**Meet the editors**

Rolando Chamy is a Biochemical Engineer and Ph.D. in Chemical Engineering from the University of Santiago de Compostela, Spain. He holds a professor chair at the Pontificia Universidad Católica de Valparaíso, and is the Director of the Biotechnology Center of Curauma, a center of applied research, which belongs to the same university. Currently, he also leads the research line

in Renewable Bioresource at Fraunhofer Chile Research. His main lines of research are environmental biotechnology, renewable energy, climate change, and innovation management.

Additionally, he has developed an extensive body of work in the field of applied research, with more than 100 as project director, principal investigator or co-investigator, supported with more than 100 ISI publications and 19 contributions to book chapters.

Francisca Rosenkranz is a Biochemical Civil Engineer (Pontificia Universidad Católica de Valparaíso) and PhD in Biotechnology (Pontificia Universidad Católica de Valparaíso and Universidad Técnica Federico Santa María) with the co-supervision of the University of Santiago de Compostela, Spain. Her specialization areas include biogas production, anaerobic treatment of hard biodegradable compounds and studies of microbial communities in anaerobic systems. Her actual positions are at Fraunhofer Chile Research, as research project coordinator in the bioenergy area, and at Biotechnology Center of Curauma, as Chief of Operations.

Contents

**Preface VII**

**Section 1 Biodegradation of Hydrocarbons 1**

Santiesteban-López

Magdalena Urbaniak

**Digestion 139**

Chapter 1 **Biodegradability of Water from Crude Oil Production 3**

Chapter 2 **Emulsification of Hydrocarbons Using Biosurfactant Producing**

Chapter 3 **Microbial Hydrocarbon Degradation: Efforts to Understand Biodegradation in Petroleum Reservoirs 47**

Chapter 5 **Crude Oil Biodegradation in the Marine Environments 101** Mehdi Hassanshahian and Simone Cappello

Chapter 7 **Advanced Monitoring and Control of Anaerobic Digestion in**

Isabel Natalia Sierra-Garcia and Valéria Maia de Oliveira

**Strains Isolated from Contaminated Soil in Puebla, Mexico 25** Beatriz Pérez-Armendáriz, Amparo Mauricio-Gutiérrez, Teresita Jiménez-Salgado, Armando Tapia-Hernández and Angélica

Álvaro Torres, Fernando G. Fermoso, Bárbara Rincón, Jan Bartacek,

Edixon Gutiérrez and Yaxcelys Caldera

Chapter 4 **Biodegradation of PCDDs/PCDFs and PCBs 73**

**Section 2 Biodegradation and Anaerobic Digestion 137**

Rafael Borja and David Jeison

**Bioreactor Landfills 161**

Chapter 6 **Challenges for Cost-Effective Microalgae Anaerobic**

Mohamed Abdallah and Kevin Kennedy

## Contents

## **Preface XI**

Chapter 8 **Sustainable Post Treatment Options of Anaerobic Effluent 191**

Abid Ali Khan, Rubia Zahid Gaur, Absar Ahmad Kazmi and Beni Lew

Preface

plied to the optimization of biodegradation processes.

The book has three main sections classified as:

This book contains a collection of different research activities where several technologies have been ap‐

**A) Hydrocarbons Biodegradation** This section includes chapters that mention the following topics: Bio‐ logical and physicochemical systems as efficiency alternatives for the treatment of water from crude oil production; Hydrocarbons degradation capacity and bio-surfactant production by microorganisms iso‐ lated from hydrocarbon-contaminated soil as efficient candidates for the recovery of soils; Implementa‐ tion of culture-independent molecular methods to allow the access to the microbial diversity and metabolic potential of microorganisms and bring novel information about microbial diversity and new pathways involved in biodegradation processes taking place in petroleum deposits; Biodegradation of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlori‐

**B) Biodegradation and anaerobic digestion**This section contains the followings topics: Microalgae anaerobic digestion; Monitoring and Control of Anaerobic Digestion in Landfills; Anaerobic digestion post-treatment; Anoxic and anaerobic biodegradability characteristics of sulfamethoxasole; and the last topic: Effects of this compound on microbial communities and different processes involved in manure biodegradation, both the emissions that are produced as well as how biodegradation can be used to

**C) Biodegradation and sustainability**The last sections contain the following: Fundamental aspects of corrosion of magnesium-based alloys in bodily fluids and review of the various techniques that can be used to tune up their degradation rate. The time-dependent evolution of their mechanical properties during the biodegradation process is also outlined; Nitrogen Budget for a Commercial Recirculating Aquaculture Facility; Application of the treatment sequence build up by ozonation followed by biode‐ gradation. This combined treatment is tested in two cases: the first one uses a model with water samples containing some chlorinated phenol derivatives (4-chlorophenol and 2,4-dichlorophenol) and the sec‐ ond case considers real wastewater samples obtained from the pulp and paper industry (Kraft process in bleaching); Emerging Trend in Natural Resource Utilization for Bioremediation of Oil - Based Drill‐ ing Wastes in Nigeria; and the last topic is an overview of the current biodegradable polymer matrices and some of the most used reinforcements are described as well as the properties and applications of

**Rolando Chamy and Francisca Rosenkranz**

Pontificia Universidad Católica de > Valparaíso, Chilr

Pontificia Universidad Católica de > Valparaíso, Chile

Biotechnology Center of Curauma

School of Biochemical Engineering

nated biphenyls (PCBs) and the last topic; crude oil biodegradation in marine environments.

treat both the manure and the residues from manure management.

the bio-composites obtained.

Chapter 9 **Determination of Anaerobic and Anoxic Biodegradation Capacity of Sulfamethoxasole and the Effects on Mixed Microbial Culture 223**

Zeynep Cetecioglu, Bahar Ince, Samet Azman, Nazli Gokcek, Nese Coskun and Orhan Ince

	- **Section 3 Biodegradation and Sustainability 275**

## Preface

Chapter 8 **Sustainable Post Treatment Options of Anaerobic**

Chapter 9 **Determination of Anaerobic and Anoxic Biodegradation**

Chapter 10 **Biodegradation in Animal Manure Management 251**

Abid Ali Khan, Rubia Zahid Gaur, Absar Ahmad Kazmi and Beni Lew

Zeynep Cetecioglu, Bahar Ince, Samet Azman, Nazli Gokcek, Nese

Matthieu Girard, Joahnn H. Palacios, Martin Belzile, Stéphane

**Capacity of Sulfamethoxasole and the Effects on Mixed**

**Effluent 191**

**VI** Contents

**Microbial Culture 223**

Coskun and Orhan Ince

Godbout and Frédéric Pelletier

**Section 3 Biodegradation and Sustainability 275**

**Aquaculture Facility 341** S. Sandu and E. Hallerman

Philip D. Shekwolo

Chapter 11 **Methods for Separation, Recycling and Reuse of Biodegradation Products 277**

Ganapati D. Yadav and Jyoti B. Sontakke

Chapter 12 **Biodegradation and Mechanical Integrity of Magnesium and Magnesium Alloys Suitable for Implants 313** S. González, E. Pellicer, S. Suriñach, M.D. Baró and J. Sort

Chapter 13 **Biodegradation of Nitrogen in a Commercial Recirculating**

Chapter 15 **Emerging Trend in Natural Resource Utilization for**

Chapter 14 **Aerobic Biodegradation Coupled to Preliminary Ozonation for the Treatment of Model and Real Residual Water 365** P. Guerra, J. Amacosta, T. Poznyak, S. Siles, A. García and I. Chairez

Chapter 16 **Biocomposites: Influence of Matrix Nature and Additives on the Properties and Biodegradation Behaviour 433**

Derval dos Santos Rosa and Denise Maria Lenz

**Bioremediation of Oil — Based Drilling Wastes in Nigeria 389** Iheoma M. Adekunle, Augustine O. O. Igbuku, Oke Oguns and

This book contains a collection of different research activities where several technologies have been ap‐ plied to the optimization of biodegradation processes.

The book has three main sections classified as:

**A) Hydrocarbons Biodegradation** This section includes chapters that mention the following topics: Bio‐ logical and physicochemical systems as efficiency alternatives for the treatment of water from crude oil production; Hydrocarbons degradation capacity and bio-surfactant production by microorganisms iso‐ lated from hydrocarbon-contaminated soil as efficient candidates for the recovery of soils; Implementa‐ tion of culture-independent molecular methods to allow the access to the microbial diversity and metabolic potential of microorganisms and bring novel information about microbial diversity and new pathways involved in biodegradation processes taking place in petroleum deposits; Biodegradation of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlori‐ nated biphenyls (PCBs) and the last topic; crude oil biodegradation in marine environments.

**B) Biodegradation and anaerobic digestion**This section contains the followings topics: Microalgae anaerobic digestion; Monitoring and Control of Anaerobic Digestion in Landfills; Anaerobic digestion post-treatment; Anoxic and anaerobic biodegradability characteristics of sulfamethoxasole; and the last topic: Effects of this compound on microbial communities and different processes involved in manure biodegradation, both the emissions that are produced as well as how biodegradation can be used to treat both the manure and the residues from manure management.

**C) Biodegradation and sustainability**The last sections contain the following: Fundamental aspects of corrosion of magnesium-based alloys in bodily fluids and review of the various techniques that can be used to tune up their degradation rate. The time-dependent evolution of their mechanical properties during the biodegradation process is also outlined; Nitrogen Budget for a Commercial Recirculating Aquaculture Facility; Application of the treatment sequence build up by ozonation followed by biode‐ gradation. This combined treatment is tested in two cases: the first one uses a model with water samples containing some chlorinated phenol derivatives (4-chlorophenol and 2,4-dichlorophenol) and the sec‐ ond case considers real wastewater samples obtained from the pulp and paper industry (Kraft process in bleaching); Emerging Trend in Natural Resource Utilization for Bioremediation of Oil - Based Drill‐ ing Wastes in Nigeria; and the last topic is an overview of the current biodegradable polymer matrices and some of the most used reinforcements are described as well as the properties and applications of the bio-composites obtained.

> **Rolando Chamy and Francisca Rosenkranz** Biotechnology Center of Curauma Pontificia Universidad Católica de > Valparaíso, Chilr

> School of Biochemical Engineering Pontificia Universidad Católica de > Valparaíso, Chile

**Section 1**

**Biodegradation of Hydrocarbons**

**Biodegradation of Hydrocarbons**

**Chapter 1**

**Biodegradability of Water from Crude Oil Production**

According to Gutiérrez *et al.* (2007) the waters of formation (WOF), are those that are naturally in the rocks and are present before the perforation of the well. Their composition depends on the origin of the water and the modification that could happen as soon as they enter in contact with the environment of the subsoil. WOF must be obtained from the bottom of the well; nevertheless, for costs reason the samples are taken at the surface level, in the head of the well. As they rise in the column from the well up to the surface, their characteristics change due to the changes of pressure, temperature and composition of the gases. For this reason the name adapted for these samples of waters is water associated with crude oil production. Other researchers name these waters as water from petroleum, water from oil field production, oily waters, effluent from the extraction of oil, water from petroleum. In this work they are named

Among the characteristics of WCP are their high content of free and emulsified crude oil and hydrocarbons, suspended solid, H2S and mercaptans (Gutiérrez *et al.,* 2002), aromat‐ ic, poliaromatic and phenols compounds (Rincón *et al.,* 2008), high temperature and high salinity (Guerrero *et al.,* 2005; Li *et al.,* 2005), saturated, aromatics, resins and asphaltenes compounds (SARA) (Díaz *et al.,* 2007), and metal traces Na, Ca, Mg, Fe, Sr, Cr, As and Hg (Gutiérrez *et al.,* 2009). According to García *et al.* (2004) among the pollutants with a major potential impact related to the petroleum industry are polycyclic aromatic hydrocarbons (PAH), voltaic organic compounds (VOC) and total hydrocarbons of the oil (THO). The first ones have high carcinogenic, mutagenic and teratogenic potential in aquatic organ‐ isms; the second ones contribute to the greenhouse effect and are involved in the direct ozone formation on the soil level and indirectly on the acid rain, besides some individual

> © 2013 Gutiérrez and Caldera; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Gutiérrez and Caldera; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Edixon Gutiérrez and Yaxcelys Caldera

http://dx.doi.org/10.5772/56328

**1. Introduction**

Additional information is available at the end of the chapter

waters associated with crude oil production (WCP).

## **Biodegradability of Water from Crude Oil Production**

Edixon Gutiérrez and Yaxcelys Caldera

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56328

## **1. Introduction**

According to Gutiérrez *et al.* (2007) the waters of formation (WOF), are those that are naturally in the rocks and are present before the perforation of the well. Their composition depends on the origin of the water and the modification that could happen as soon as they enter in contact with the environment of the subsoil. WOF must be obtained from the bottom of the well; nevertheless, for costs reason the samples are taken at the surface level, in the head of the well. As they rise in the column from the well up to the surface, their characteristics change due to the changes of pressure, temperature and composition of the gases. For this reason the name adapted for these samples of waters is water associated with crude oil production. Other researchers name these waters as water from petroleum, water from oil field production, oily waters, effluent from the extraction of oil, water from petroleum. In this work they are named waters associated with crude oil production (WCP).

Among the characteristics of WCP are their high content of free and emulsified crude oil and hydrocarbons, suspended solid, H2S and mercaptans (Gutiérrez *et al.,* 2002), aromat‐ ic, poliaromatic and phenols compounds (Rincón *et al.,* 2008), high temperature and high salinity (Guerrero *et al.,* 2005; Li *et al.,* 2005), saturated, aromatics, resins and asphaltenes compounds (SARA) (Díaz *et al.,* 2007), and metal traces Na, Ca, Mg, Fe, Sr, Cr, As and Hg (Gutiérrez *et al.,* 2009). According to García *et al.* (2004) among the pollutants with a major potential impact related to the petroleum industry are polycyclic aromatic hydrocarbons (PAH), voltaic organic compounds (VOC) and total hydrocarbons of the oil (THO). The first ones have high carcinogenic, mutagenic and teratogenic potential in aquatic organ‐ isms; the second ones contribute to the greenhouse effect and are involved in the direct ozone formation on the soil level and indirectly on the acid rain, besides some individual

© 2013 Gutiérrez and Caldera; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Gutiérrez and Caldera; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

compounds are toxic, carcinogenic, mutagenic or bioaccumulative, and the last ones present diverse effects on the flora an fauna.

