BIODEGRADATION OF LANDFILL LEACHATE BY BIOAUGMENTATION

A Seminar Paper

In Partial Fulfilment of the Requirements

in the Course

Field Trips & Seminars

Second Semester
S.Y. 2017-2018

By:

Bose, Judy Ann D.

Leneses, Catherine B.

Ramos, Melanie B.

Suor, Alexa C.

Engr. Caesar P. Llapitan

Instructor

May 2018
(This page is intentionally left blank.)
 Table of Contents

Chapter 1 .................................................................................................................................. 1

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

1.1. Background of the Study .......................................................................................... 1

1.2. Objectives ................................................................................................................... 2

1.3. Significance of the Study........................................................................................... 2

Chapter II ................................................................................................................................. 3

Review of Related Literature .................................................................................................. 3

2.1. Different techniques for Biodegradation................................................................. 3

2.1.1. Biostimulation .................................................................................................... 3

2.1.2. Bioremediation ................................................................................................... 4

2.1.3. Bioaugmentation ................................................................................................ 5

2.2. Contaminants that can be Treated with Biodegradation ...................................... 6

2.2.1. Heavy Metals ...................................................................................................... 6

2.2.2. Hydrocarbons ..................................................................................................... 7

2.2.3. Organic Contaminants ...................................................................................... 7

2.3. Advantages and limitations of Bioaugmentation.................................................... 8

2.4. Microorganisms used in Landfill Leachate Bioaugmentation .............................. 9

2.5. Methodology of Bioaugmentation .......................................................................... 14

2.6. Orthogonal Experimental Design .......................................................................... 16

Chapter III .............................................................................................................................. 17

Synthesis of Current Research ............................................................................................. 17

3.1. Recent study on Biodegradation of Landfill leachate by Bioaugmentation ...... 17

3.2. Major findings, contradictions and confirmations across the studies ................ 18

Chapter IV .............................................................................................................................. 20

Applications and Implications .............................................................................................. 20

4.1. Remodeled Flow Diagram for Bioaugmentation of Landfill leachate ................ 20

Chapter V ............................................................................................................................... 25

Summary and Conclusions.................................................................................................... 25

5.1. Summary and Conclusions ......................................................................................... 25

References ............................................................................................................................... 26
 LIST OF FIGURES

Figure 1. Flow diagram of Landfill leachate Bioaugmentation ............................................... 15

Figure 2. Remodelled Flow diagram of Bioaugmentation of Landfill leachate ...................... 20

LIST OF TABLES

Table 1. Functional Microbial Ecology in LFL treatment processes (Zhang, et al., 2016) ....... 9
 Chapter 1

Introduction

1.1. Background of the Study

Sanitary landfills are the most common way of eliminating municipal solid wastes

(MSW) because it is common, economical and environmentally acceptable. During the

stabilization of landfilled waste, most organic materials are broken down into simpler

compounds by aerobic and anaerobic microorganisms, leading to the formation of landfill

gas and leachate (Ruo, et al., 2005). Generation of heavily polluted leachates, presenting

significant variations in both volumetric flow and chemical composition, constitutes a

major drawback. Landfill leachate contains contaminants that pose a negative impact on

the environment (Sumaiya, et al., 2014). The heterogeneous nature of leachate is often a

concern because it may contain monocyclic aromatic hydrocarbons, xenobiotic organics,

polychlorinated biphenyls (PCB), heavy metals or metalloids, and other pollutants. The

leachate problem is worsened by the fact that many landfills lack an appropriate bottom

liner or collection system; increasing the possibility of dissipation of leachate through the

landfill layers to contaminate ground water (Kanmani, et al., 2013). Year after year, the

recognition of landfill leachate impact on environment has forced authorities to fix more

and more stringent requirements for pollution control. Bioaugmentation is one of the

technique in which strains of natural or genetically engineered bacteria with unique

metabolic profiles are used to treat sewage or contaminated water or soil.

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1.2. Objectives

The main objective of this study is to develop a method of implementing

bioaugmentation of landfill leachate through review and analysis from recent studies.

Specifically, it is intended:

1. To present the technologies for biodegradation and determine the nature of

contaminants in landfills.

2. To evaluate the different techniques for demonstrating biodegradation in landfill

leachate.

3. To conduct a review of bioaugmentation from recent studies

4. To develop a method of implementing an efficient bioaugmentation of landfill

leachate.

1.3. Significance of the Study

Results of this study would be particularly useful to the community, environment and

natural resources planning officers and conservationists, and policy makers at the local level.

Environmental management and waste management offices may use this study in facilitating

the planning and implementation of appropriate and sustainable landfill leachate treatment.

Further, this study would be significant to other waste management academe, research

and environment related government and non-government institutions and use the strategies

to support groups in environmental undertakings and entice to make use of biodegradation

processes in the treatment of the different contaminants generated by landfills.

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 Chapter II

Review of Related Literature

2.1. Different techniques for Biodegradation

Biodegradation is the use of microorganisms to degrade environmental

contaminants into less toxic forms. Microorganisms destroy organic contaminants in the

course of using the chemicals for their own growth and reproduction. This includes

bacteria (aerobic and anaerobic), fungi, and actinomycetes (filamentous bacteria).

