewater. Environmental federation. Hobson, P. N and Show, B.G. (1976). Inhibition of Methane Production by Methanobacterium Formicicum. Water Research, 7,437. Hutnan, M., Drtil, M., Marfkova, L., Derco, J., and Buday, J. (1999). Comparison of Startup and Anaerobic Wastewater Treatment in UASB, hybrid and baffled reactor. Bioprocess Engineering, 21, 439-454. Jayanth K.S. and Ramanujam T.K. (1995). Start-up criteria for an Up-flow Anaerobic sludge Blanket (UASB) reactor. Bioprocess Engineering, 13(1995), 307-310 Lettinga G. (1996). Sustainable Integrated Biological Wastewater Treatment. Water Science and Technology, 33(3):85-98 Lettinga, G., Van Velsen, A.F.M., Hobma, S. W., De Zeeuw, W., and Klapwijk. (1980). Use of Upflow Sludge Blanket Reactor Concept for Biological Wastewater Tratment, especially for anaerobic treatment. Biotechnol. Bioengineer,22, 699-734. Lin, K.C., and Yang, Z. (1991). Technical Review on The UASB Process. International Environmental studies,39, 203-222.

45 Linsley, R., Franzini, J., Freyberg, D., and Tchobanoglaus, G. (1992). Water Resources Engineering, (4th. Ed.) New York :McGraw-Hill. Liu, S.J., Hu, J.C., and Gu, X.S. (1991). Anaerobic. Sludge-Granaltuem In A UASB Reactor Treating Protein-Containing Wastewater. Environmental Science, 12(3), 7-12 Martinez, J.R., Garza, I.R., Flores, E.P., Flores, E.P., Balageurusamy, N., Santillan, G.S., and Garcia, Y.G. (2002). Kinetics of Anaerobic treatment of Slaughterhouse Wastewater In Batch and Up-flow Anaerobic Sludge Blanket Reactor. Bioresource Technology, 84, (235-241). Mckinney, R. (1962). Microbiology for sanitary Engineers, (1st. ed.) New York : Mc Graw-Hill. Metcalf and Eddy (2003). Wastewater engineering: Treatment and reuse, (4th ed) New Yourk : McGraw-Hill. Mohan, S.V., Prakasham, R.S., Satiyavathi, B., Annapurna, J., and Ramakrishna, S.V. (2001). Biotreatability Studies of Pharmaceutical Wastewater Using An Anaerobiv Suspended Film Contact Reactor. Water science and Technology, 43 (2), 271-276. Ping, Z., Lan, H.B., (2002). Start-up strategies of UASB Reactor for Treatment of Pharmaceutical wastewater. Environmental Sciences, 14 (2), 250-255. Price, E.C., and Cheremisinoff, P.N. (1981). Biogas Production and Utilization: Ann Arbor Science Publishers, Inc. Sandberg, M., and Ahring, B.K. (1992). Applied Microbiology and Biotechnology, 36 (6), 800-804. Tagawa, T., Tarahashi, H., Sekiguchi, Y., Ohashi, A., and Harada, H. (2002). PilotPlant Study on Anaerobic Treatment of A Lipid- And protein- Rich Food Industrial Wastewater By A Thermophilic Multi-Staged (UASB) Reactor. Water Science and Technology, 45(10), 225-230 Torkian, A., Amin, M., Movadian, H., Hasheman, S., and Salehi, M.S. (1997,b). Performance evaluation of UASB system treating slaughter house wastewater, Fourth interntional conference on civil engineering, May 4-6 , 1997, Sharif University of Technology. Torkian, A., Eqbali, A., and Hashemian, S. (2003). The effect of organic Loading rate on the performance of UASB reactor treating slaughterhouse effluent. Resources Conservation and Recycling, 40 (2003): 1-11. Wageningen university (1999). Biological Wastewater Treatment Part 1 Anaerobic Wastewater Treatment, Netherland, Lettinga, G., Hulshoff pol, W., and Zeeman, G. Auther

46 Yu, H.Q., and Fang, H.H.P. (2003). Acidogenesis of Gelatin-Rich Wastewater In An Up-flow Anaerobic Reactor: Influence of PH and Temperature. Water Research, 37,55-66.