**2. Results**

the three cuts (WCPC).

WCP.

**2. Results**

the petroleum industry in the last years.

industry in the last year.

**2.1. Origin and composition of the waters associated with the crude oil production**

The WCP samples were obtained from the Ulé tank farm, located on the east coast of Maracaibo Lake, Tía Juana, Zulia state, Venezuela (Figure 1). The water samples come from the segrega‐ tions: Tía Juana light (TJL), Urdaneta heavy (UH), Tía Juana medium (TJM), and the dehydra‐ tions of the Punta Gorda tank farm (Rosa medium-RM), Shell Ulé (F-6/h-7) and lacustrine terminal of La Salina (LTLS). These waters were obtained from the separation of the water associated with the extraction of light crude oil (>31.8ºAPI) WCPL, from the water associated with the extraction of medium crude oil (22ºAPI-29.9ºAPI) WCPM, from the water associated with the extraction of heavy crude oil (10ºAPI-21.9ºAPI) WCPH, classified according to the American Petroleum Institute. Also, water samples were taken from the converged point of

In this investigation was reviewed a several papers from studies conducted at the Universidad del Zulia during 2002 to 2012, to analyze the efficiency of biological and physicochemical systems BR, UASB, SBR and RBC, and the physicochemical treatment as coagulation and flotation (DAF), which have been evaluated to remove COD, hydrocarbons, SARA and phenols, present in the

Biodegradability of Water from Crude Oil Production

http://dx.doi.org/10.5772/56328

5

The instrument used was a matrix register of the treatment, considering criteria like WCP type, system of treatments, operation conditions, organic load, retention times, temperature, pollutant contents and dose of coagulant. The efficiency of the treatments

The WCP samples were obtained from the Ulé tank farm, located on the east coast of Maracaibo Lake, Tía Juana, Zulia state, Venezuela (Figure 1). The water samples come from the segregations: Tía Juana light (TJL), Urdaneta heavy (UH), Tía Juana medium (TJM), and the dehydrations of the Punta Gorda tank farm (Rosa medium-RM), Shell Ulé (F-6/h-7) and lacustrine terminal of La Salina (LTLS). These waters were obtained from the separation of the water associated with the extraction of light crude oil (>31.8ºAPI) WCPL, from the water associated with the extraction of medium crude oil (22ºAPI-29.9ºAPI) WCPM, from the water associated with the extraction of heavy crude oil (10ºAPI-21.9ºAPI) WCPH, classified according to the American Petroleum

The Tables 1, 2, 3 and 4 present the principal characteristics of WCPL, WCPM, WCPH and WCPC, respectively. In general, it is observed that the physicochemical characteristics of the WCP are different depending on the contact of these waters with the crude oil associated. They are waters with high pollutant contents and they do not comply with the Venezuelan environmental regulations to be discharged into water bodies (Gaceta Oficial, 1995). On the other hand, the differences in the characteristics reported by the researchers, might be related to the changes that have been given in the productive processes of the petroleum

The Tables 1, 2, 3 and 4 present the principal characteristics of WCPL, WCPM, WCPH and WCPC, respectively. In general, it is observed that the physicochemical characteristics of the WCP are different depending on the contact of these waters with the crude oil associated. They are waters with high pollutant contents and they do not comply with the Venezuelan environmental regulations to be discharged into water bodies (Gaceta Oficial, 1995). On the other hand, the differences in the characteristics reported by the research‐ ers, might be related to the changes that have been given in the productive processes of

Institute. Also, water samples were taken from the converged point of the three cuts (WCPC).

Figure 1. Geographical location of the Ulé tank farm, Tía Juana Zulia state, Venezuela.

**Díaz**  *et al.* **(2005a)**

**Díaz**  *et al.* **(2005b)**

pH 7.9 8.0 8.3 7.99 NR Alkalinity (mg CaCO3/L) 2933 2215 2670 2412 NR COD soluble (mg/L) 1065.2 799 1400 1105 106.2 Phenols (mg/L) 19.36 1.73 NR 16.8 NR Nitrogen NTK (mg/L) 23.82 28.8 20 21.2 23.82 Phosphorous (mg/L) 1.07 1.0 2.2 1.57 1.07 Hydrocarbons (mg/L) NR 91 224.2 78.0 NR Chlorines (mg/L) NR NR NR NR NR TSS (mg/L) NR NR 104 NR NR VSS (mg/L) NR NR 54 NR NR H and O (mg/L) NR NR 66 100.7 NR

**Gutiérrez** *et al.* **(2012)**

**González**  *et al.* **(2007)**  **Rincón** *et al.* **(2008)**

**Parameters**

**Figure 1.** Geographical location of the Ulé tank farm, Tía Juana Zulia state, Venezuela.

**2.1. Origin and composition of the waters associated with the crude oil production**

was compared considering the parameters COD, phenols, hydrocarbons and SARA.

Gives that the WCP volumes generated in the Ulé tank farm, on the east cost of Maracaibo Lake, Venezuela, belonging to the petroleum industry in Venezuela, would exceed the needs for secondary recovery and the systems of reinjection would be rapidly saturated, different research works were realized to present alternatives to the petroleum industry, to diminish the potential pollutant of WCP.

In this aspect, some proposals for the treatment of WCP are aerobic and anaerobic biological processes, physicochemical treatment and some new technologies as constructed wetlands. Among the anaerobic processes are the batch reactors (BR) and the upflow anaerobic sludge blanket reactors (UASB).

The biological mesophilical and thermophilical anaerobic systems have been successful in the treatment of complex waters, with low, moderate and high organic load (Lettinga, 2001). In the case of UASB, these reactors are outlined by their capacity to retain biomass, to form granular sludge with high properties of sedimentation, to handle high organic load to short hydraulic retention time (HRT), produce biogas and remove high concentration of biodegrad‐ able organic matter (Lepisto and Rintala, 1990; Lettinga, 2005).

On the other hand, the aerobic systems have been efficient for the treatment of wastewater containing chemical compounds resistant to be biodegraded. Among these systems are the sequential biological reactors (SBR), which have showed excellent results in the degradation of toxic compounds present in industry effluents (Díaz *et al.,* 2005a; González *et al.,* 2007). As well as, the rotating biological contactor reactors (RBC), which produce good quality effluents including total nitrification, low costs and ease of operation and maintenance (Behling *et al.,* 2003).

Among the physicochemical treatment applied to reduce the pollutants in wastewater are the dissolve air flotation (DAF) and the coagulation. The most applied products to treat natural water and wastewater by coagulation and flocculation are iron and aluminium salts. However, the cationic polymers have demonstrated their efficiency in the removal of oils and phenols from industrial wastewater (Renault *et al.,* 2009; Ahmad *et al.,* 2006).

In this investigation was reviewed a several papers from studies conducted at the Universidad del Zulia during 2002 to 2012, to analyze the efficiency of biological and physicochemical systems BR, UASB, SBR and RBC, and the physicochemical treatment as coagulation and flotation (DAF), which have been evaluated to remove COD, hydrocarbons, SARA and phenols, present in the WCP.

The instrument used was a matrix register of the treatment, considering criteria like WCP type, system of treatments, operation conditions, organic load, retention times, temperature, pollutant contents and dose of coagulant. The efficiency of the treatments was compared considering the parameters COD, phenols, hydrocarbons and SARA.

## **2. Results**

compounds are toxic, carcinogenic, mutagenic or bioaccumulative, and the last ones present

Gives that the WCP volumes generated in the Ulé tank farm, on the east cost of Maracaibo Lake, Venezuela, belonging to the petroleum industry in Venezuela, would exceed the needs for secondary recovery and the systems of reinjection would be rapidly saturated, different research works were realized to present alternatives to the petroleum industry, to diminish

In this aspect, some proposals for the treatment of WCP are aerobic and anaerobic biological processes, physicochemical treatment and some new technologies as constructed wetlands. Among the anaerobic processes are the batch reactors (BR) and the upflow anaerobic sludge

The biological mesophilical and thermophilical anaerobic systems have been successful in the treatment of complex waters, with low, moderate and high organic load (Lettinga, 2001). In the case of UASB, these reactors are outlined by their capacity to retain biomass, to form granular sludge with high properties of sedimentation, to handle high organic load to short hydraulic retention time (HRT), produce biogas and remove high concentration of biodegrad‐

On the other hand, the aerobic systems have been efficient for the treatment of wastewater containing chemical compounds resistant to be biodegraded. Among these systems are the sequential biological reactors (SBR), which have showed excellent results in the degradation of toxic compounds present in industry effluents (Díaz *et al.,* 2005a; González *et al.,* 2007). As well as, the rotating biological contactor reactors (RBC), which produce good quality effluents including total nitrification, low costs and ease of operation and maintenance (Behling *et al.,*

Among the physicochemical treatment applied to reduce the pollutants in wastewater are the dissolve air flotation (DAF) and the coagulation. The most applied products to treat natural water and wastewater by coagulation and flocculation are iron and aluminium salts. However, the cationic polymers have demonstrated their efficiency in the removal of oils and phenols

In this investigation was reviewed a several papers from studies conducted at the Universidad del Zulia during 2002 to 2012, to analyze the efficiency of biological and physicochemical systems BR, UASB, SBR and RBC, and the physicochemical treatment as coagulation and flotation (DAF), which have been evaluated to remove COD, hydrocarbons, SARA and

The instrument used was a matrix register of the treatment, considering criteria like WCP type, system of treatments, operation conditions, organic load, retention times, temperature, pollutant contents and dose of coagulant. The efficiency of the treatments was compared

able organic matter (Lepisto and Rintala, 1990; Lettinga, 2005).

from industrial wastewater (Renault *et al.,* 2009; Ahmad *et al.,* 2006).

considering the parameters COD, phenols, hydrocarbons and SARA.

diverse effects on the flora an fauna.

4 Biodegradation - Engineering and Technology

the potential pollutant of WCP.

blanket reactors (UASB).

phenols, present in the WCP.

2003).

## **2.1. Origin and composition of the waters associated with the crude oil production**

The WCP samples were obtained from the Ulé tank farm, located on the east coast of Maracaibo Lake, Tía Juana, Zulia state, Venezuela (Figure 1). The water samples come from the segrega‐ tions: Tía Juana light (TJL), Urdaneta heavy (UH), Tía Juana medium (TJM), and the dehydra‐ tions of the Punta Gorda tank farm (Rosa medium-RM), Shell Ulé (F-6/h-7) and lacustrine terminal of La Salina (LTLS). These waters were obtained from the separation of the water associated with the extraction of light crude oil (>31.8ºAPI) WCPL, from the water associated with the extraction of medium crude oil (22ºAPI-29.9ºAPI) WCPM, from the water associated with the extraction of heavy crude oil (10ºAPI-21.9ºAPI) WCPH, classified according to the American Petroleum Institute. Also, water samples were taken from the converged point of the three cuts (WCPC). In this investigation was reviewed a several papers from studies conducted at the Universidad del Zulia during 2002 to 2012, to analyze the efficiency of biological and physicochemical systems BR, UASB, SBR and RBC, and the physicochemical treatment as coagulation and flotation (DAF), which have been evaluated to remove COD, hydrocarbons, SARA and phenols, present in the WCP. The instrument used was a matrix register of the treatment, considering criteria like WCP type, system of treatments, operation conditions, organic load, retention times, temperature, pollutant contents and dose of coagulant. The efficiency of the treatments was compared considering the parameters COD, phenols, hydrocarbons and SARA. **2. Results**

The Tables 1, 2, 3 and 4 present the principal characteristics of WCPL, WCPM, WCPH and WCPC, respectively. In general, it is observed that the physicochemical characteristics of the WCP are different depending on the contact of these waters with the crude oil associated. They are waters with high pollutant contents and they do not comply with the Venezuelan environmental regulations to be discharged into water bodies (Gaceta Oficial, 1995). On the other hand, the differences in the characteristics reported by the research‐ ers, might be related to the changes that have been given in the productive processes of the petroleum industry in the last years. **2.1. Origin and composition of the waters associated with the crude oil production**  The WCP samples were obtained from the Ulé tank farm, located on the east coast of Maracaibo Lake, Tía Juana, Zulia state, Venezuela (Figure 1). The water samples come from the segregations: Tía Juana light (TJL), Urdaneta heavy (UH), Tía Juana medium (TJM), and the dehydrations of the Punta Gorda tank farm (Rosa medium-RM), Shell Ulé (F-6/h-7) and lacustrine terminal of La Salina (LTLS). These waters were obtained from the separation of the water associated with the extraction of light crude oil (>31.8ºAPI) WCPL, from the water associated with the extraction of medium crude oil (22ºAPI-29.9ºAPI) WCPM, from the water associated with the extraction of heavy crude oil (10ºAPI-21.9ºAPI) WCPH, classified according to the American Petroleum Institute. Also, water samples were taken from the converged point of the three cuts (WCPC). The Tables 1, 2, 3 and 4 present the principal characteristics of WCPL, WCPM, WCPH and WCPC, respectively. In general, it is

> observed that the physicochemical characteristics of the WCP are different depending on the contact of these waters with the crude oil associated. They are waters with high pollutant contents and they do not comply with the Venezuelan environmental regulations to be discharged into water bodies (Gaceta Oficial, 1995). On the other hand, the differences in the characteristics reported by the researchers, might be related to the changes that have been given in the productive processes of the petroleum

*et al.* **(2005a)**

*et al.* **(2005b)**

pH 7.9 8.0 8.3 7.99 NR Alkalinity (mg CaCO3/L) 2933 2215 2670 2412 NR COD soluble (mg/L) 1065.2 799 1400 1105 106.2 Phenols (mg/L) 19.36 1.73 NR 16.8 NR Nitrogen NTK (mg/L) 23.82 28.8 20 21.2 23.82 Phosphorous (mg/L) 1.07 1.0 2.2 1.57 1.07 Hydrocarbons (mg/L) NR 91 224.2 78.0 NR Chlorines (mg/L) NR NR NR NR NR TSS (mg/L) NR NR 104 NR NR VSS (mg/L) NR NR 54 NR NR H and O (mg/L) NR NR 66 100.7 NR

**Gutiérrez** *et al.* **(2012)**

**González**  *et al.* **(2007)**  **Rincón** *et al.* **(2008)**

**Díaz Díaz Figure 1.** Geographical location of the Ulé tank farm, Tía Juana Zulia state, Venezuela.

Figure 1. Geographical location of the Ulé tank farm, Tía Juana Zulia state, Venezuela.