According to the definition by the International Union of Pure and Applied

Chemistry, the term biodegradation is “Breakdown a substance catalysed by enzymes in-

vitro or in-vivo. This may be characterized for the purpose of hazard assessment such as

alteration of the chemical structure of a substance resulting in loss of a specific property

of that substance, biodegradation to such an extent as to remove undesirable properties

of the compound, and complete breakdown of a compound to either fully oxidized or

reduced simple molecules (Porto, et al., 2012). Biodegradation of hydrocarbons by

natural populations of microorganisms is the most eco-friendly and economically

viable method for the management of petroleum contaminated sites.

2.1.1. Biostimulation

Biostimulation involves the modification of the environment to stimulate

existing bacteria capable of bioremediation. This can be done by the addition of

various forms of rate limiting nutrients and electron acceptors, such as

phosphorus, nitrogen, oxygen or carbon (wikipedia.org/Biostimulation). It can be

improve pollutant degradation by optimizing conditions such as aeration,

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 addition of nutrients, pH and temperature. The primary advantage of

biostimulation is that bioremediation will be undertaken by already present native

microorganisms that are well-suited to the subsurface environment, and are well

distributed spatially within the subsurface. The primary challenge is that the

delivery of additives in a manner that allows the additives to be readily available

to subsurface microorganisms is based on the local geology of the subsurface.

Tight impermeable subsurface lithology make it difficult to spread additives

throughout the affected area. Fractures in the subsurface create preferential

pathways in the subsurface which additives preferentially follow, preventing

even distribution of additives (Adams, et al., 2015).

2.1.2. Bioremediation

Bioremediation is a process which uses microorganisms and their

biodegradative capacity to remove contaminants from the soil. In particular,

native soil microorganisms play a key role in soil bioremediation. They perform

as bio-geochemical agents to transform complex organic compounds into simple

inorganic compounds or into their constituent elements. This process is termed

rnineralization. The microorganisms (bacteria) are adsorbed to soil particles by

the mechanism of ionic exchange. In general soil particles have a negative

charge, and soil and bactena can hold together by a ionic bond involving

polyvalent cations (Killham, 1994).

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 2.1.3. Bioaugmentation

Bioaugmentation is the addition of microorganisms to enhance a specific

biological activity. It is the addition of pollutant-degrading microorganisms

(natural/exotic/acclimatized/genetically engineered) to augment the

biodegradative capacity of indigenous microbial populations. It involves the

introduction of microorganisms isolated from the contaminated sites, from a

historical site or carefully selected and genetically modified to support the

remediation of contaminated sites based on the assumption or confirmation that

indigenous organisms within the impacted site cannot biodegrade the

contaminants. Successful bioaugmentation treatments depend on the use of

inocula consisting of microbial strains or microbial consortia that have been well

adapted to the site to be decontaminated. Foreign microorganisms (those in

inocula) have been applied successfully but their efficiency depends on ability to

compete with indigenous microorganisms, predators and various abiotic factors.

Factors affecting proliferation of microorganisms used for bioaugmentation

including the chemical structure and concentration of pollutants, the availability

of the contaminant to the microorganisms, the size and nature of the microbial

population and the physical environment should be taken into considerations

when screening for microorganisms to be applied (Adams, et al., 2015).

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2.2. Contaminants that can be Treated with Biodegradation

2.2.1. Heavy Metals

One of the most hazardous components in leachate is heavy metals. Heavy

metals from leachate are persistent pollutants in soil, especially when landfills

lack liners and basic structural system that prevents overflow of leachate beyond

restricted areas (Fauziah, et al., 2017). There is a growing concern regarding the

build-up of heavy metals in soil and ground water. Different kinds of wastes are

responsible for the presence of heavy metals in the landfills. Sources such as

electronic waste, painting waste and used batteries increase heavy metals content

in landfills (Adeolu, et al., 2011). The concentrations of heavy metals, like many

organic contaminants, are found to be variable from site to site as well as with the

age of landfill. The landfill passes through a series of stages in terms of chemical

and biochemical reactions until it "stabilizes” (Ray, et al., 2015). Among the

various components of the leachate, heavy metals are significantly important

because soil is a main sink for metals and can induce associated toxic impacts on

the soil ecosystem. However, the situation may be problematic because metals

are not chemically and microbiologically degraded like most organic

contaminants that are oxidized by microbial interactions to CO2. Hence, the

inability to degrade is the reason for the long time persistence of total metals

concentration whenever the soil core is contaminated with metal-induced

leachate (Adriano, 2003). Even changes in the chemical forms and bioavailability

of the metals occur in metal-contaminated soil. Therefore, excess deposition of

heavy metals to soil poses environmental risk. The most commonly implicated

metals in contaminated sites are arsenic (As), cadmium (Cd), chromium (Cr),

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 lead (Pb), mercury (Hg), nickel (Ni), zinc (Zn) and even iron (Fe) (Evanko, et al.,

1997).

2.2.2. Hydrocarbons

A transparent petroleum-derived liquid that is popularly referred to as petrol

called gasoline is used primarily as a fuel in internal combustion engines. It is

utilized in enormous number of internal combustion engines used in modern

transport and industry and has significant impact on the environment

(Nwankwegu, et al., 2017). During the production and transportation of gasoline,

unsuitable operation and leakage may result in contamination of soil with

petroleum hydrocarbons. Petroleum contamination causes significant

environmental impacts and presents substantial hazards to human health (Xu, et

al., 2010).