Internet Sites:
Web sites retrieved August 8,2004, from http://www.gelatin.co.za/g1tn1.htm1.
http://www.singaporescienceCenterScienceNetPhysicalSciencesGeneralChemistry.htm.

http://www.united-tech.com/wd-anaerobicdigestion.html.

47

Appendix A. Tests Procedures Appendix A1. COD Test Procedure


Reagents: Standard Potassium Dichromate solution, 0.25M: 12.259 g of K2Cr2O7, previously dried at 103C for 2hr, 167ml conc. H2SO4 (95-97)% and 33g HgSO4 (99%) are added to 500mL distilled water, and are cooled at room temperature and diluted to 1000mL. Sulfuric Acid reagent: 5.59 Ag2SO4 was dissolved and added to 1Kg Conc. H2SO4 (95-97)%. Ferrion Indicator solution: 1.485g of 1.10-Phenantheroline monohydrate and 695 mg FeSO4. 7H2O dissolved in distilled water and dilute to 100ml. Standard Ferrous Ammonium Sulfate (FAS) Titrant, 0.1M: 39.2g of Fe (NH4)2 . 6(H2O) (98%) was dissolved in distilled water and 20ml Conc. H2SO4 (95-97)% were added and diluted to 1000ml. Molarity of FAS solution was calculated after titration by using equation: M= 5 Volume FAS used in titration, mL 0.25

5 mL of K2Cr2O7 Standard solution that was added to the flash to make titration, 20mL of H2SO4 was added and diluted to 100mL. Apparatus: Reflux apparatus, consisting borosilicate tubes that is placed in a heating block (digester), the set point temperature is 150C and the digestion time continued for two hours. Sampling: Analysis must not be delayed if the sample is not ready, if this happen the sample preserved by acidification to PH<2 using conc. H2CO4.

48 A preliminary dilutions made for wastes containing a high COD to reduce the error inherent in measuring small sample volumes. Analytical Procedure: In this test 5mL of influent and effluent samples was added to the digestion tube. Digestion solution was added, 5mL of H2SO4 reagent was added to the digestion tube. The sample was diluted 100 times by taking 5mL of the raw wastewater and is added to 500mL volumetric flask of distilled water. 5 Pumic stones granules are added to the digestion tubes to prevent splashing. Then the tubes are covered with their cap and placed in the block digester that is preheated to 150C and reflux for 2 hours. Samples are cooled after digestion to room temperature and then transferred to conical flasks and 3 drops of ferrion indicator is added and titrating with 0.1M FAS. The end point is a sharp color change from bluegreen to reddish brown. A blank Sample is prepared in the same manner by taking a volume of distilled water aqual to that of the sample and digestion solutions are added. The COD is calculated as (mg O2/L) using the equation: COD = (A-B) x M x 8000dilution mL sample

Where: A= mL FAS used for blank, B= mL FAS used for sample, and M= molarity of FAS.

49 Appendix A3. Biodegrdability Test Procedure To conduct this test following components are added: 1. Micro nutrients that are illustrated in Table 15. Table 15. Micro nutrients Micro nutrients (mg/L) FeCl3 6H2O (99%) (1134) CoCL2. 6 H2O (97%) (1000) MnCL2. 4 H2O (98%) (250) CuCL2 . 2H2 O (98%) (15 ) ZnCL2 (98%) (25) H3BO3 (99.5%) (25) ALCL3 (99%) (25) (NH4)6 Mo7O24. 4H2O (99%) (45) Na2SeO3 (99%) (33) NiCL2. 6H2O (98%) (5) EDTA (98%) (500) HCL ( 36%) (1mL/L) Manufacturer Scharlau Chemie S.A. Company Park scientific Ltd. Company Fisher scientific company BDH chemicals Ltd poole England company Scharlau chemie S.A company BDH chemicals Ltd Poole England company Fluka chemical Company Park scientific Ltd. company BDH chemicals Ltd Poole England company Fluka chemical Company BDH chemicals Ltd. Poole England Company