**Parameters**

industry in the last year.

**Parameters**

**Table 2.** Physicochemical parameters of WCPM from tank farm of Ulé

**Díaz** *et al.* **(2005a)**

\*Values in (%), NR: No register

\*Values in (%). NR: No register

**Table 3.** Physicochemical parameters of WCPH from tank farm of Ulé

**Parameters**

**Díaz** *et al.* **(2005a)**

Aromatics (mg/L) NR 5.86\* 9.77 50.34 Resins (mg/L) NR 6.49\* 5.30 33.22 Asphaltenes (mg/L) NR 5.99\* 5.30 16.10

> **Gutiérrez et al. (2012)**

pH 8.0 8.2 8.3 7.08 8.41 Alkalinity (mg CaCO3/L) 885 1000 885 NR 803.33 COD soluble (mg/L) 307<PHONE_NUMBER> 259.6 Phenols (mg/L) 2.70 NR 2.5 NR 0.83 Nitrogen NTK (mg/L) 10.61 15.7 9.2 8.26 5.60 Phosphorous (mg/L) 2.68 2.0 9.8 0.013 3.01 Hydrocarbons (mg/L) NR 52.7 78 35.0 123.21 Chlorines (mg/L) NR NR NR NR 1101.21 TSS (mg/L) NR NR NR NR 573.33 VSS (mg/L) NR NR NR NR 220.00 Color (CU) NR NR NR NR 718.80 Turbidity (NTU) NR NR NR NR 140.00 Chrome (mg/L) NR NR NR 4.75 NR Lead (mg/L) NR NR NR 4.35 0.0 Sodium (mg/L) NR NR NR 89.94 NR Zinc (mg/L) NR NR NR 2.50 0.30 O&G (mg/L) NR NR 113.3 NR NR Saturated (mg/L) NR 23.97\* NR NR NR Aromatic (mg/L) NR 6.15\* NR NR NR Resins (mg/L) NR 64.7\* NR NR NR Asphaltenes (mg/L) NR 5.14\* NR NR NR

**González et al. (2007)**

**Gutiérrez et al. (2012)**

**Rincón et al. (2008)**

Biodegradability of Water from Crude Oil Production

**Gutiérrez et al. (2009)**

**Castro et al. (2008)** 7

http://dx.doi.org/10.5772/56328

**Caldera et al. (2011)**

**Table 1.** Physicochemical parameters of WCPL from tank farm of Ulé

\*Values in (%), NR: No register

**Parameters**

6 Biodegradation - Engineering and Technology

\*Values in (%), NR: No register

**Parameters**

**Table 1.** Physicochemical parameters of WCPL from tank farm of Ulé

**Díaz** *et al.* **(2005a)**

pH 8.0 8.5 NR 8.04 Alkalinity (mg CaCO3/L) 3440 2800 NR 2906 COD soluble (mg/L) 782.6 933 782.6 880 Phenols (mg/L) 1.40 NR NR NR Nitrogen NTK (mg/L) 39.20 15.1 39.20 NR Phosphorous (mg/L) 1.05 3.5 1.05 NR Hydrocarbons (mg/L) NR 148.7 NR NR Chlorines (mg/L) NR NR NR NR TSS (mg/L) NR NR NR 82.57 VSS (mg/L) NR NR NR 69.71 Saturated (mg/L) NR 25.32\* 5.73 0.24

**Gutiérrez et al. (2012)**

**Rincón et al. (2008)** **Castro et al. (2008)**

**Díaz** *et al.* **(2005a)**

**Díaz** *et al.* **(2005b)**

pH 7.9 8.0 8.3 7.99 NR Alkalinity (mg CaCO3/L) 2933 2215 2670 2412 NR COD soluble (mg/L) 1065.2 799 1400 1105 106.2 Phenols (mg/L) 19.36 1.73 NR 16.8 NR Nitrogen NTK (mg/L) 23.82 28.8 20 21.2 23.82 Phosphorous (mg/L) 1.07 1.0 2.2 1.57 1.07 Hydrocarbons (mg/L) NR 91 224.2 78.0 NR Chlorines (mg/L) NR NR NR NR NR TSS (mg/L) NR NR 104 NR NR VSS (mg/L) NR NR 54 NR NR O&G (mg/L) NR NR 66 100.7 NR Saturated (mg/L) NR NR 76.6\* NR 1.24 Aromatics (mg/L) NR NR 7.04\* NR 17.64 Resins (mg/L) NR NR 6.34\* NR 8.51 Asphaltenes (mg/L) NR NR 7.73\* NR 7.49

**Gutiérrez et al. (2012)**

**González et al. (2007)**

**Rincón et al. (2008)**

**Table 2.** Physicochemical parameters of WCPM from tank farm of Ulé

\*Values in (%). NR: No register

**Table 3.** Physicochemical parameters of WCPH from tank farm of Ulé

**2.3. Biological treatment applied to the waters associated with crude oil production**

**Characteristics of the**

RBC of 9.5 L, with 50 circular disc of PVC, 0.8 cm separation, supported in an axis of carbon steel 3/8 " diameter, rotation speed of 2.5 rpm. The discs were immersed 40 % in the effluent. The area of contact was 2.44 m2. The water volume was 7.5 L

The SBR of 4 L were constructed in material of plastic and cylindrical form, with a volume of operation of 2 L, in which 600 mL sludge and 1.4 L of WCP. At the bottom of the reactors were located air diffusers connected to

The SBR of 2 L was constructed in material of plastic, with 600 mL of sludge and 1.4 L of WCPM. They gave oxygen to the reactor by means of a compressor.

a compressor.

treatments to WCP.

**Kind of WCP**

WCPC (WCPL, WCPM and WCPH)

WCPL, WCPM and WCPH

WCPM SBR

**Treatment systems**

RBC

SBR

**Researcher, year**

Behling *et al.* (2003)

Díaz *et al.* (2005a)

Díaz *et al.* (2005b)

The Tables 5 and 6 show a resume of the aerobic and anaerobic biological treatments applied to WCP, and Table 8 shows the operation conditions of the combined system aerobic-anaerobic applied to WCP. Among the aerobic biological systems are the rotating biological contactor reactors (RBC), the sequential biological reactors (SBR) and the continuous flow reactors (CR); and among the anaerobic biological treatments are the batch reactors (BR) and the upflow anaerobic sludge blanket reactors (UASB), working under mesophilic and thermophilic conditions. Likewise, Table 9and Table 10 present a summary of the results of applying these

**experimental equipment Operation conditions**

The RBC worked under mesophilic condition. The organic load average applied was 2.04 ± 0.7 g COD/m2d and 5.2 mL/min, TRH of 24 h, temperature 27-32ºC.

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9

After acclimated and stabilized, they worked with HRT of 16 hours with sequence of 15 hours of ventilation, 30 minutes of sedimentation and 30 minutes for capture of sample and recharges of the reactor. The temperature was mesophilic (37 ºC). The SBR-1, SBR-2, SBR-3 operated with organic charges of 1.6; 1.17 and 0.46 kg/m3d for the WCPL, WCPM and WCPH, respectively.

After acclimated and stabilized, they were operated at the first stage of 15 hours the HRT and time of cellular retention of 15-20 days with sequence of 14 hours for mixed, ½ hour of rest and ½ hour for discharge and load. Whereas in the second stage the HRT was 24 hours with sequence of 23 hours for mixed and ventilation and one hour of discharge and load. The temperature was 37 ºC. The

**Parameters evaluated**

pH COD TSS VSS Total alkalinity

pH Alkalinity COD Phenols

COD Hydrocarbons Phenols

a Combination of light, medium and heavy crude oil, and exit of the clarifier

b Combination of medium and heavy crude oil, API 5.

c Combination of light, medium and heavy crude oil, and in of the clarifier

NR: No register

**Table 4.** Physicochemical parameters of WCPC from tank farm of Ulé

#### **2.2. Treatment of the waters associated with crude oil production**

The Tables 5, 6, 7 and 8 show a summary of the methodology used by each researcher, showing the operational conditions for each system. On the other hand, Table 9 and Table 10 compare the different treatments: physicochemical treatments, aerobic and anaerobic biological treatment, and combined treatments.

## **2.3. Biological treatment applied to the waters associated with crude oil production**

**Parameters Behling et al.**

8 Biodegradation - Engineering and Technology

Combination of light, medium and heavy crude oil, and exit of the clarifier

Combination of light, medium and heavy crude oil, and in of the clarifier

**Table 4.** Physicochemical parameters of WCPC from tank farm of Ulé

**2.2. Treatment of the waters associated with crude oil production**

The Tables 5, 6, 7 and 8 show a summary of the methodology used by each researcher, showing the operational conditions for each system. On the other hand, Table 9 and Table 10 compare the different treatments: physicochemical treatments, aerobic and anaerobic biological

b Combination of medium and heavy crude oil, API 5.

treatment, and combined treatments.

a

c

NR: No register

**(2003)a**

**Rincón** *et al.* **(2004)a**

pH 7.72 8 7.74 8.03

Alkalinity (mg CaCO3/L) 2460 2238 2477 2635 COD soluble (mg/L) 823 NR NR 1391.85 COD total (mg/L) NR 700 NR NR Phenols (mg/L) NR 5 NR 2.14 Nitrogen NTK (mg/L) 12.92 NR NR 17.55 Phosphorous (mg/L) 1.40 NR NR 3.67 Hydrocarbons (mg/L) NR 100 NR 276.68 Chlorine (mg/L) NR NR 1802 1404.87 TSS (mg/L) 170 NR 122 550 VSS (mg/L) 50 NR NR 82.35 Sulfides (mg/L) NR NR NR 7.32 Turbidity (NTU) NR NR 480 NR Chrome (mg/L) NR NR NR 0.31 Lead (mg/L) NR NR NR 0.17 Sodium (mg/L) NR NR NR 8880.32 Nickel (mg/L) NR NR NR 0.20 Zinc (mg/L) NR NR NR 0.32 Copper (mg/L) NR NR NR 0.19 O&G (mg/L) NR 181 737 NR

**Rojas** *et al.* **(2008)b**

**Blanco** *et al.* **(2008)c**

The Tables 5 and 6 show a resume of the aerobic and anaerobic biological treatments applied to WCP, and Table 8 shows the operation conditions of the combined system aerobic-anaerobic applied to WCP. Among the aerobic biological systems are the rotating biological contactor reactors (RBC), the sequential biological reactors (SBR) and the continuous flow reactors (CR); and among the anaerobic biological treatments are the batch reactors (BR) and the upflow anaerobic sludge blanket reactors (UASB), working under mesophilic and thermophilic conditions. Likewise, Table 9and Table 10 present a summary of the results of applying these treatments to WCP.

**Researcher, year**

Gutiérrez *et al.* (2009)

Rincón *et al.* (2002)

Díaz *et al.* (2005a)

WCPL

WCPL WCPM WCPH

UASB reactors

locality.

UASB reactors

**Kind of WCP**

WCPM and WCPH

Batch reactors

**Treatment systems**

**Characteristics of the experimental**

They placed three (3) reactors of 500 mL each one, containing 20 % of the useful volume mesophilic granular sludge proceeding from a beer industry, and 80 % of effluent to treat. The reactors were immersed in a thermal bath that allowed controlling the temperature. The produced biogas was meter by water

There were employed at a UASB reactor of 4 L, 0.098 m of diameter, 0.67 m high and 0.53 m high of water, inoculated with 30 % of granular sludge from a UASB reactor that treats residual waters of a brewery of the locality.

They worked with 3 UASB reactors of 4 L, inoculated with 1.2 L of granular sludge from an UASB reactor treating residual waters of a brewery of the

displacement.

**equipment Operation conditions**

Initially the reactors were loaded, for ten days, with D +glucose on an equivalent concentration in COD of 1500 mg/L and solution of nutrients, for a retention time (RT) of 24 hours. Later they added to two reactors WCPM and WCPH with concentrations of 1876.9 and 1029.0 mgCOD/L, respectively. The third reactor worked with glucose (D+glucose). To reach the thermophilic conditions (55ºC ± 1ºC) the temperature was increased from the mesophilic conditions (37ºC ± 1ºC) at the reason of 1ºC/day. The RT in all the cases was 24

Biodegradability of Water from Crude Oil Production

hours.

1ºC).

24 h.