2.2.3. Organic Contaminants

Generally, leachate can be simply classified depending on the age of the

landfill into young and old leachate. The concentration of organic pollutants (as

COD) is above 10000 mg/l in young leachate, while COD is below 3000 mg/l in

leachate from landfills older than 10 years (Chang, 1989). Meanwhile, the

BOD5/COD ratio at the level of 0.4 and higher is showed for the former. And

the BOD5/COD ratio at the level of 0.1 and lower is showed for the latter

(Marttinen, et al., 2003). In fact, for all landfills concerned, leachates generated

within most of the time are mixture of young leachate and old leachate.

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 Currently biological method is the main process for the treatment of leachate.

So leachate biodegradability has a direct effect on the types and effectiveness of

leachate treatment processes.

Among the major sources of organic pollutants are Alcanes, the major source

of both normal and cyclic alcanes are petroleum and its derivatives. In addition

to petroleum, food grade oils also contaminate some soils; however both their

spread and toxicity are much smaller compared to those of petroleum. One of

the main reasons for the pollution by petroleum products globally is the fact that

the biggest oil producers are not the biggest consumers which means that huge

amounts of oil have to be transported. (Karamanev, 1999)

2.3. Advantages and limitations of Bioaugmentation

Bioaugmentation process will result in an increase in the biodegradability of

municipal solid waste (MSW) and a high degree of waste stabilization. However, if the

MSW contains a high proportion of easily digestible materials, the increased level of

biodegradation associated with leachate recirculation can result in the imbalance in the

growth rates of fast-growing acid genic bacteria and slow-growing methanogens in the

first phase of MSW decomposition. Bioaugmentation are highly effective in the

treatment of young landfill leachate, while their impact is reduced in old leachate

treatment due to the resistance of contaminants (Hasar, et al., 2009). It has been

realized that biodegradation mechanism depends upon the age and origin of the

landfill, and the type and operation of the treatment system (Hasar, et al., 2009).

Leachate recirculation is a more efficient method of increased levels of biodegradation to

obtain higher methane yields and lower treatment costs (He, et al., 2005).

Bioaugmentation has been proposed as an effective and low-cost bioremediation

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 technology to remove POPs, such as simazine and atrazine, and to minimize their

dispersion to non-agricultural environments. (V, et al., 2009).

2.4. Microorganisms used in Landfill Leachate Bioaugmentation

Table 1. Functional Microbial Ecology in LFL treatment processes (Zhang, et al., 2016)

Processes LFL parameters Running Functional Related Phylum

and (mg l-1) Condition microbes and and Percentage

references percentage

(Class, genus,

species)

Upflow COD: 12350-47800 pH: 4.5 Methanosaeta Euryarchaeota

hybrid bed NH3-N:1500-2680 T: 35±3˚C

Aged COD:200-3200 T: 10-20˚C Pseudomonadaceae Pseudomonadaceae

refuse BOD5:200-320 , , Bacteriodetes,

bioreactor NH4-N:1700-2700 Xanthomonadaceae DeinococcusTherm

TN:1800 -2 , Alcaligenaceae, us,

Sphingomonadacea Gemmatimonadetes

e, ,

Rhodobacteraceae, Planctomycetaceae

Hyphomicrobiacea

e, Rhodocyclaceae,

Comamonadaceae,

Flavobacteriaceae,

Flexibacteraceae,

Cryomorphaceae,

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 Crenotrichaceae,

Deinococcaceae,

Trueperaceae,

Gemmatimonadace

ae, unclassified,

Nitroso-bacteria,

Nitro-bacteria

Aged COD:1925-3100 H:7.6e8.2 AnAOB Proteobacteria

refuse BOD5:200-320 T: 30 ± 1 C (0.11%e0.52%) (34.9%-46.4%),

bioreactor VFA:130-1240 Nitrosomonas, Nitrospira (0.05%

Nitrosospira, and -0.17%),

Nitrosococcus Chloroflexi

(0.49%-1.78%), (14.7%-17.2%),

Nitrobacter Actinobacteria

(0.05%), Nitrospira (7.2%-16.8%),

(0.05%-0.17%), Bacteroidetes

g-Proteobacteria (3.0%-10.3%),

(12.4%), b- Gemmatimonadetes

Proteobacteria . (2.3%-6.8%),

(11.3%), Firmicutes (2.4%

Achromobacter, -7.3%),

Azoarcus, Planctomycetes

Comamonas, (3.19%-5.37%)

Halomonas,

Hyphomicrobium,

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 Neisseria,

Pseudomonas,

Rhodobacter,

Rhodoplanes,

Thauera, and

Thiobacillus,

Flexibacter,

Flavobacterium,

Sphingobacterium,

Bacillus, Pirellula,

Rhodopirellula, and

Phycisphaera;

Candidatus

Kuenenia (less than

0.01%)

Unclassified

(2.36%)

ANAMMO COD:303-954 pH:7.5-8.0 Bacillus sp. (45%), Firmicutes,

X DOC:288 T: 30 ± 1˚C Paenibacillus sp. Bacteroidetes

(anaerobic AHS:228 (25%),

ammonium NH3eN:506-885 Staphylococcus

oxidation) NO2 --N: 441-1011 sp. (13%),

reactor Bacteroides sp

AnDMBR COD:13000±750 pH:7.6±0.2 Clostridia (27.9%), Firmicutes

(anaerobic VFA:7133.0±1027. T: 37 ± 1 C Bacteroidia (29.7%),

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dynamic 8 (20.2%), Bacilli, Bacteroidetes

membrane) NH3eN:3199.4±13 Erysipelotrichi (27.9%), TM6

6.6 Minor, Mollicutes, (31.3%);

Sphingobacteriia, Chloroflexi,

VadinHA17, SB-1, Actinobacteria and

Caldilineae, Proteobacteria

Anaerolineae, (6.06%);