2- Macro nutrients that are illustrated in Table 16. Table 16. Macro nutrients Micro nutrients (g/L) NH4CL (99%) (85) KH2PO4 (98-101%) (19) CaCL2 (90%) (3) MgSO4. 7H2O (99%) (6) Manufacturer BDH chemicals Ltd. Poole England Company BDH chemicals Ltd. Poole England Company Grainland Chemical Company Grainland Chemical Company

50 3- 2.5g sludge VSS. 4- 0.1g yeast extract. Analytical Procedure: After adding all the previous, bottles are closed and the headspace of each bottle is flushed for 2-3 minutes with nitrogen gas to create anaerobic conditions. Bottles are incubated at 33 C. The gas production in time is measured every two to three days using the Liquid displacement test. The Liquid displacement test is used for the biodegradability and methanogenic activity test that is illustrated in appendix A4. To determine the CH4 gas produced by Liquid displacement test this can be done as follows: the prepared bottles are connected to Mariott flasks in which the produced biogas is led through a NaOH solution of pH 12, the CO2 will dissolve. The remaining CH4 will increase the internal pressure of the bottle and some Liquid will be pushed out off the bottle. Liquid will be collected and weighed. The amount of (CH4-COD) is calculated as the following equation: m= Where: m= P= V= M= R= T= Produced CH4 (g) Air-pressure (Amman = 0.897 atm) Volume collected NaOH solution (L) also it equals volume of CH4 (L) Molar mass of CH4 (16 g/mole) Gas constant (0.0821 atm.L/mole k) Temperature (k) = (33C+273) = 306K PVM RT

Then the COD of methane produced can be calculated by multiplying (m) by a conversion factor of (4 g COD/g CH4). At t=0, COD of the wastewater is measured So, The amount of COD that is digested can be calculated by the amount of produced methane, using the equation:

51 CH 4,t=t CH4b,t=t COD total degraded = COD tot,t=0 100%

CH4b is for the blank that is prepared in the same manner of the wastewater sample but, tap water is used instead of wastewater

52 Appendix A2. Total And Volatile Solids Tests Procedure Total Solids (TS): Procedure It is conducted by evaporating a well mixed sample of non volume in a weighed dish and dried in an oven at 103-105C for 24 hours. The sample is then cooled in a desiccator to balance temperature. Usually it was taken 10 ml sample of influent and effluent. Eaton et al., (1995). Total Solids is calculated by using the following equation: mg Total Solids/L = Where: A = Weight of dried residue + dish, mg. B = Weight of dish, mg Volatile Solids (VS) After making the (TS) test dish is transferred to furnace at 550, and ignite for 2 hours, then the dish is cooled in desiccators to balance temperature and then is weighed. Eaton et al., (1995) The volatile solids is calculated by using the following equation: mg volatile solids/L = Where: A: Weight of dried residue + dish before ignition, mg. B: Weight of dish after ignition, mg. (A-B) x 100 . Sample Volume, ml (A-B) 1000 Sample Volume, ml

53 Appendix A4. Activity Test Procedure To Perform The Methanogenic Activity test we need the following: 1- Sludge: The used sludge for this test was 1.25 g sludge VSS 2- Substrate: The used substrate for this test was sodium Acetate its concentration is 2.5g COD/L and this is prepared by dissolving 45.95g of sodium Acetate in 250mL distilled water. 3- Buffer The concentration of the used buffer was 0.01 M NaH2Po4 in serum bottle and this is prepared by dissolving 15g (99)% in 250 mL distilled water and 0.016 M K2 HPO4 in serum bottle and this is prepared by dissolving 35.25 g (98)% in 250 mL distilled water, pH is kept at 7.0. 4- Sodium hydroxide: It is needed to estimate the produced methane by Liquid displacement test that was previously explained in the wastewater Biodegradability test in appendix A3. 5- Micro nutrients: They were mentioned previously in the wastewater Biodegradability test in appendix A3. 6- Macro nutrients They were mentioned previously in the wastewater Biodegradability test. 7- 0.1g yeast extract Analytical Procedure: Two serum bottles are used and the previous chemicals and solutions were added to each serum bottle. Bottles were filled up with tap water till 0.5 Liter. After closing the

54 bottles they were flushed with nitrogen gas for three minutes. The bottles were incubated at a temperature of 33C. pH must kept between (6.3-7.8). The methane production was measured once a day by using Liquid displacement that was explained in Biodegradability test in appendix A3. The activity was measured by Plotting the acaccumulative methane as (COD) production per amount of sludge VS as a function of time. The Maximum activity is equal to the steepest slope of this plot.