Initially, the reactor was fed with residual synthetic water that was containing glucose as the only source of carbon (1 g/L) and nutrients. Later, it was operated for 275 days with HRT from 38 to 5 h. The reactors were evaluated for organic loads of 0.78; 1.20; 1.46; 1.64; 1.90; 3.17 and 4.70 kg COD/m3d for HRT of 36, 24, 21, 17, 11, 8 and 6 hours, respectively. They worked under mesophilic conditions (37ºC ±

Initially, the reactor was fed with residual synthetic water that was containing glucose as the only source of carbon (850 mg/L) and nutrients. Later, the reactors UASB-1, UASB-2 and UASB-3 were fed by WCPL, WCPM and WCPH why organic loads of 1.06; 0.78 and 0.31 kg COD/m3d, respectively. They worked under mesophilic conditions (37ºC ± 1ºC) during 1 month with HRT of **Parameters evaluated**

11

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pH COD TSS and VSS Alkalinity VFA Methane

pH Alkalinity COD Phenols

pH Alkalinity COD SARA

**Table 5.** Methodology for aerobic treatment of WCPM

**Researcher, year**

González *et al.* (2007)

Castro *et al.* (2008)

**Researcher, year**

Gutiérrez *et al.* (2007)

**Kind of WCP**

10 Biodegradation - Engineering and Technology

WCPL and WCPH

WCPM

**Kind of WCP**

WCPL WCPM and WCPH

Batch rectors

**Treatment systems**

SBR

Batch reactor

**Table 5.** Methodology for aerobic treatment of WCPM

**Treatment systems**

**Characteristics of the**

by a compressor.

The reactor was a receptacle adjusted as Plexiglas of 3 L, provided with a porous circular stone and a hose connected to the tubes for the supply of compressed air. As effective volume of 0.3 L of bacterial suspension and 0.7 L of WCPM.

**Characteristics of the experimental**

They placed four (4) reactors of 500 mL each one, containing 20 % of the useful volume of mesophilic granular sludge proceeding from a beer industry, and 80 % of effluent to treat. The reactors were immersed in a thermal bath that allowed controlling the temperature. The produced biogas was meter by water

displacement.

**equipment Operation conditions**

The SBR of 2 L was constructed in material of plastic, in cylindrical form, in which they added 600 mL of sludge and 1.4 L of WCP. They gave oxygen to the reactor

**experimental equipment Operation conditions**

organic load applied was between

HRT of 8 hours and time of cellular retention of 20 days. Nutrients were added. The COD in the inflow was 1105 and 320 mg/L for WCPL and WCPH, respectively.

They used several functional groups and consortiums of bacteria. The systems were operated under mesophilic conditions (27 ºC) and HRT of 144 h. The COD of feeding was 880

Initially the reactors were loaded, for ten days, with D +glucose on an equivalent concentration in COD of 1500 mg/L and solution of nutrients, for a retention time (RT) of 24 hours. Later they added to three reactors WCPL, WCPM and WCPH with concentrations of 1200-1300 mgCOD/L, 857-960 mgCOD/L and 860-870 mgCOD/L, respectively. The fourth reactor worked with glucose (D+ glucose). To reach the thermophilic conditions (55ºC ± 1ºC) the temperature was increased from the mesophilic conditions (37ºC ± 1ºC) at the reason of 1ºC/day. The RT in all the cases was 24

mg/L.

hours.

0.89 and 0.51 kg/m3d

**Parameters evaluated**

COD Hydrocarbons Phenols

pH COD TSS VSS Alkalinity

**Parameters evaluated**

pH COD TSS and VSS Alkalinity VFA Methane

**Table 6.** Methodology for anaerobic treatment of WCP

**Treatment systems**

Coagulation and DAF

WCPH Coagulationflocculation

WCPH Coagulationflocculation

**Characteristics of the**

The DAF, consisted of a pressurization cell or saturation camera, constructed in material of transparent plastic of 90 mm of external diameter and 270 mm high. Inside the camera was finding a manual agitator of stainless steel and a filter that worked as diffuser; in addition, a series of connections and valves of the distribution and pressure

of the water and air.

They used a Jar Test model JLT6; adding 1 L of WCP, to each of six precipitation jar of 1000 mL, taking one of these as a control.

They used a Jar Test model JLT6; adding 1 L of WCP, to each of six precipitation jar of 1000 mL, taking one of these as a control.

**experimental equipment Operation conditions**

of 25ºC.

They worked with pressures of 30, 40 and 50 psi and recycle of 30%, 40% and 50%, and temperature

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They evaluated a cationic flocculants of high molecular weight, in concentration of 0.006 % in volume (3.54 mg/L)

They simulated coagulation, flocculation, and sedimentation processes to 100 rpm for rapid agitation for 1 minute and 30 rpm for slow agitation by 20 minutes. The sedimentation was 30 minutes. The initial turbidity of the

water was 140 NTU. They used as coagulant

%. They worked with concentrations of 24, 30, 36, 42 and 48 mg/L of solution of LCH and CCH, respectively.

was 52 NTU.

They simulated coagulation, flocculation and sedimentation processes to 100 rpm for rapid agitation for 2 minutes, and 100 rpm for slow agitation for 30 minutes. The sedimentation was 30 minutes. The turbidity initial

As coagulant agent was used commercial chitosan (CCH) dissolved in acetic acid 0.10 M, preparing solutions of 1.0%. The concentrations evaluated were 40,

commercial chitosan (CCH) (Sigma Chemical Co.) and chitosan obtained in the laboratory (LCH) to 100 ºC dissolved in acetic acid 0.10 M, preparing solutions of 0.6

**Parameters evaluated**

13

TSS Turbidity O&G

pH COD TSS VSS Turbidity Color O&G Hydrocarbons

pH COD TSS VSS Turbidity Color O&G Hydrocarbons

**Kind of WCP**

WCPC (WCPM and WCPH)

**Researcher, year**

Rojas *et al.* (2008)

Caldera *et al.* (2009)

Caldera *et al.* (2011)

**Table 6.** Methodology for anaerobic treatment of WCP

**Researcher, year**

Gutiérrez *et al.* (2006)

Caldera *et al.* (2007)

Rincón *et al.* (2008)

WCPL

WCPL WCPM UASB reactors

locality.

UASB reactor

**Kind of WCP**

12 Biodegradation - Engineering and Technology

WCPL

**Treatment systems**

UASB reactors

industry.

**Characteristics of the experimental**

They used a UASB reactor constructed in Plexiglas with volume of 2.5 L, inoculated with anaerobic mesophlic granular sludge (30 % of the useful volume) proceeding from a beer industry. The reactor was provided with a jacket, supporting the temperature of 55 ± 1°C for recirculation of warm water.

They used two UASB reactors of 2.5 L, inoculated with 0.75 L of granular sludge from an UASB reactor treating residual waters of a brewery of the

They used two UASB reactors constructed in Plexiglas with volumes of 1.7 and 2.5 L, operating under temperatures of 37 ± 1ºC and 55 ± 1°C, respectively. The reactors were provided with a jacket, supporting the temperature for recirculation of warm water. Both reactors were inoculated with mesophilic anaerobic granular sludge (20 % of the useful volume) proceeding from a beer

**equipment Operation conditions**

Initially the reactors were load, for two days, with D+glucose on an equivalent concentration in COD of 1500 mg/L and solution of nutrients and TRH of 24 h; then WCPL was added. Later to reach the thermophilic conditions (55ºC ± 1ºC) in the thermophilic reactor, the temperature was increased from the mesophilic condition (37ºC ± 1ºC) to a rate of 1ºC/day. The reactors were evaluated for organic loads of 1.4, 1.9, 2.8 and 5.6 kg COD/m3d and RTH of 24, 18, 12 and 6 hours, respectively.

Initially the reactor was loaded, for two days, with D+glucose on an equivalent concentration in COD of 1500 mg/L and solution of nutrients, and HRT of 24 h; then WCPL was added. Later to reach the thermophilic condition (55ºC ± 1ºC), in the thermophilic reactor, the temperature was increased from the mesophilic condition (37ºC ± 1ºC) to a rate

The reactor was evaluated for 42 days, with HRT of 24 and 12 and organic loads of 1.4 and 2.8 kg COD/m3d, respectively.

Initially, the reactors were fed with residual synthetic water that was containing glucose as the only source of carbon (850 mg/L) and nutrients. Later, the reactors UASB-1 and UASB-2 were fed with WCPL and WCPM APPL and organic load of 1.06 and 0.78 kg COD/m3d respectively. They worked mesophilic conditions (37ºC ± 1ºC) during 1 month with HRT of

of 1ºC/day.

24 h.

**Parameters evaluated**

pH Alkalinity COD VFA Methane Enzymes

pH Alkalinity COD VFA Methane

pH Alkalinity COD SARA

**Researcher year**

**Rojas et al. (2008)**

**Caldera et al. (2009)**

**Caldera et al. (2011)**

**Behling et al. (2003)**

**Díaz et al. (2005a)**

**Díaz et al. (2005b)**

**González et al. (2008)**

**Castro et al. (2008)**

**Researcher year**

**Gutiérrez et al. (2007)**

**Gutiérrez et al. (2009)**

a and b: different HRT

**Treatment systems / WCP**

Coagulation and DAF WCPC

Coagulationflocculation WCPH

Coagulationflocculation WCPH

> RBC WCPC

SBR WCPL, WCPM and WCPH

> SBR WCPM

SBR WCPL and WCPH

Batch reactor WCPM

**Table 9.** Results of the treatment of WCP

**Treatment systems / WCP**

Batch reactors WCPL, WCPM and WCPH

Batch reactors WCPM and WCPH

88.8 65.2 62.9

65.1a 60.9b

> 88 66

62.4-89. 8

63.3-9. 5

**COD (%) VSS (%) Hydro-**

70.7 59.9 62.1

68.2-69. 2 55.9-50. 4

**COD (%)**

**TSS (%)** **VSS (%)**

**Hydrocarbons (%)**

\_\_ \_\_ \_\_ \_\_ 96.8

79.5

73.8

**carbons (%)**

**Phenols (%)**

\_\_ \_\_ 7.6

\_\_ \_\_ 8.2

\_\_ \_\_ 76.8

\_\_ \_\_ 84.4

**O&G (%) Phenols**

\_\_ 77 \_\_ \_\_ 90 \_\_ 69 \_\_ \_\_

50.7 \_\_ \_\_ 70.1 \_\_ \_\_ 90.7 8.0-8.2 \_\_

12.5 55-61 41-63 70-90 39-59 \_\_ 76-78 7.9 \_\_

76.1 < 4 < 3 \_\_ \_\_ \_\_ \_\_ 8.9 2343

55.5 62.4 89.2 82.8

87.5 92

\_\_ \_\_ \_\_ \_\_ \_\_ 7.4-6.6 \_\_

**pH Alkalinity**

7.6 7.2

7.5

**(mgCaCO3/L)**

2673.7 2620.0 936.7

\_\_ 95.6 79.4 \_\_ 9.0-9.9 9.0-9.6 8.9-9.4

\_\_ \_\_ \_\_

\_\_ \_\_ \_\_

**SARA (%)**

\_\_ \_\_ \_\_

**Methane content (%)**

> 51.9 54.1

\_\_ 73.1

**(%)**

**Turbidity (%)**

Biodegradability of Water from Crude Oil Production

**pH Alkalinity (mgCaCO3/L)** 15

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\_\_

**Table 7.** Methodology for physicochemical treatment of WCP

**Table 8.** Methodology for combined treatment of WCP

a and b: different HRT

**Researcher, year**

**Researcher year**

Rincón *et al.* (2004)

Paz *et al.* (2012)

Blanco *et al.* (2008)

**Kind of WCP**

14 Biodegradation - Engineering and Technology

**Kind of WCP**

WCPL WCPC

WCPC

WCPC

Subsuperficial constructed wetlands (SSCW)

**Table 8.** Methodology for combined treatment of WCP

**Treatment systems**

**Table 7.** Methodology for physicochemical treatment of WCP

**Treatment systems**

UASB-SBR system

Superficial constructed wetlands (SCWFF)

plant.

**Characteristics of the**

**Characteristics of the**

They used two types of reactors placed in series, a reactor UASB of 2.5 L of useful volume and a SBR. The reactor UASB was inoculated by sludge from an UASB reactor treating residual waters of a brewery. While the SBR reactor was inoculated with aerobic sludge from a wastewater treatment

They used two superficial constructed wetlands of free flow (SCWFF) to pilot scale. The support material was gravel and soil, and aquatic emergent plants that counted of support of gravel and soil, and aquatic emergent plants (*Cyperus luzulae y Cyperus ligulari* – SCWFF I, y *Cyperuz feraz, Paspalum sp. y Typha dominguesis* – SCWFF II), and a control (C) without plants.

The system SSCW consisted of three polyethelene tray of 1.28 m long for 0.45 m wide and 0.45 m high, one that of them as control (without plants) and the others two with emergent aquatic plants *Cyperus luzulae, Cyperus feraz L.C*, *Cyperus ligularis L.* y *Typha dominguensis* (SSCW I y SSCW II). The beds of the tray were constituted by 86400 cm3 of gravel as support and a water level of 1.5 L to simulate a natural system of

wetland.

**experimental equipment Operation conditions**

**experimental equipment Operation conditions**

42, 44, 46 and 48 mg/L of CCH

The system worked 195 days, in two stages. The first was feeding with WCPL from 1100 to 1230 mg COD/L (133 days) and the second one with WCPC of 176 and 264 mg COD/L (66 days). The effluent treated in the UASB was fed in the SBR. The HRT was 24 hours and the temperatures were UASB 37ºC and

They placed 30 plants for each species. The depth of the support was 0.25 cm with 7 % of gravel and 93 % of soil, and a water layer of 0.05 m of water. The flow fed was 8 mL/min, with a HRT of 7 days and organic load of 23.5 g COD/m2d. The samples were collected weekly for 80 days.

The systems worked to continue flow, without recirculation of the effluent with an organic load of 29.42 g/m2d, a flow of 10 mL/min

and HRT of 7 days.

solution.

SBR 28 ºC.