Acidimicrobiia, Synergistetes,

Actinobacteria, b- Planctomycetes,

Proteobacteria, OD1, OP9 and RF3

Synergistia, (2.48%);

Planctomycetacia, Lentisphaerae,

Lentisphaeria, Tenericutes and

Mollicutes, Spirochaetae

Spirochaetes (2.56%);

Methanomicrobia Euryarchaeota

(98.6%), (99.9%)

Methanobacteria,

Thermoplasmata

PN-SBR COD:8545±2120 pH:8.34±0. AOB (increase Bacteriodetes 52%,

(partial BOD5:746±96 33 from 5% to 33%) Proteobacteria

nitritation- VFA:130-1240 T: 35 C NOB (decrease

sequencing NH4 þ-N: 2479 ± from 2.5% to lower

batch 314 than 0.25%)

reactor) TN:2910±363 Alphaproteobacteri

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 Alkalinity (HCO3 - a and

): Betaproteobacteria

2543 ± 467

Stabilizatio TCOD:2529±603 pH:9.16±0. Methanogenic Euryarchaeota

n SBOD5:820±300 1 Archaea groups Proteobacteria

pond NH4 þ-N:920 ± 94 T:26.7±3.1˚ (25%), SRB groups

system C (25%),

DO: 0.5 ± Nitrosomonas,

0.2 Nitrobacter and b-

proteobacteria

(10%),

Pseudomonas sp

Microbes are essential in bioreactor for degrading organic pollutants, removing

nitrogen and producing energy. The interactions between microbes are affected by the

bioreactor's running condition, then influence the bioreactor's performance. The

knowledge of microbial interactions is necessary for engineers to operate the

bioreactor in an efficient way. LFL containing high strength ammonium and complex

organic substances stimulate highly diverse microbial community, which make the

microbial interaction more complicate. High strength of ammonium is still the major

concern in LFL biotreatment processes. ANAMMOX process has been proved to be a

promising way for nitrogen removal from LFL. The nitrogen loading rate of

ANAMMOX reactor in LFL treatment (<2 kgN/m3/d) is lower than that in sludge

digestion wastewater (10kgN/m3/d) .One of influencing factors is organic substances.

The effects of organic substances can be (i) toxic to AnAOB and inhibiting the

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 ANAMMOX activity; (ii) improving the growth of heterotrophic bacteria that compete

living space and substrates with AnAOB. In LFL, organic substances are composed of

VFAs, AHS and XOCs. The effects of them on ANAMMOX reaction, especially the

effects of AHS, are not clear enough. Biodegradable fractions of organic substances in

LFL can stimulate the growth of heterotrophic denitrifying bacteria and influence the

interactions of microbes responsible for nitrogen removal. It is necessary to investigate

the effects of organic substances in LFL on the interaction between AnAOB and

heterotrophic denitrifying bacteria. The knowledge will be helpful for managing the

AnAOB and heterotrophic denitrifying bacteria in good cooperation, improving nitrogen

removal efficiency through multiple ways simultaneously, and reducing the LFL

treatment cost (Zhang, et al., 2016).

2.5. Methodology of Bioaugmentation

In industry, often the biomass present needs reinforcements to increase its

degradation efficiency. Bioaugmentation is the method currently in use. It is the addition

of pollutant-degrading microorganisms (natural/exotic/acclimatized/genetically

engineered) to augment the biodegradative capacity of indigenous microbial populations.

For enhancement of biodegradation, bioaugmentation (which is the process of

adding microorganisms with the potential of pollution biodegradation to the bio-slurry

reactor) was applied and results of the experiment show that bioaugmentation may be

considered as an effective method to enhance the bioremediation (Nasseri, et al., 2010).

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 Leachate sample
Collection and
Chemicals

Microorganism
Acclimation and
Isolation

Identification of
isolated Strains

Bioaugmentation Degradation
with Domesticated Bacterial
Strains

Data Analysis

Figure 1. Flow diagram of Landfill leachate Bioaugmentation

The samples taken from storage facilities contain many different strains of

bacteria, so the particular strain needed must be isolated. The method of isolation is to

create an environment containing the organic compounds of interest and see which

strains are able to survive and grow in their presence.

After the candidate strains are isolated, they are tested to see which strains in the

processing of degradation consume the most oxygen, oxidize the target compound, and

produce the largest quantities of gas. The next phase of the process is to remove minor

strains to increase overall efficiency.

From the sample strains remaining, the concentration of the target compound can

be increased or more of these strains can be isolated to obtain the desired activity. A
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 concern with bioaugmentation is the saprophytic bacteria, which is found in the majority

of the strains isolated. Some of the bacteria, under certain conditions, are pathogens that

can cause infection. The safety method used to combat this problem is to subject the

strains to antibiotics, to see if these remedies will work.

2.6. Orthogonal Experimental Design

The orthogonal array (often referred to as the Taguchi method) is a well-known

technique that provides a systematic and efficient methodology for process optimization.

Instead of having to test all possible combinations, this method tests pairs of

combinations and uses only a fraction of all possible factor or level combinations to

reduce the number of experiments, which allows the simultaneous effect of several

process parameters. This method has been used to achieve the best result under the given

removal conditions and to improve the bioaugmentation efficiency. The quantitative

evaluation and statistical analysis of the effects of degradation conditions were

investigated through an orthogonal experiment design (Yu, et al., 2014).