55

Appendix B. Calculations
Appendix B1. Calculation of COD removal % An example to calculate the COD removal percentage COD in = 9014, COD out = 1442 COD (mg O2/L) = (Eaton et al., 1995). A = Volume of FAS used for the blank = 5 ml. B = Volume of FAS used for the inlet sample = 4.5 ml. B = Volume of FAS used for the outlet sample = 4.96 ml. Vt = Volume of AS used for titrant Molatity of FAS : M = M = 0.239 Dilution = 100 CODin (mg O2/L) = (A-B) 8000x M dilution Sample volume (mL) = (5-4.5) 8000x 0.239 100 5 = 9104 mg COD/l 5x0.25 Vt (A-B) 8000x M dilution Sample volume (mL)

CODout (mg O2/L) = (5-4.96) 8000x 0.239 100 = 1442 mg COD/l 5 COD removal percentage = CODin-CODout 100% = 9104-1442 100% COD in 9104 = 85%

56

Appendix B2. Calculation of Biodegradability


CODdegraded = CH4t=t cH4 blank,t=t 100% CODt=o

CH4 t=t (as g COD) = PV Mwt 4 g COD RT gCH4 0.338L CH4 0.895 16 atm 4 0.08206 (atm.L/moL.K)x (273+33) gCH g COD 4 = 0.771 g COD mol CH4 g CH4

CH4 blank t=t ( as g COD ) = PV Mwt 4 gCOD = RT g CH4 0.327L CH4 0.895 16 atm 4 0.08206 (atm.L/moL.K)x (273+33) gCH g COD 4 = 0.746 g COD mol CH4 g CH4

g COD t=o = ( g COD ) t=o volume of water used g COD t=o = 7.5 g/ L 0.4375L=0.0328125 g COD(sample diluted 100 times) 100 CODdegraded = CH4t=t cH4 blank,t=t 100% CODt=o = 0.771-0.746 100% = 0.0328125 = 76.2%

57 Appendix C. Gelatin Properties Limed Bone Gelatin Raw Material Name Suppler Code No. No 12 3 4 5 6 7 8 9 10 11 12 13 Description Solubility Identification Appearance of solution Odor and water-insoluble substances Residue on ignition Heavy metals Sulphur dioxide Arsenic Viscosity Viscosity Breakdown Klett value Particle size Test B.P. 98 B.P. 98 B.P. 98 B.P. 98 U.S.P. 95 U.S.P. 95 < 2.0% B.P. 98 < 50 ppm B.P. 98 < 200 ppm B.P. 98 < 0.8 ppm 45-55 mp < 24% N.L.T. 75% 70% passes a U.S#10 screen (2.0mm) N.M.T. 20% passes a U.S# 20 screen (850 um) N.M.T. 5% passes a U.S.# 40 screen (425um). B.P. 98 3.8 7.6 235-265 B.P. 98 < 15% B.P. 98 N.M.T 100 ppm 4.7-5.2 <1000 cfu/gm <1000 spore/gm Negative / 5gm Negative / 5gm Negative / 5gm Negative / 5gm Limed Bone Gelatin Date Suppler Batch No. Q.C. Serial No. Limit Result

14 15 16 17 18 19

HP (1%sol.) Gel strength Loss on drying Peroxides Isoelectric point Microbial Limit test - Total microbial count - Molds and yeast count - Escherichla coli - Pseudomonas aeruginosa - Staphylococcus aureus - Salmonella species

- Gelatin Liquefying Bacteria Negative

58

. . . 10000-8000/ . : , . 32 36 " 6.8 . 7.2 . 19.2-15.4


3

%85-%78 30/ 3 %46 65 .% 49.7

59

. 29.9-23.9 : / . 3 , 0.25/ 8 .

Potrebbero piacerti anche