**Parameters evaluated**

**Parameters evaluated**

pH Alkalinity COD Hydrocarbons Phenols

COD pH Sulphide Phenols TSS VSS DO

pH Alkalinity COD VSS Hydrocarbons Phenols

**Table 9.** Results of the treatment of WCP

the system. On the other hand, Gutiérrez *et al.* (2006) indicated that for the same temperature conditions, the HRT optimal was 18 hours with COD removal of 80%; they indicate also that for thermophilic conditions, the optimal HRT was 18 hours with COD removal of 84%,

Biodegradability of Water from Crude Oil Production

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17

When the efficiency of COD removal of WCPL in UASB reactors under mesophilic and thermophilic conditions were compared, major percentages of COD removal under thermo‐ philic conditions for HRT under at 15 hours were observed. This removal of COD can be associated to high temperature accelerate the enzymatic biological systems. Nevertheless, there were not significant differences (p>0.05) between the values obtained for mesophilic and

When combined systems were used, the COD removal of the system was higher than those

The maximum COD removal reached for the systems applied to WCPL were between 67% and 95%. In this aspect, the petroleum industry has alternatives to treat the WCPL; however, the final decision will be an economic decision between the temperature, the size of the reactor and energy costs. In the case of thermophilical route, it is necessary to considerate the cost of raising the temperature of the water, because the WCPL is at atmospheric conditions. For

**2.5. Biological treatment of the waters associated with medium crude oil production**

It is observed in the Table 9 and Table 10 that the WCPM presented lower biodegradability than WCPL, for both aerobic and anaerobic systems. In discontinuous batch aerobic systems, Castro *et al.* (2008) report that the COD removal was between 62.4% and 89.8%; while for SBR reactors was between 60.9% and 65.2% (Díaz *et al.,* 2005 a; Díaz *et al.,* 2005b). On the other hand, in batch anaerobic reactors under thermophilic conditions, the COD removal was between 59.9% and 69.2% (Gutiérrez *et al.* 2007; Gutiérrez *et al.,* 2009). In UASB reactors, the COD

In the different treatment systems it is observed that the COD removal for WCPM was between

In the case of the waters associated with heavy crude oil production (WCPH), the behavior was similar to WCPM. In SBR systems the COD removal was between 62.9% (Díaz *et al.,* 2005a) and 66% (González *et al.,* 2007). While in anaerobic batch reactor systems under thermophilic conditions, the COD removal was between 50.4% and 62.1% (Gutiérrez *et al.,* 2007; Gutiérrez *et al.,* 2009). On the other hand, in UASB reactors under mesophilic conditions,

**2.7. Biological treatment of the combination of waters associated with crude oil production** The WCPC represent the combination of the waters in contact with different fractions of crude oil, whether produced in plant or by the researchers. The biodegradability of these waters has

**2.6. Biological treatment of the waters associated with heavy crude oil production**

the COD removals were lower than 40% (Díaz *et al.,* 2005a; Rincón *et al.,* 2008).

been studied in RBC and combined systems UASB-SBR (Table 8).

maintaining good COD efficient removal for HRT of 6 hours (67%).

thermophilic temperature conditions, for the HRT from 12 to 24 hours.

aerobic processes, the costs of the energy associated must be considered.

removal was between 23.5% (Díaz *et al.,*2005a) and 26% (Rincón *et al.,* 2008).

obtained in each separated system (Rincón *et al.,* 2004).

23.5% and 89.8%.

**Table 10.** Results of the treatment of WCP

#### **2.4. Biological treatment of the waters associated with light crude oil production**

The waters associated with the production of light crude oil (WCPL) are biodegradable in aerobic biological treatments, in anaerobic biological treatments and in a combination of these treatments. Díaz *et al.* (2005a) report that the COD removal in SBR was 88.8%, and the removal of phenols was 96.8%.

Likewise, the WCPL showed be biodegradable in anaerobic conditions in batch and continuous systems, under mesophilic conditions (37ºC) and thermophilic conditions (55ºC). In batch systems the COD removal reached 70.7% under mesophilic conditions (Gutiérrez *et al.,* 2007), while in UASB reactors under both temperature conditions, the efficiency of COD removal reached over 75%.

In UASB reactors the HRT influenced in the COD removal; so, Rincón *et al.* (2002) reported that under mesophilic conditions the optimal HRT was between 15 and 10 hours, with COD removal above 80%, but for HRT under 10 hours the system did not allow the methanogenic microorganisms to be able to transform volatile fatty acid (VFA), provoking the inhibition of the system. On the other hand, Gutiérrez *et al.* (2006) indicated that for the same temperature conditions, the HRT optimal was 18 hours with COD removal of 80%; they indicate also that for thermophilic conditions, the optimal HRT was 18 hours with COD removal of 84%, maintaining good COD efficient removal for HRT of 6 hours (67%).

**Rincón et al. (2002)**

**Díaz et al. (2005a)**

**Gutiérrez et al. (2006)**

**Caldera et al. (2007)**

**Rincón et al. (2008)**

**Rincón et al. (2004)**

Paz *et al.* (2012)

**Blanco et al. (2008)** UASB WCPL

16 Biodegradation - Engineering and Technology

UASB WCPL, WCPM and WCPH

> UASB WCPL

> UASB WCPL

UASB WCPL and WCPM

> UASB-SBR WCPL-WCPC

Superficial constructed wetlands WCPC

Sub-superficial constructed wetlands WCPC

**Table 10.** Results of the treatment of WCP

of phenols was 96.8%.

reached over 75%.

M: Mesophilic T: Thermophilic; a and b : different HRT

23.8-86. 1

> 81.7 23.5 35.7

40-80M 67-84T

> 78a 77b

> > 93 26

> > 95 79

31.4-65. 7

45.2-91. 9

42-73 52-67

\_\_ 74

**2.4. Biological treatment of the waters associated with light crude oil production**

The waters associated with the production of light crude oil (WCPL) are biodegradable in aerobic biological treatments, in anaerobic biological treatments and in a combination of these treatments. Díaz *et al.* (2005a) report that the COD removal in SBR was 88.8%, and the removal

Likewise, the WCPL showed be biodegradable in anaerobic conditions in batch and continuous systems, under mesophilic conditions (37ºC) and thermophilic conditions (55ºC). In batch systems the COD removal reached 70.7% under mesophilic conditions (Gutiérrez *et al.,* 2007), while in UASB reactors under both temperature conditions, the efficiency of COD removal

In UASB reactors the HRT influenced in the COD removal; so, Rincón *et al.* (2002) reported that under mesophilic conditions the optimal HRT was between 15 and 10 hours, with COD removal above 80%, but for HRT under 10 hours the system did not allow the methanogenic microorganisms to be able to transform volatile fatty acid (VFA), provoking the inhibition of

82

\_\_ \_\_ 55.1

74.7 92.5

\_\_ \_\_ 7.4-8.5

99.9 90

61.3

\_\_ \_\_ \_\_ 8.0

\_\_ \_\_ \_\_ 7.5

\_\_ \_\_ 10-59 7.6-8.0 2500-2800 24-95

\_\_ \_\_ \_\_

84 54

\_\_ 53-79 54-80

\_\_

\_\_ \_\_

87 77

1960-2633 2190-2454

> 2413 2945

> 1955 2520

> 2468 2405

7.3-8.6 7.1-8.5 7.2-8.4

7.9-8.0

8.2

7.8

9 9

9.13-10.5 8.84-9.93

77.5 94.7 8.9 2508 \_\_ \_\_

When the efficiency of COD removal of WCPL in UASB reactors under mesophilic and thermophilic conditions were compared, major percentages of COD removal under thermo‐ philic conditions for HRT under at 15 hours were observed. This removal of COD can be associated to high temperature accelerate the enzymatic biological systems. Nevertheless, there were not significant differences (p>0.05) between the values obtained for mesophilic and thermophilic temperature conditions, for the HRT from 12 to 24 hours.

When combined systems were used, the COD removal of the system was higher than those obtained in each separated system (Rincón *et al.,* 2004).

The maximum COD removal reached for the systems applied to WCPL were between 67% and 95%. In this aspect, the petroleum industry has alternatives to treat the WCPL; however, the final decision will be an economic decision between the temperature, the size of the reactor and energy costs. In the case of thermophilical route, it is necessary to considerate the cost of raising the temperature of the water, because the WCPL is at atmospheric conditions. For aerobic processes, the costs of the energy associated must be considered.

## **2.5. Biological treatment of the waters associated with medium crude oil production**

It is observed in the Table 9 and Table 10 that the WCPM presented lower biodegradability than WCPL, for both aerobic and anaerobic systems. In discontinuous batch aerobic systems, Castro *et al.* (2008) report that the COD removal was between 62.4% and 89.8%; while for SBR reactors was between 60.9% and 65.2% (Díaz *et al.,* 2005 a; Díaz *et al.,* 2005b). On the other hand, in batch anaerobic reactors under thermophilic conditions, the COD removal was between 59.9% and 69.2% (Gutiérrez *et al.* 2007; Gutiérrez *et al.,* 2009). In UASB reactors, the COD removal was between 23.5% (Díaz *et al.,*2005a) and 26% (Rincón *et al.,* 2008).

In the different treatment systems it is observed that the COD removal for WCPM was between 23.5% and 89.8%.

#### **2.6. Biological treatment of the waters associated with heavy crude oil production**

In the case of the waters associated with heavy crude oil production (WCPH), the behavior was similar to WCPM. In SBR systems the COD removal was between 62.9% (Díaz *et al.,* 2005a) and 66% (González *et al.,* 2007). While in anaerobic batch reactor systems under thermophilic conditions, the COD removal was between 50.4% and 62.1% (Gutiérrez *et al.,* 2007; Gutiérrez *et al.,* 2009). On the other hand, in UASB reactors under mesophilic conditions, the COD removals were lower than 40% (Díaz *et al.,* 2005a; Rincón *et al.,* 2008).

## **2.7. Biological treatment of the combination of waters associated with crude oil production**

The WCPC represent the combination of the waters in contact with different fractions of crude oil, whether produced in plant or by the researchers. The biodegradability of these waters has been studied in RBC and combined systems UASB-SBR (Table 8).

Behling *et al.* (2003) commented that the COD removal in RBC system used to treat WCPC was 76.1%, while Rincón *et al.* (2004) studied a UASB-SBR system and reported that the COD removal reached 79%, indicating that was important removals of phenols and hydrocarbons were obtained.

Also the researchers have presented biodegradability percentages of different types of WCP under anaerobic conditions. They report values for mesophilic and thermophilic anaerobic systems of 80% and 78%, 45% and 86%, and 20% and 0%, for WCPL, WCPM and WCPP respectively in batch reactors (Gutiérrez and Caldera, 2011; Gutiérrez *et al.,* 2007; Rincón *et*

Biodegradability of Water from Crude Oil Production

http://dx.doi.org/10.5772/56328

19

In regard to phenols concentration, the studies mention that the consortium of microorganisms developed in mesophilic UASB reactors were influenced by the initial phenols concentrations, indicating that the phenols removal might be associated with the presence of different phenols compounds in the different types of WCP, with varied resistance to degradation and metab‐

Additionally, the studies indicate that the alkalinity values in the WCP were between 900 and 3000 mg CaCO3/L. It has been commented that the difference of COD removal might be due to the acidity-basicity conditions in the WCP. The WCPH presented lower values of alkalinity (642.9-580.4 mg CaCO3/L) and lower COD removal than WCPM. The alkalinity of WCPM was superior to 2000 mg CaCO3/L. As for the pH, the WCP presented basic pH (7-10) for the

In other cases, it is mentioned that the presence of metals in the WCP makes the treatment more complex. However, the metals K, Na, Fe, Cr, Pb and Zn can be used by thermophilic microorganisms or can be removed from the WCP and reach to the sludge by diverse mech‐

In relation to degrading microorganisms present in the WCP, some have been isolated, and identified the genus *Aeromonas, Klebsielle, Xanthomona, Bacteroides* and *Acinetobacter*, as well as a consortium of them, that resulted to be effective in COD decrease (Castro *et al.,* 2008).

The Table 7 shows that WCP has been treated by coagulation-flocculation at laboratory level using chitosane as a coagulating agent in concentrations of 24 to 38 mg/L of solution of commercial chitosane (CCH), and by dissolved air flotation (DAF) using a cationic flocculants

Rojas *et al.* (2008) reported that the TSS removal and the turbidity in the WCPC were 77% and 69% respectively. On the other hand, Caldera *et al.* (2009, 2011) commented that the turbidity removal in the WCPH was 90.7%, accompanied of COD removal of 50.7%. In any case, the hydrocarbons removal and oils removal by physicochemical methods were between 70% and 90%, concluding that the cationic polymers represent an alternative to remove oily compounds

Table 8 shows other alternatives applied to treat WCP. In constructed sub-superficial wetlands COD removal of WCPC was between 31.4% and 65.7%, while in constructed superficial wetlands there was no COD removal. Both systems showed efficiency to remove more than

The application of ozone also has been proposed to increase the biodegradability of the WCP. According to Gutiérrez *et al.* (2002), the application of ozone improves considerably the biodegradability of the WCP, with an increase of up to 87%. They concluded that the applica‐

60% of the hydrocarbons present in the WCPC (Paz *et al.*, 2012; Blanco *et al.*, 2008).

*al.*, 2006).

olism (aerobic/anaerobic).

different treatments.

anisms (Gutiérrez *et al.*, 2009).

of high molecular weight.

in the WCP.

## **3. Discussion**

Comparing the biodegradability of WCPL, WCPM and WCPH, it is observed that the WCPL present the major biodegradability in the different treatment systems and operating conditions studied.

The biodegradability of the WCP has been associated to diverse factors as SARA composition, phenols concentration, alkalinity, organic load, metals concentration and temperature.

Some researchers (Rincón *et al.,* 2002; Gutiérrez *et al.,* 2006; Gutiérrez *et al.,* 20007) argue that the WCPL biodegradability is good in anaerobic systems under mesophilic conditions and under thermophilic conditions. The final decision between the temperature used and size of the reactor will be economical, because the WCPL are at atmospheric temperature and the termophilic route implicates to consider the costs associated of warming the water. In the cases of WCPM and WCPH the studies realized up to the moment are not conclusive.