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 Chapter III

Synthesis of Current Research

3.1. Recent study on Biodegradation of Landfill leachate by Bioaugmentation

In a recent study where mature landfill leachate, which is characterized by a high

concentration of ammonia nitrogen (NH3-N) and humic acid (HA) was studied where

bioaugmentation technology is applied in landfill leachate degradation by introducing a

domesticated NH3-N and HA resistant bacteria strain, which was identified as Bacillus

cereus (abbreviated as B. cereus Jlu) and Enterococcus casseliflavus (abbreviated as E.

casseliflavus Jlu), respectively. The isolated strains exhibited excellent tolerant ability for

NH3-N and HA and improved the COD (chemical oxygen demand), NH3-N and HA

removal rate, and efficiency of bioaugmentation degradation of landfill leachate. Only 3

days was required for the domesticated bacteria to remove about 70.0% COD, compared

with 9 days’ degradation for the undomesticated (autochthonous) bacteria to obtain a

similar removal rate. An orthogonal array was then used to further improve the COD and

NH3-N removal rate. Under the optimum condition, the COD removal rate in leachate by

using E. casseliflavus Jlu and B. cereus Jlu increased to 86.0% and 90.0%, respectively,

after 2 days of degradation. The simultaneous removal of NH3-N and HA with more than

50% and 40% removal rate in leachate by employing the sole screened strain was first

observed. Considering the effectiveness, energy consumption, and operability,

bioaugmentation treatments of unsterilized landfill leachate were performed by using E.

casseliflavus Jlu and B. cereus Jlu. Four factors, including temperature (A), pH (B),

inoculum size (C), and phosphorus supplement (D), were investigated at five levels to

optimize the bioaugmentation treatment of landfill leachate by using E. casseliflavus Jlu

and B. cereus Jlu based on orthogonal experimental design (Yu, et al., 2014).

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 Another investigation was designed to evaluate the effects of bioaugmentation on

maintaining the system stability under shock loading conditions, standardizing the

effluent, and improving the sludge settlement. In this study, impacts of bioaugmentation

with the best isolated microorganism on the system performance facing sudden toxic

shock were investigated after acclimatizing the system with phenol, isolating the phenol

degrading microorganisms, and selecting the best phenol degrading strain. Results

indicated that this method improved the efficiency of the system under shock loading

from 30% to 94% and SVI from 333 ml/g to 80 ml/g. The system efficiency was located

in an average range of 99.4-99.9%. The sludge growth was good, even under high organic

loading rates after bioaugmentation (Amiri, et al., 2007).

From a study where a landfill leachate is treated, after mixing with chemical

manufacturing wastewaters, by activated carbon adsorption, excellent treatment

efficiency was consistently achieved under a variety of operating conditions: wastewater

TOC, feed rate, hydraulic retention time, MLSS, organic loading, temperature, and cycle

time. Biodegradation rates for some of the more persistent wastewater constituents were

enhanced in batch bioreactors which were supplemented with strains of bacteria isolated

from the landfill site (Ying, et al., 1986).

3.2. Major findings, contradictions and confirmations across the studies

From the study by Yu, et al., the success of bioaugmentation depends, to a large

degree, on the ability of the introduced microbes to survive and display their activities in

the mixed culture The study shows that the maximum COD, NH3-N, and HA removal

efficiencies were much higher by using domesticated bacteria compared with those of

autochthonous bacteria. By employing orthogonal design in the methodology, the results

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 showed that the bioaugmentation efficiency was further improved by the orthogonal

experimental design. (Yu, et al., 2014).

In a study by Amiri, et al., application of bioaugmentation resulted in reducing the

inhibitory action of phenol to activated sludge activity, increasing system stability during

the shock loading condition, standardizing the effluent, and improving activated sludge

settling. It was found that bioaugmentation is a practical means to increase the resistance

of a Conventional Activated sludge (CAS) system against shock loadings of recalcitrant

compounds, and to remove hazardous compounds according to standards recommended

(Amiri, et al., 2007).

In a study of the treatment of a mixed landfill leachate and chemical industry

wastewater in SBRs it was concluded that "supplementations of the right kinds of bacteria

were shown to have enhanced biodegradation rates for many of the more persistent

wastewater constituents (Ying, et al., 1986).

Vogel stated that engineering design requires an understanding of the parameters

that control the bioaugmentation process. He also said that a good engineering design

needs to be applied to bioaugmentation process design at the experimental level.

Bioaugmentation, according to him, clearly provides certain advantages over

biostimulation in cases where pollutant toxicity or a lack of appropriate microorganisms

is important (Vogel, 1996).

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 Chapter IV

Applications and Implications

4.1. Remodeled Flow Diagram for Bioaugmentation of Landfill leachate

Leachate Sample Collection and Chemical

Analysis

Microorganism Acclimation and Isolation

Identification of Isolated Strains

Determination of Optimum Conditions for

Bioaugmentation

Bioaugmentation Degradation with

Domesticated Bacterial Strains in an
ANAMMOX Bioreactor

Data Analysis

Figure 2. Remodelled Flow diagram of Bioaugmentation of Landfill leachate

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 Leachate Sample Collection and Chemical Analysis

Prior to the experimental activities, the first step was to conduct a review of literature

on raw leachate samples from landfills to obtain an in-depth knowledge of pollutants

distribution and the physicochemical properties of the sampled leachate. The study will

use two sources of soil samples. The first soil source was landfill soil polluted with raw

leachate, and used for the isolation of persistent bacteria in the polluted soil. Another

source of soil will be the excavated soil samples from non-contaminated garden and will

be used for the bioremediation of the induced experimental setup. Collection of soil

samples (0–20 cm from surface) will be carried out according to 2004 Standard

guidelines for conducting terrestrial soil-core microcosm test. Portion of soil samples

meant for the experimental step up will be oven dried and will be used for metal analyses

to obtain the initial metal concentrations.