Other researchers (Gutiérrez *et al.,* 2007; Gutiérrez *et al.,* 2001; Gutiérrez *et al.,* 2012) share that the biodegradability of WCP is associated to the SARA composition present in these waters, as product of the contact with the crude oil associated. The difference of composition of these fractions confer characteristics that influence in their biodegradability, because the SARA fractions change in relation to the crude oil that is in contact with the WCP, being the WCPL the waters with the biggest percentages of saturated, considered more biodegradable than WCPM and WCPH.

When the organic fractions present in the WCP are compared, it is observed that WCPM and WCPH present a similar content of organic fractions (p>0.05). The opposite case was observed with the WCPL, which organic fractions are different in saturated, aromatics and resins, in comparing to WCPM and WCPH (p>0.05).

On the other hand, there is a tendency to increase the saturated fractions in WCP (r=0.871) with the increase of the API gravity of the crude oil with the water associated, following the order WCPL>WCPM>WCPH. In relation to the resins, it was observed that it increases with regard to the decrease of the API gravity of the crude oil with the WCP were associated following the order WCPL<WCPM<WCPH.

A study realized by Díaz *et al.* (2007) with WCPM from other tank farm of the Venezuelan petroleum industry, indicated that the SARA fractions can be removed from the WCP using UASB reactors. They obtained removals of 72% of saturated, 91% of resins and 71% of asphaltenes, and did not obtain removals of aromatics. They associated these results with the increases of the aromatic fractions for degradation of the fractions like resins and asphaltenes to aromatics.

Also the researchers have presented biodegradability percentages of different types of WCP under anaerobic conditions. They report values for mesophilic and thermophilic anaerobic systems of 80% and 78%, 45% and 86%, and 20% and 0%, for WCPL, WCPM and WCPP respectively in batch reactors (Gutiérrez and Caldera, 2011; Gutiérrez *et al.,* 2007; Rincón *et al.*, 2006).

Behling *et al.* (2003) commented that the COD removal in RBC system used to treat WCPC was 76.1%, while Rincón *et al.* (2004) studied a UASB-SBR system and reported that the COD removal reached 79%, indicating that was important removals of phenols and hydrocarbons

Comparing the biodegradability of WCPL, WCPM and WCPH, it is observed that the WCPL present the major biodegradability in the different treatment systems and operating conditions

The biodegradability of the WCP has been associated to diverse factors as SARA composition, phenols concentration, alkalinity, organic load, metals concentration and temperature.

Some researchers (Rincón *et al.,* 2002; Gutiérrez *et al.,* 2006; Gutiérrez *et al.,* 20007) argue that the WCPL biodegradability is good in anaerobic systems under mesophilic conditions and under thermophilic conditions. The final decision between the temperature used and size of the reactor will be economical, because the WCPL are at atmospheric temperature and the termophilic route implicates to consider the costs associated of warming the water. In the cases

Other researchers (Gutiérrez *et al.,* 2007; Gutiérrez *et al.,* 2001; Gutiérrez *et al.,* 2012) share that the biodegradability of WCP is associated to the SARA composition present in these waters, as product of the contact with the crude oil associated. The difference of composition of these fractions confer characteristics that influence in their biodegradability, because the SARA fractions change in relation to the crude oil that is in contact with the WCP, being the WCPL the waters with the biggest percentages of saturated, considered more biodegradable than

When the organic fractions present in the WCP are compared, it is observed that WCPM and WCPH present a similar content of organic fractions (p>0.05). The opposite case was observed with the WCPL, which organic fractions are different in saturated, aromatics and resins, in

On the other hand, there is a tendency to increase the saturated fractions in WCP (r=0.871) with the increase of the API gravity of the crude oil with the water associated, following the order WCPL>WCPM>WCPH. In relation to the resins, it was observed that it increases with regard to the decrease of the API gravity of the crude oil with the WCP were associated following the

A study realized by Díaz *et al.* (2007) with WCPM from other tank farm of the Venezuelan petroleum industry, indicated that the SARA fractions can be removed from the WCP using UASB reactors. They obtained removals of 72% of saturated, 91% of resins and 71% of asphaltenes, and did not obtain removals of aromatics. They associated these results with the increases of the aromatic fractions for degradation of the fractions like resins and asphaltenes

of WCPM and WCPH the studies realized up to the moment are not conclusive.

were obtained.

18 Biodegradation - Engineering and Technology

**3. Discussion**

WCPM and WCPH.

comparing to WCPM and WCPH (p>0.05).

order WCPL<WCPM<WCPH.

to aromatics.

studied.

In regard to phenols concentration, the studies mention that the consortium of microorganisms developed in mesophilic UASB reactors were influenced by the initial phenols concentrations, indicating that the phenols removal might be associated with the presence of different phenols compounds in the different types of WCP, with varied resistance to degradation and metab‐ olism (aerobic/anaerobic).

Additionally, the studies indicate that the alkalinity values in the WCP were between 900 and 3000 mg CaCO3/L. It has been commented that the difference of COD removal might be due to the acidity-basicity conditions in the WCP. The WCPH presented lower values of alkalinity (642.9-580.4 mg CaCO3/L) and lower COD removal than WCPM. The alkalinity of WCPM was superior to 2000 mg CaCO3/L. As for the pH, the WCP presented basic pH (7-10) for the different treatments.

In other cases, it is mentioned that the presence of metals in the WCP makes the treatment more complex. However, the metals K, Na, Fe, Cr, Pb and Zn can be used by thermophilic microorganisms or can be removed from the WCP and reach to the sludge by diverse mech‐ anisms (Gutiérrez *et al.*, 2009).

In relation to degrading microorganisms present in the WCP, some have been isolated, and identified the genus *Aeromonas, Klebsielle, Xanthomona, Bacteroides* and *Acinetobacter*, as well as a consortium of them, that resulted to be effective in COD decrease (Castro *et al.,* 2008).

The Table 7 shows that WCP has been treated by coagulation-flocculation at laboratory level using chitosane as a coagulating agent in concentrations of 24 to 38 mg/L of solution of commercial chitosane (CCH), and by dissolved air flotation (DAF) using a cationic flocculants of high molecular weight.

Rojas *et al.* (2008) reported that the TSS removal and the turbidity in the WCPC were 77% and 69% respectively. On the other hand, Caldera *et al.* (2009, 2011) commented that the turbidity removal in the WCPH was 90.7%, accompanied of COD removal of 50.7%. In any case, the hydrocarbons removal and oils removal by physicochemical methods were between 70% and 90%, concluding that the cationic polymers represent an alternative to remove oily compounds in the WCP.

Table 8 shows other alternatives applied to treat WCP. In constructed sub-superficial wetlands COD removal of WCPC was between 31.4% and 65.7%, while in constructed superficial wetlands there was no COD removal. Both systems showed efficiency to remove more than 60% of the hydrocarbons present in the WCPC (Paz *et al.*, 2012; Blanco *et al.*, 2008).

The application of ozone also has been proposed to increase the biodegradability of the WCP. According to Gutiérrez *et al.* (2002), the application of ozone improves considerably the biodegradability of the WCP, with an increase of up to 87%. They concluded that the applica‐ tion of doses of ozone to WCP in the order of 30 mg/L of ozone, would affect favorably in the later biological processes applied.

[2] Behling, E, Marín, J, Gutiérrez, E, & Fernández, N. (2003). Aerobic treatment of two industrial effluents using a rotating biological contactor reactor. Multiciencias, 3 (2),

Biodegradability of Water from Crude Oil Production

http://dx.doi.org/10.5772/56328

21

[3] Blanco, E, Gutiérrez, E, Caldera, Y, Núñez, M, & Paz, N. (2008). Tratamiento de aguas de producción a través de humedales construidos de flujo subsuperficial a es‐ cala piloto. Memorias del XXXI Congreso Interamericano de Ingeniería Sanitaria y

[4] Caldera, Y, Rodriguez, Y, Oñate, H, Prato, J, & Gutiérrez, E. (2011). Efficiency of chi‐ tosan as coagulant during treatment of low turbidity water associated crude oil pro‐

[5] Caldera, Y, Clavel, N, Briceño, D, Nava, A, Gutiérrez, E, & Mármol, Z. (2009). Chito‐ san as a coagulant during treatment of water from crude oil production. Boletín del

[6] Caldera, Y, Gutiérrez, E, Madueño, P, Griborio, A, & Fernández, N. (2007). Anaerobic biodegradability of industrial effluents in an UASB reactor. Impacto Científico, 2 (1),

[7] Castro, F, Fernández, N, & Chávez, M. (2008). Diminution of the COD in formation

[8] Díaz, A, Rincón, N, López, F, Fernández, N, Chacín, E, & Debellefontaine, H. (2005b). The biological treatment in sequencing batch reactor (SBR) of effluents from the ex‐

[9] Díaz, A, Rincón, N, Marín, J, Behling, E, Chacín, E, & Fernández, N. (2005a). Degra‐ dation of total phenols during biological treatment of oilfields produced water. Cien‐

[10] Díaz, V, Bauza, R, Cepeda, N, Behling, E, Díaz, A, Fernández, N, & Rincón, N. (2007). Development of micro-SARA method for organic fractions of crude oil determination on the oil extraction production waters with anaerobic treatment. Ciencia, 15, (1),

[11] Gaceta Oficial 5021 de la República de Venezuela ((1995). Caracas 18 de diciembre de 1995. 5021, Extraordinaria. Decreto 883. Normas para la clasificación y el control de

[12] García, A, Arreguín, F, Hernández, S, & Lluch, D. (2004). Impacto ecológico de la in‐ dustria petrolera en sonda de Campeche, México, tras tres décadas de actividad: Una

[13] González, Y, Rincón, N, López, F, & Díaz, A. (2007). Organic matter removal from the petroleum effluents by a sequencing batch reactor (SBR). Rev. Téc. Ing. Univ. Zu‐

waters using bacterial stocks. Rev. Téc. Ing. Univ. Zulia, 31 (3), 256-265.

traction of medium oil production. Multiciencias, 5 (2), 150-156.

las aguas de los cuerpos de agua y vertidos o efluentes líquidos.

Ambiental, AIDIS, 12 al 15 de octubre, Santiago de Chile, Chile.

duction. Revista Tecnocientífica URU, 1 (1), 54-60.

Centro de Investigaciones Biológicas, 43 (4), 541-555.

126-135.

11-23.

cia, 13 (3), 281-291.

revisión. Interciencia, 29(6), 311-319.

lia, 30 (Edición Especial), 82-89.

95-104.

## **4. Conclusions**

The WCP from the different cuts: light (WCPL), medium (WCPM), heavy (WCPH) and combinations of them (WCPC), have different characteristics and their biodegradability or treatment are associated on the SARA compositions, organic matters concentration, hydro‐ carbons and phenols concentrations, and the operation conditions (HRT and temperature).

The biodegradability of the WCP followed the order WCPL>WCPM>WCPH.

The COD removal in biological systems changed between 67%-95%, 23.5%-89.8% and 35%-66% for WCPL, WCPM and WCPH, respectively.

The physicochemical treatment DAF and coagulation, removed hydrocarbons and oils between 70% and 90%.

Other parameters like phenols, hydrocarbons and SARA fractions, can be removed from the WCP by biological treatments.

It is necessary to analyze other parameters and operating conditions, as well as to conduct an economic evaluation before the treatment selection.

## **Author details**

Edixon Gutiérrez1 and Yaxcelys Caldera2

\*Address all correspondence to<EMAIL_ADDRESS><EMAIL_ADDRESS>
1 Centro de Investigación del Agua. Facultad de Ingeniería, Universidad del Zulia, Maracai‐ bo, estado Zulia, Venezuela

2 Laboratorio de Investigaciones Ambientales, Núcleo Costa Oriental del Lago, Universidad del Zulia. Cabimas, estado Zulia, Venezuela

## **References**

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[2] Behling, E, Marín, J, Gutiérrez, E, & Fernández, N. (2003). Aerobic treatment of two industrial effluents using a rotating biological contactor reactor. Multiciencias, 3 (2), 126-135.

tion of doses of ozone to WCP in the order of 30 mg/L of ozone, would affect favorably in the

The WCP from the different cuts: light (WCPL), medium (WCPM), heavy (WCPH) and combinations of them (WCPC), have different characteristics and their biodegradability or treatment are associated on the SARA compositions, organic matters concentration, hydro‐ carbons and phenols concentrations, and the operation conditions (HRT and temperature).

The COD removal in biological systems changed between 67%-95%, 23.5%-89.8% and 35%-66%

The physicochemical treatment DAF and coagulation, removed hydrocarbons and oils

Other parameters like phenols, hydrocarbons and SARA fractions, can be removed from the

It is necessary to analyze other parameters and operating conditions, as well as to conduct an

1 Centro de Investigación del Agua. Facultad de Ingeniería, Universidad del Zulia, Maracai‐

2 Laboratorio de Investigaciones Ambientales, Núcleo Costa Oriental del Lago, Universidad

[1] Ahmad, A, Sumathi, S, & Hameed, B. (2006). Coagulation of residue oil and suspend‐ ed solid in palm oil milk effluent by chitosan, alum and PAC. Chemical Engineering

The biodegradability of the WCP followed the order WCPL>WCPM>WCPH.

later biological processes applied.

20 Biodegradation - Engineering and Technology

for WCPL, WCPM and WCPH, respectively.

economic evaluation before the treatment selection.

and Yaxcelys Caldera2

\*Address all correspondence to<EMAIL_ADDRESS><EMAIL_ADDRESS>
**4. Conclusions**

between 70% and 90%.

**Author details**

Edixon Gutiérrez1

**References**

bo, estado Zulia, Venezuela

del Zulia. Cabimas, estado Zulia, Venezuela

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[21] Lepisto, R, & Rintala, J. (1999). Extreme thermophilic (70ºC), VAF-FED UASB reactor; performance, temperature response, load potential and comparation with 35 and

[22] Lettinga, G. (2005). A good life environment for all through conceptual, technological and social innovations. Memorias del VIII Taller y Simposio Latinoamericano sobre

[23] Lettinga, G. (2001). Digestion and degradation, air for life. Water Science Tech., 44

[24] Li, Q, Kang, C, & Zhang, C. (2005). Wastewater produced from an oilfield and con‐ tinuous treatment with an oil-degrading bacterium. Process Biochemistry, 40 (2),

[25] Paz, N, Blanco, E, Gutiérrez, E, Núñez, M, & Caldera, Y. (2012). Pilot scale superficial flow constructed wetlands for sulfide and phenols removals from oil field producer

[26] Renault, F, Sancey, B, Bodot, P, & Crini, G. (2009). Chitosan for coagulation/floccula‐ tion process- An eco-friendly approach. European Polymer Journal, 45 (5), 1337-1348.

[27] Rincón, N, Behling, E, & Cepeda, N. (2004). Combinación de tratamientos anaerobioaerobio de aguas provenientes de la industria petrolera venezolana. Memorias del

ción de crudo en sistemas biológicos anaerobios. Intellectus, 1 (1), 11-21.

tín del Centro de Investigaciones Biológicas, 40 (3), 242-256.

actors. Rev. Téc. Ing. Univ. Zulia, 30 (2), 111- 117.

del Centro de Investigaciones Biológicas, 43 (1), 145-160.

55ºC UASB reactor. Water Research, 33 (14), 3162-3170.

Digestión Anaerobia. Punta del Este, Uruguay, 1-15.

water. Rev. Téc. Ing. Univ. Zulia, 35 (1), 71-79.

27-33.

22 Biodegradation - Engineering and Technology

nocientífica URU, 2 (2), 31-37.

ciencias, 2 (1), 50-54

(8), 157-176.

873-877.

**Chapter 2**

**Emulsification of Hydrocarbons Using Biosurfactant**

**Puebla, Mexico**

Beatriz Pérez-Armendáriz, Amparo Mauricio-Gutiérrez, Teresita Jiménez-Salgado,

http://dx.doi.org/10.5772/56143

high incidence of contamination.

metabolites and respiration rate.

**1. Introduction**

Armando Tapia-Hernández and Angélica Santiesteban-López

Additional information is available at the end of the chapter

**Producing Strains Isolated from Contaminated Soil in**

Among Mexico's main riches are its oil and the great expanses of land used to grow food. A large number of pipelines pass through Mexico's agricultural region carrying diesel, gasoline or crude oil, however, lack of maintenance of the pipeline installations, fuel theft, vehicle transport and even the topographical, terrain and hydrological conditions of the site cause a

Petrolic activities have generated extensive pollution of soils worldwide, mainly in those regions where petroleum is explored, extracted, and refined. The composition of hydrocarbons on polluted soil varies according to environmental conditions and natural degradation processes. In México there are soil impacted by weathered hydrocarbons, which are predom‐ inantly saturated and aromatic, become more recalcitrant if polluted soils are not remediated,

Hydrocarbon spills on agricultural soil have direct repercussions on soil quality and its function. Some authors [1] indicate that hydrocarbon contamination reduces food crop growth by preventing water and nutrient absorption through the roots, and reducing the transport of

> © 2013 Pérez-Armendáriz et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Pérez-Armendáriz et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

affecting the underground water, food chains, and diverse human activities.

## **Emulsification of Hydrocarbons Using Biosurfactant Producing Strains Isolated from Contaminated Soil in Puebla, Mexico**

Beatriz Pérez-Armendáriz, Amparo Mauricio-Gutiérrez, Teresita Jiménez-Salgado, Armando Tapia-Hernández and Angélica Santiesteban-López

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56143

**1. Introduction**

Among Mexico's main riches are its oil and the great expanses of land used to grow food. A large number of pipelines pass through Mexico's agricultural region carrying diesel, gasoline or crude oil, however, lack of maintenance of the pipeline installations, fuel theft, vehicle transport and even the topographical, terrain and hydrological conditions of the site cause a high incidence of contamination.

Petrolic activities have generated extensive pollution of soils worldwide, mainly in those regions where petroleum is explored, extracted, and refined. The composition of hydrocarbons on polluted soil varies according to environmental conditions and natural degradation processes. In México there are soil impacted by weathered hydrocarbons, which are predom‐ inantly saturated and aromatic, become more recalcitrant if polluted soils are not remediated, affecting the underground water, food chains, and diverse human activities.

Hydrocarbon spills on agricultural soil have direct repercussions on soil quality and its function. Some authors [1] indicate that hydrocarbon contamination reduces food crop growth by preventing water and nutrient absorption through the roots, and reducing the transport of metabolites and respiration rate.

© 2013 Pérez-Armendáriz et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Pérez-Armendáriz et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The recovery of hydrocarbon-contaminated agricultural soil in Mexico is a complex theme because the producers harvest the crops for sustenance or sale. A remedy is therefore needed that uses sustainable biological technologies which do not pose a risk for the products of the harvests. The production of biosurfactants to recover agricultural soil used for food production is a viable alternative because of their biodegradability. Furthermore, biosurfactants have been used in the oil industry to recover oils from hydrocarbons, in the emulsification of heavy hydrocarbon fractions and in the degradation of polychlorinated biphenyls [2] and polycyclic aromatic hydrocarbons (PAH's) [3].

[11]. Several authors have reported bacterial strains isolated from hydrocarbon-contaminated soil and water which present emulsifying activity and which are capable of growing in oil using it as sole carbon source. The reported microorganisms are: *Pseudomonas aeruginosa*, *P. mendocina*, *P. aureofasciens*, *Listonella damsela*, *Bacillus sphaericus*, *B. brevis, Enterobacter cloacae, Acinetobacter calcoaceticus* var. *anitratus, Hafnia alvei, Citrobacter freundii, C. amalonaticus, Sphingobacterium multivorum, Staphylococcus* sp*, Neisseria* sp*, Micrococcus* sp*, Serratia rubidae, Alcaligenes, Flavobacterium, Nocardia, Achromobacter, Arthrobacter* [12-16]. There has been a recent rise in the study of biosurfactant for their antimicrobial characteristics as fungicide [17,

Emulsification of Hydrocarbons Using Biosurfactant Producing Strains Isolated from Contaminated Soil in Puebla…

http://dx.doi.org/10.5772/56143

27

The use of biosurfactants for the bioremediation of hydrocarbon contaminated soil has been studied intensely since the last decade [2-3, 20]. Biosurfactants have been used by the oil industry to enhanced oil recovery [21, 22], in the emulsification of heavy hydrocarbon fractions [23], and in the treatment of wastewater with insoluble substances. They have also been used in the degradation of polychlorinated biphenyls [2]. Chemical surfactants have the advantage of being non-toxic, environmentally friendly, and biodegradable and can be produced from

Biosurfactants can be used as additives to stimulate bioremediation; however, the concentra‐ tion of these can also be increased by the addition of bioemulsifier-producing bacteria. Bioemulsifier-producing bacteria can participate in the biodegradation of hydrocarbons and, alternatively, function as a family of bacteria that supply emulsifiers to another group of

A mixture of biosurfactants including cellular lipids produced during the degradation of heavy hydrocarbons, and additives increases solubility and facilitates hydrocarbon degradation. Cellular lipids have excellent surfactant properties and can form micelles at low concentra‐ tions, but these surfactants do not release the solubilized organic compounds to degrade them [25]. An increase in the apparent solubility of naphthalene has been observed when the concentration of glycolipids excreted by *Pseudomonas areuginosa* 19SJ exceeds the critical

Biosurfactants have different chemical compositions depending on the microorganism that produces them and may be lipopeptides, lipoproteins, fatty acids or phospholipids [27]. The production of biosurfactants depends on physicochemical factors (aeration, pH, substrate availability) and their evaluation will depend on kinetic factors (substrate consumption, product formation, and biomass production). Knowing the kinetics of biosurfactant produc‐ tion will allow the proposal of sustainable oil hydrocarbon recovery technologies for aqueous

Mexico has large areas of soil contaminated by oil activities; especially agricultural soils have few alternatives of sustainable technologies, therefore in this work different microorganisms were isolated from hydrocarbons-contaminated soil and the kinetics of biosurfactant produc‐ tion was studied to generate a proposal for the recovery of oil hydrocarbons as Maya crude

18] and as, zoospore inhibitors [19].

agricultural substrates [10].

bacteria that degrade the contaminants [24].

micellar concentration (CMC) [26].

or solid systems.

oil.

## **2. Approach to the problem**

In the agricultural fields of Puebla, Mexico two hydrocarbon spills have been reported due to lack of pipeline maintenance. In 2002, a crude oil spill in the town of Acatzingo, Puebla affected a large expanse of agricultural land (approximately 50 hectares) [4]. And in San Martin Texmelucan, Puebla on December 19, 2010, the explosion caused by a crude oil spill took 30 human lives and greatly affected the agricultural land of the population [5]. The inhabitants of the affected regions still perceive damage to the soil and do not consider the land to be fully recovered [4].

In Mexico, the environmental impact of oil industry activities is rigorously controlled by the authorities (Federal Environmental Protection Agency, *Procuraduría Federal de Protección al Medio Ambiente*, PROFEPA) and therefore recuperation should take only a short time. Biore‐ mediation processes have not given the expected results: expanses of contaminated land are heterogeneous as far as climate, water availability and oxygen availability, and the biostimu‐ lation of microbial populations is insufficient due to competing autochthonous microorgan‐ isms and inadequate nutritional balance [6, 7].

Mexico relies mainly on micro-encapsulation technology for the restoration of hydrocarboncontaminated land, according to the National Ecology Institute (*Instituto Nacional de Ecologia*, INE) [6] using chemical substances which encapsulate hydrocarbons and prevent biodegra‐ dation. Surfactants have also been used to restore marine sediment with a recovery of 45,000 t [8]. Chemical surfactants, however, are not always environmentally biodegradable [9] and so there is a need to use biosurfactants to recover oil hydrocarbons in impacted soils.

## **3. Area of application**

Biosurfactants are molecules with a polar region and a non-polar region, and are hence considered amphipathic, produced by extracellular or intracellular microorganisms, also can reduce surface tension at the air-water interface between two immiscible liquids or between the solid-water interface [10].

Biosurfactants have other advantages over chemical detergents since they are non-toxic and ecologically acceptable [10]. They are also highly effective at breaking down surface tension [11]. Several authors have reported bacterial strains isolated from hydrocarbon-contaminated soil and water which present emulsifying activity and which are capable of growing in oil using it as sole carbon source. The reported microorganisms are: *Pseudomonas aeruginosa*, *P. mendocina*, *P. aureofasciens*, *Listonella damsela*, *Bacillus sphaericus*, *B. brevis, Enterobacter cloacae, Acinetobacter calcoaceticus* var. *anitratus, Hafnia alvei, Citrobacter freundii, C. amalonaticus, Sphingobacterium multivorum, Staphylococcus* sp*, Neisseria* sp*, Micrococcus* sp*, Serratia rubidae, Alcaligenes, Flavobacterium, Nocardia, Achromobacter, Arthrobacter* [12-16]. There has been a recent rise in the study of biosurfactant for their antimicrobial characteristics as fungicide [17, 18] and as, zoospore inhibitors [19].

The recovery of hydrocarbon-contaminated agricultural soil in Mexico is a complex theme because the producers harvest the crops for sustenance or sale. A remedy is therefore needed that uses sustainable biological technologies which do not pose a risk for the products of the harvests. The production of biosurfactants to recover agricultural soil used for food production is a viable alternative because of their biodegradability. Furthermore, biosurfactants have been used in the oil industry to recover oils from hydrocarbons, in the emulsification of heavy hydrocarbon fractions and in the degradation of polychlorinated biphenyls [2] and polycyclic

In the agricultural fields of Puebla, Mexico two hydrocarbon spills have been reported due to lack of pipeline maintenance. In 2002, a crude oil spill in the town of Acatzingo, Puebla affected a large expanse of agricultural land (approximately 50 hectares) [4]. And in San Martin Texmelucan, Puebla on December 19, 2010, the explosion caused by a crude oil spill took 30 human lives and greatly affected the agricultural land of the population [5]. The inhabitants of the affected regions still perceive damage to the soil and do not consider the land to be fully

In Mexico, the environmental impact of oil industry activities is rigorously controlled by the authorities (Federal Environmental Protection Agency, *Procuraduría Federal de Protección al Medio Ambiente*, PROFEPA) and therefore recuperation should take only a short time. Biore‐ mediation processes have not given the expected results: expanses of contaminated land are heterogeneous as far as climate, water availability and oxygen availability, and the biostimu‐ lation of microbial populations is insufficient due to competing autochthonous microorgan‐

Mexico relies mainly on micro-encapsulation technology for the restoration of hydrocarboncontaminated land, according to the National Ecology Institute (*Instituto Nacional de Ecologia*, INE) [6] using chemical substances which encapsulate hydrocarbons and prevent biodegra‐ dation. Surfactants have also been used to restore marine sediment with a recovery of 45,000 t [8]. Chemical surfactants, however, are not always environmentally biodegradable [9] and

Biosurfactants are molecules with a polar region and a non-polar region, and are hence considered amphipathic, produced by extracellular or intracellular microorganisms, also can reduce surface tension at the air-water interface between two immiscible liquids or between

Biosurfactants have other advantages over chemical detergents since they are non-toxic and ecologically acceptable [10]. They are also highly effective at breaking down surface tension

so there is a need to use biosurfactants to recover oil hydrocarbons in impacted soils.

aromatic hydrocarbons (PAH's) [3].

26 Biodegradation - Engineering and Technology

**2. Approach to the problem**

isms and inadequate nutritional balance [6, 7].

**3. Area of application**

the solid-water interface [10].

recovered [4].