Microorganism Acclimation and Isolation

Usually, microbe is isolated by inoculating domesticated bacteria with the

contaminated soil. To augment the heavy metal degrading bacteria a two layered medium

comprising lower layer of minimal nitrogen medium (nitrogen source) covered with

leachate layer (ammonium source) can be used. The bacterial identification can be done

by standard biochemical tests, fatty acid analysis or by rDNA analysis. Care should be

taken that the selected organisms are non-pathogenic.

Identification of Isolated Strains

Further confirmation of the biodegradation ability of the selected strains should be

done by inoculating leachate laden sterile soil with the selected culture and incubating it

for about 15 days. Total bacterial count of the samples can be monitored to select the

most efficient strains.

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 Determination of Optimum conditions for Bioaugmentation through Orthogonal

Experimental Design

According to the preliminary experiment and references, four factors were

investigated to optimize the bioaugmentation, including temperature, pH, inoculums size,

and the phosphorus supplement, which were found to have great effects on the

degradation efficiency of landfill leachate. To simplify the experiments, the interactions

between these four factors were not taken into account. The orthogonal table will be

designed, in which two blank columns will be designated for the error evaluation. The

four factors will be at five levels as follows: temperature of 20˚C, 25˚C, 30˚C, 37˚C, and

40˚C; pH of 5, 6, 7, 8, and 9; inoculum size of 0, 0.1, 0.2, 0.3, and 0.4 ml; and phosphorus

supplement of 0, 0.6, 1.2, 1.8, and 2.4 mg/ml. The orthogonal experimental table with

investigation factors, the corresponding levels, and the designated boundary

values will be shown in Table form. The data analysis will be conducted after the

orthogonal array. The process/system design phase involves deciding the best

values/levels for the control factors. The signal-to-noise (S/N) ratio, which results in

minimization of the quality characteristic variation due to the uncontrollable parameter, is

an ideal metric for that purpose. The S/N ratio also indicates the degree of predictable

performance in the presence of noise factors. S/N represents the magnitude of the mean of

a process compared with its variation, serves as objective functions for optimization, and

helps in data analysis and prediction of optimum results. For analyzing the data of the

orthogonal experiment, the S/N number will be used to measure the quality characteristics

deviating from the desired values will be calculated for each experiment by using

Assistant for Orthogonal Experimental Design. The average S/N value will be calculated

for each factor and level. The mean S/N ratio for each variable at each level will be

calculated by averaging the S/N ratios for all the experiments. The S/N value ratio will be

22 | P a g e
 classified into the smaller-the-better type, the larger-the-better type, and nominal-the-best

type, based on the type of objective function.

Bioaugmentation Degradation with Domesticated Bacterial Strains in a

ANAMMOX Bioreactor

Detailed characteristics of the raw leachate and operative scheme of Anaerobic

Reactor (AR) could be seen in the previous report by (Liang, et al., 2008). At time of

continuous running, raw leachate will be fed to the reactor for the removal of

contaminants and acquirement of mixture with proper nitrite/ammonium ratio. The

effluent from the unit will be further purified in the AR. Finally the AR effluent will be

fed to USIS for advanced treatment. AR, a bench scale fixed biofilm reactor, will be

strictly airproofed for preventing oxygen introduction from air and coated with silver

paper for avoiding illumination. During the entire operational period, temperature in the

reactor will be maintained at 30 ± 1˚C, the pH value will be controlled at the range of

7.5–8.0, and the oxidization–reduction potential (ORP) will be continuously monitored

with an ORP meter (Liang, et al., 2008).

Data analysis

The removal rates of COD, HA, and NH3-N will be used to evaluate the effectiveness

of bioaugmentation treatment of landfill leachate in this study. The COD concentration

will be determined by the dichromate closed refluxed and colorimetric assay method

according to the Standard Methods for the Examination of Water and Wastewater.

Samples will be diluted (if necessary) and will be added to the standard COD ampules.

COD samples will be then incubated. After allowing the COD tubes to cool to room

temperature, COD levels will be determined by measuring the absorbance of the digested

23 | P a g e
 assay solution at λ = 600 nm on a spectrophotometer. A 1 cm path-length will be

maintained by using a standard cuvette. The NH3-N concentration will be measured by a

reagent colorimetric method. The HA concentration will measured by an ultraviolet–

visible spectrometer model at 300 nm (HA exhibited the maximum UV adsorption at this

wavelength). The bacterial cell biomass will be detected as the optical density of samples

at 600 nm. All experiments will be performed in triplicate and the average values will be

informed (Yu, et al., 2014).

24 | P a g e
 Chapter V

Summary and Conclusions

5.1. Summary and Conclusions

The heterogeneous nature of leachate is often a concern because it may contain

monocyclic aromatic hydrocarbons, xenobiotic organics, polychlorinated biphenyls

(PCB), heavy metals or metalloids, and other pollutants. The different technologies

for biodegradation (e.g. Biostimulation, Bioremediation, and Bioaugmentation) were

discussed and evaluated. Bioaugmentation is selected as an efficient technique for the

biodegradation of landfill leachate. Also, the nature of the different contaminants that

can be treated with bioaugmentation were discussed in this paper.

A review and analysis from different recent studies of bioaugmentation was

conducted to develop a method of implementing an efficient bioaugmentation of

landfill leachate. It is found that implementing orthogonal experimental design in

understanding of the parameters that control the bioaugmentation process increases

the efficiency of the bioaugmentation process. Also, it is found that ANAMMOX

Bioreactor is the most effective reactor in the bioaugmentation of landfill leachate.

From these analyses, a method of implementing an efficient bioaugmentation of

landfill leachate was developed integrating orthogonal experimental design in the

process.

25 | P a g e
References

Adams G. [et al.] Bioremediation, Biostimulation and Bioaugmentation: A Review

[Journal]. - 2015.

Adeolu A.O. [et al.] Assessment of groundwater contamination by leachate near a municipal

solid waste landfill [Journal]. - [s.l.] : African Journal of Environmental Science and

Technology, 2011.

Adriano D.C. Trace elements in terrestrial environments: Biogeochemistry, Bioavailability

and Risks of Metals [Book]. - [s.l.] : Springer, 2003. - Vol. 2nd Ed..

Amiri F., Yaghmaei S. and Samie S. Application of Bioaugmentation Technology to

Improve the activated Sludge Treatment process in removal of Aromatic compounds

[Journal]. - [s.l.] : Iranian Journal of Chemical Engineering, 2007. - Vol. IV.

Ayotamuno M. J. [et al.] Bioremediation of a crude-oil polluted agricultural-soil at Port

Harcourt, Nigeria [Journal] // Applied energy. - 2006. - pp. 1249-1257.

Chang J. E. Treatment of landfill leachate with an upflow anaerobic reactor combining a

sludge bed and a filter [Journal] // Water Science Technology. - 1989.

Costa R.S. [et al.] Critical perspective on the consequences of the limited availability of

kinetic data in metabolic dynamic modelling [Journal] // IET System Biology. - 2011. - pp.

157-163.

Das A. J. and Kumar R. Bioremediation of petroleum contaminated soil to combat toxicity

on Withania somnifera through seed priming with biosurfactant producing plant growth

promoting rhizobacteria [Journal] // Journal of environmental management. - 2016. - pp. 79-

86.

26 | P a g e
Dias R. L. [et al.] 6. Dias, R. L., Ruberto, L., Hernández, E., Vázquez, S. C.,

BalboBioremediation of an aged diesel oil-contaminated Antarctic soil: evaluation of the “on

site” biostimulation strategy using different nutrient sources [Journal] // International

Biodeterioration & Biodegration. - 2012. - pp. 96-103.

en.m.wikipedia.org/wiki/Biostimulation [Report].

Evanko C.R. and Dzombak D.A Remediation of metals-contaminated soils and

groundwater [Book]. - 1997.

Fauziah S.H. [et al.] Assessing the Bioaugmentation of Individual Isolates from Landfill on

Metal-Polluted Soil [Journal]. - 2017.

Goryanin I. and Demin O. Kinetic Modelling in Systems Biology [Journal]. - 2009.

Hasar H. [et al.] Stripping/Flocculation/Membrane Bioreactor/Reverse Osmosis Treatment

of Municipal Landfill Leachate [Journal] // Journal of Hazardous Materials. - 2009.

He R., Shen D. and Wang J. Biological degradation of MSW in a methanogenic reactor

[Journal]. - 2005. - p. 3666.

Kanmani S. and Gandhimathi R. Assessment of heavy metal contamination in soil due to

leachate migration from an open dumping site [Journal]. - [s.l.] : Applied Water Science,

2013.

Karamanev Dimitre G. Biodegradation of Soil Contaminants [Book]. - 1999.

Killham [Journal]. - 1994.

Klipp E. [et al.] Systems Biology in Practice [Journal]. - 2005.

Liang Z., Liu J.X. and Li J. Decomposition and mineralization of aquatic humic substances

(AHS) in treating landfill leachate using the Anammox process [Journal]. - 2008.

27 | P a g e
Marttinen S.K., Kettunen R.H. and Rintala J.A. Occurrence and removal of organic

pollutants in sewages and landfill leachates [Journal] // Science Total Environment. - 2003.

Morgante V. et al Bioaugmentation with Pseudomonas sp. strain MHP41 promotes simazine

attenuation and bacterial community changes in agricultural soils [Journal]. - 2009. - p. 13.

Nasseri S. [et al.] INFLUENCE OF BIOAUGMENTATION IN BIODEGRADATION OF

PAHs-CONTAMINATED SOIL IN BIO-SLURRY PHASE REACTOR [Journal]. - 2010.

Nasseri S. [et al.] INFLUENCE OF BIOAUGMENTATION IN BIODEGRADATION OF

PAHs-CONTAMINATED SOIL IN BIO-SLURRY PHASE REACTOR. [Journal]. - 2010.

Nwankwegu A. S. and & Onwosi C. O. Bioremediation of gasoline contaminated

agricultural soil by bioaugmentation [Journal] // Environmental Technology & Innovation. -

2017. - pp. 1-11.

Nwankwegu A. S. and Onwosi C. O. Bioremediation of gasoline contaminated agricultural

soil by bioaugmentation. [Journal] // Environmental Technology & Innovation. - 2017.

Porto A. [et al.] Biodegradation of Pesticides [Journal]. - 2012.

Porto A. [et al.] Biodegradation of Pesticides. [Journal]. - 2012.

Poyntner Caroline Bioremediation of waste gas and soil by black extremotolerant fungi

[Journal]. - 2014. - p. 111.

Rahman K. S. M. [et al.] Bioremediation of gasoline contaminated soil by bacterial

consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant [Journal] //

Bioresource technology. - 2002. - pp. 25-32.

Ray C. and Chan P. HEAVY METALS IN LANDFILL LEACHATE [Journal]. - [s.l.] :

1986 Gordon and Breach, Science Publishers, Inc, 2015.

28 | P a g e
Renou S. [et al.] Landfill leachate treatment: Review and opportunity [Journal]. - [s.l.] : J

Hazard Matter, 2007.

Reyes Milagros Philippine Petroleum Industry [Journal]. - 1997.

Ruo H., Dong-sheng S. and Jun-qin W. Biological degradation of MSW in a methanogenic

reactor using treated leachate recirculation [Journal]. - [s.l.] : Process Biochemistry, 2005.

Silva Elisabeth , Falho, Arsenio M [Journal]. - 2004.

Sumaiya A. [et al.] Assessment of Heavy Metals in Leachate of an Unlined Landfill in the

Sultanate of Oman [Journal]. - [s.l.] : International Journal of Environmental Science and

Development, 2014.

Tahseen Sayara. et al Bioremediation of PAHs-contaminated soil through composting:

Influence of Bioaugmentation and Biostimulation on contaminant biodegradation [Journal]. -

2011.

V Morgante, A Lopez and C Flores Bioaugmentation with Pseudomonas sp. strain MHP 41

promotes simazine attenuation and bacterial community changes in agricultural soils

[Journal]. - 2009.

Vogel T. M. Bioaugmentation as a soil bioremediation approach [Journal] // Current opinion

in biotechnology. - 1996. - pp. 311-316.

wikipedia.org/Biostimulation [Report].

Xu Y. and Lu M. Bioremediation of crude oil-contaminated soil: comparison of different

biostimulation and bioaugmentation treatments [Journal]. - 2010.

Xu Y. and Lu M. Bioremediation of crude oil-contaminated soil: comparison of different

biostimulation and bioaugmentation treatments. [Journal]. - 2010.

29 | P a g e
Ying W.C. [et al.] Biological treatment of a landfill leachate in sequencing batch reactors

[Journal]. - 1986.

Yu D. [et al.] Bioaugmentation Treatment of Mature Landfill Leachate by New Isolated

Ammonia Nitrogen and Humic Acid Resistant Microorganism [Journal]. - 2014.

Yu D. [et al.] Bioaugmentation Treatment of Mature Landfill Leachate by New Isolated

Ammonia Nitrogen and Humic Acid Resistant Microorganism [Journal]. - [s.l.] : J.

Microbiol. Biotechnol, 2014.

Zhang D. [et al.] Microbes in biological processes for municipal landfill leachate treatment:

Community function and interaction [Journal]. - [s.l.] : International Biodeterioration &

Biodegradation, 2016.

Zhongyun M Bioremediation of Petroleum Hydrocarbon Contaminated soil using

Indigenous Culture [Journal]. - 1998. - p. 111.

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 RUBRICS for SEMINAR PAPERS

Name: Bose, Judy Ann D. Leneses, Catherine B.

Ramos, Melanie B. Suor, Alexa C.

Title of Seminar Paper: BIODEGRADATION OF LANDFILL LEACHATE BY BIOAUGMENTATION

Emerging Competent Mastering

CRITERIA
1–2
Little or no evidence that related
Problem contemporary issues and/or
Clarification professional challenges were taken into
consideration.
No identification of related ethical
considerations.
Impact Analysis
Little or no consideration of the impact
the design will have on contexts beyond
meeting client needs.
Little or no evidence of problem
decomposition.
Design Synthesis:
Concept
Generation
Little or no evidence of organization in
the concept exploration process.
Visuals (charts, tables, Gantt charts,
diagrams, schematics and photos, etc.)
are frequently inappropriate, difficult to
Communication decipher and may even detract from
written communication.
Frequent errors obscure and/or
misrepresent the content.
Points
3–4 5–6
The problem is clearly articulated with
The problem is adequately articulated
well-defined parameters that
but teams may have not fully considered
realistically consider contemporary
related contemporary issues or
issues, along with professional
professional challenges.
challenges.
Relevant ethical considerations are
Relevant ethical considerations are
identified, but not addressed fully in the
identified and addressed in the design.
design.
Some consideration of the impact the
Impacts of the design are examined and
design will have on two or more relevant
weighed in all relevant contexts.
contexts.
The team decomposes the problem into
a set of sub functions; a set of
The team conducts a cursory problem
subsystems; a sequence of actions;
decomposition process.
and/or the set of primary client
preferences.
Strong evidence that the exploration
The team struggles to organize the
process has been skillfully managed in
exploration process in a way that best
ways that organize and guide the
guides the creative energies and
creative energies and technical expertise
technical expertise of the team.
of the team.
Visuals (charts, tables, Gantt charts,
Visuals (charts, tables, Gantt charts,
diagrams, schematics and photos, etc.)
diagrams, schematics and photos, etc.)
generally support the written
are clear, concise, and have been chosen
component, but some may be overly
for their ability to support and extend
complex/simplistic or unclear due to
the written component.
improper resolution.
Errors exist, but do not distract from or Writing is polished, professional, and
misrepresent the content. virtually error free.
TOTAL