The use of biosurfactants for the bioremediation of hydrocarbon contaminated soil has been studied intensely since the last decade [2-3, 20]. Biosurfactants have been used by the oil industry to enhanced oil recovery [21, 22], in the emulsification of heavy hydrocarbon fractions [23], and in the treatment of wastewater with insoluble substances. They have also been used in the degradation of polychlorinated biphenyls [2]. Chemical surfactants have the advantage of being non-toxic, environmentally friendly, and biodegradable and can be produced from agricultural substrates [10].

Biosurfactants can be used as additives to stimulate bioremediation; however, the concentra‐ tion of these can also be increased by the addition of bioemulsifier-producing bacteria. Bioemulsifier-producing bacteria can participate in the biodegradation of hydrocarbons and, alternatively, function as a family of bacteria that supply emulsifiers to another group of bacteria that degrade the contaminants [24].

A mixture of biosurfactants including cellular lipids produced during the degradation of heavy hydrocarbons, and additives increases solubility and facilitates hydrocarbon degradation. Cellular lipids have excellent surfactant properties and can form micelles at low concentra‐ tions, but these surfactants do not release the solubilized organic compounds to degrade them [25]. An increase in the apparent solubility of naphthalene has been observed when the concentration of glycolipids excreted by *Pseudomonas areuginosa* 19SJ exceeds the critical micellar concentration (CMC) [26].

Biosurfactants have different chemical compositions depending on the microorganism that produces them and may be lipopeptides, lipoproteins, fatty acids or phospholipids [27]. The production of biosurfactants depends on physicochemical factors (aeration, pH, substrate availability) and their evaluation will depend on kinetic factors (substrate consumption, product formation, and biomass production). Knowing the kinetics of biosurfactant produc‐ tion will allow the proposal of sustainable oil hydrocarbon recovery technologies for aqueous or solid systems.

Mexico has large areas of soil contaminated by oil activities; especially agricultural soils have few alternatives of sustainable technologies, therefore in this work different microorganisms were isolated from hydrocarbons-contaminated soil and the kinetics of biosurfactant produc‐ tion was studied to generate a proposal for the recovery of oil hydrocarbons as Maya crude oil.

## **4. Materials and methods**

## **4.1. Isolation of biosurfactant-producing strains**

Soil sampling was done in an agricultural area of Acatzingo, Puebla, Mexico with the following geographical coordinates 18° 57' 03.0" N 97° 46' 20.5" W. Biosurfactant-producing strains were isolated using 1 g of soil in 10 mL of pre-sterilized distilled water. The culture medium was composed of (g / L): (NH4)2SO4 7.7, KH2PO4 5.7, K2HPO4 2, MgSO47H2O 2, CaCl22H2O 0.005, FeCl36H2O 0.0025, agar 15; distilled water 1,000 mL and preadapted to a petroleum environ‐ ment using the Maya petroleum provided by the Mexican State company (PEMEX). Maya petroleum was added on sterilized filter paper (3 cm2 ; with 2 g petroleum) to every lid in order to develop an atmosphere of volatile hydrocarbons inside the petri dish.

**4.4. Glucose consumption, pH and Critical Micelle Concentration (CMC)**

The Critical Micelle Concentration (CMC) was determined according to [29].

medium. If necessary it was diluted with distilled water.

was maintained close to neutrality by adding 0.1N NaOH.

**4.6. Biodegradation tests of maya crude oil**

**4.7. Viability of microorganisms**

by APIwebTM identification software (bioMerieux).

**4.5. Statistical analysis**

linear model.

The glucose was determined by the AOAC 969.39 method taking a 2 mL aliquot of culture

Emulsification of Hydrocarbons Using Biosurfactant Producing Strains Isolated from Contaminated Soil in Puebla…

http://dx.doi.org/10.5772/56143

29

The pH was determined with a potentiometer (Conductronic pH 10). In this investigation, pH

The results were adjusted to a linear model to obtain the rate of substrate consumption (g glucose h-1), the rate of biomass production (g biomass h-1) and emulsification activity (% emulsifier h-1). The slopes (rates) and correlation coefficients were obtained from regression

In addition, the average initial and final samples of emulsification activity were analyzed by variant analysis to find significant differences and Duncan-Waller multiple comparison tests.

A preculture of selected strains was grown in Banat broth at 30 °C under constant agitation (200 rpm) for 24 h. An aliquot of the selected strains was taken at an absorbance of 70 UK, inoculated in flasks with 50 mL of medium at a pH of 6.5 with 20,000 ppm of crude oil and incubated at 30 °C for 15 days. Following the incubation process, the samples were put in contact with HPLC grade hexane and agitated for 2 minutes. The mixture was then sonicated (Branson 1210 Ultrasonic Cleaner) for 10 minutes before being transferred to a 250 mL separatory funnel leaving the aqueous phase to decant for later use (Figure 1A). The organic phase, in which the hydrocarbons are found, was recovered by means of an asbestos filter and Na2SO4 anhydrous as a desiccant in a 50 mL balloon flask. The organic phase was then distilled

In addition to the hydrocarbon degradation capacity, the viability of the strains was deter‐ mined at 8, 16 and 24 days of incubation. The organic phase was therefore eliminated by centrifugation (3000 rpm for 5 minutes) and successive serial dilutions made of 10-6 and cultivated on plates of Lebac medium. Isolates strains were grown overnight in Lebac broth at 37 °C under constant agitation at 200 rpm. The biochemical characterization was carried out by the API 20 E, API 20 NE and API 50 CH systems (references No. 20160, 20050 and 50300; bioMérieux) following the manufacturer's recommendations. The identification was assessed

The statistical package used was Minitab version 13 (licensed to UPAEP, Mexico).

using a Büchi Rotavapor R11 with operating temperature of 45 °C (Figure 1B).

The bacteria were then isolated and grown in a liquid mineral medium (g / L): (NH4)2SO4 7, KH2PO4 5.7, K2HPO4 2, MgSO47H2O 2, CaCl22H2O 0.005, FeCl36H2O 0.0025, Yeast extract 0.1, glucose 20. Strains presenting biosurfactant production were identified as UPAEP 6, UPAEP 8, UPAEP 9, UPAEP 10, UPAEP 12 and UPAEP 15. The following bacteria were also bought *Arthrobacter* sp ATCC 31012, *Bacillus subtilis* ATCC 21332, *Candida petrophilum* ATCC 20226.

## **4.2. Strain selection**

The selected strains were grown in 50 mL of Lebac medium (g / L): (NH4)2SO4 7, KH2PO4 5.7, K2HPO4 2, MgSO47H2O 2, CaCl22H2O 0.005, FeCl36H2O 0.0025, Yeast extract 0.1, glucose 20, pH 7.0; in 200 mL Erlenmeyer flasks with a 200 μL aliquot of microorganisms. Twenty-four flasks of each strain were placed in an incubator (FELISA) at 37 °C under constant agitation at 200 rpm. Three flasks were removed at each interval over a 44 and 48 h kinetic.

The parameters evaluated over time were: biomass production, pH, emulsification activity on engine oil and glucose consumption.

Biomass production was determined by taking 2 mL of culture medium and passing it through a pre-dried and pre-weighed cellulose nitrate membrane filter (0.22 μm in diameter). The filter with the biomass was then dried at 100°C for 24 h until constant weight was attained; the biomass was reported in g obtained by weight difference.

## **4.3. Emulsification index**

Emulsification activity was determined by placing 6 mL of engine oil and 4 mL of culture medium with the biosurfactant-producing strains in a vortex [28]. They were agitated for 2 minutes and left to rest for 24 h. The percentage of emulsification was estimated according the following expression:

% Emulsifier = ((Total height of the mixture - Height of emulsified oil) / Total height of the mixture) \* 100

## **4.4. Glucose consumption, pH and Critical Micelle Concentration (CMC)**

The glucose was determined by the AOAC 969.39 method taking a 2 mL aliquot of culture medium. If necessary it was diluted with distilled water.

The pH was determined with a potentiometer (Conductronic pH 10). In this investigation, pH was maintained close to neutrality by adding 0.1N NaOH.

The Critical Micelle Concentration (CMC) was determined according to [29].

## **4.5. Statistical analysis**

**4. Materials and methods**

28 Biodegradation - Engineering and Technology

**4.2. Strain selection**

engine oil and glucose consumption.

**4.3. Emulsification index**

following expression:

mixture) \* 100

biomass was reported in g obtained by weight difference.

**4.1. Isolation of biosurfactant-producing strains**

petroleum was added on sterilized filter paper (3 cm2

to develop an atmosphere of volatile hydrocarbons inside the petri dish.

Soil sampling was done in an agricultural area of Acatzingo, Puebla, Mexico with the following geographical coordinates 18° 57' 03.0" N 97° 46' 20.5" W. Biosurfactant-producing strains were isolated using 1 g of soil in 10 mL of pre-sterilized distilled water. The culture medium was composed of (g / L): (NH4)2SO4 7.7, KH2PO4 5.7, K2HPO4 2, MgSO47H2O 2, CaCl22H2O 0.005, FeCl36H2O 0.0025, agar 15; distilled water 1,000 mL and preadapted to a petroleum environ‐ ment using the Maya petroleum provided by the Mexican State company (PEMEX). Maya

The bacteria were then isolated and grown in a liquid mineral medium (g / L): (NH4)2SO4 7, KH2PO4 5.7, K2HPO4 2, MgSO47H2O 2, CaCl22H2O 0.005, FeCl36H2O 0.0025, Yeast extract 0.1, glucose 20. Strains presenting biosurfactant production were identified as UPAEP 6, UPAEP 8, UPAEP 9, UPAEP 10, UPAEP 12 and UPAEP 15. The following bacteria were also bought *Arthrobacter* sp ATCC 31012, *Bacillus subtilis* ATCC 21332, *Candida petrophilum* ATCC 20226.

The selected strains were grown in 50 mL of Lebac medium (g / L): (NH4)2SO4 7, KH2PO4 5.7, K2HPO4 2, MgSO47H2O 2, CaCl22H2O 0.005, FeCl36H2O 0.0025, Yeast extract 0.1, glucose 20, pH 7.0; in 200 mL Erlenmeyer flasks with a 200 μL aliquot of microorganisms. Twenty-four flasks of each strain were placed in an incubator (FELISA) at 37 °C under constant agitation at

The parameters evaluated over time were: biomass production, pH, emulsification activity on

Biomass production was determined by taking 2 mL of culture medium and passing it through a pre-dried and pre-weighed cellulose nitrate membrane filter (0.22 μm in diameter). The filter with the biomass was then dried at 100°C for 24 h until constant weight was attained; the

Emulsification activity was determined by placing 6 mL of engine oil and 4 mL of culture medium with the biosurfactant-producing strains in a vortex [28]. They were agitated for 2 minutes and left to rest for 24 h. The percentage of emulsification was estimated according the

% Emulsifier = ((Total height of the mixture - Height of emulsified oil) / Total height of the

200 rpm. Three flasks were removed at each interval over a 44 and 48 h kinetic.

; with 2 g petroleum) to every lid in order

The results were adjusted to a linear model to obtain the rate of substrate consumption (g glucose h-1), the rate of biomass production (g biomass h-1) and emulsification activity (% emulsifier h-1). The slopes (rates) and correlation coefficients were obtained from regression linear model.

In addition, the average initial and final samples of emulsification activity were analyzed by variant analysis to find significant differences and Duncan-Waller multiple comparison tests. The statistical package used was Minitab version 13 (licensed to UPAEP, Mexico).

## **4.6. Biodegradation tests of maya crude oil**

A preculture of selected strains was grown in Banat broth at 30 °C under constant agitation (200 rpm) for 24 h. An aliquot of the selected strains was taken at an absorbance of 70 UK, inoculated in flasks with 50 mL of medium at a pH of 6.5 with 20,000 ppm of crude oil and incubated at 30 °C for 15 days. Following the incubation process, the samples were put in contact with HPLC grade hexane and agitated for 2 minutes. The mixture was then sonicated (Branson 1210 Ultrasonic Cleaner) for 10 minutes before being transferred to a 250 mL separatory funnel leaving the aqueous phase to decant for later use (Figure 1A). The organic phase, in which the hydrocarbons are found, was recovered by means of an asbestos filter and Na2SO4 anhydrous as a desiccant in a 50 mL balloon flask. The organic phase was then distilled using a Büchi Rotavapor R11 with operating temperature of 45 °C (Figure 1B).

#### **4.7. Viability of microorganisms**

In addition to the hydrocarbon degradation capacity, the viability of the strains was deter‐ mined at 8, 16 and 24 days of incubation. The organic phase was therefore eliminated by centrifugation (3000 rpm for 5 minutes) and successive serial dilutions made of 10-6 and cultivated on plates of Lebac medium. Isolates strains were grown overnight in Lebac broth at 37 °C under constant agitation at 200 rpm. The biochemical characterization was carried out by the API 20 E, API 20 NE and API 50 CH systems (references No. 20160, 20050 and 50300; bioMérieux) following the manufacturer's recommendations. The identification was assessed by APIwebTM identification software (bioMerieux).

## **4.8. Biosurfactant recovery**

The purification of biosurfactant was performed according to a modified technique described in [30]. With the strains with highest percentage of emulsifier. The strains were previously grown in 500 mL of Lebac medium. The biosurfactant was then extracted from the bacteria with isopropanol-ethanol (3:1) analytical grade (Merck, México) in a separatory flask. It was centrifuged at 1200 rpm for 30 minutes (Solbat), and the supernatant was eliminated. The sample was then filtered using cellulose paper grade 101 (Millipore 2.5μ M). The precipitate obtained was dried for 24 h at 60 °C in an oven (FELISA) and stored in an Eppendorf vial to determine the yield.

of *Bacillus subtilis* ATCC 21332 which consumed 93.6 % of glucose (Table 2). Nevertheless, the glucose consumption was inversely proportional to the biomass production during the cell growth (data not shown). The emulsification index was directly proportional at production of biomass, except *Candida petrophilum* ATCC 20226 which showed no relation. Biosurfactant synthesis and biomass production by UPAEP 6, 9, 10, and 15 (Figures 2, 4,5 and 10) strains began during the first few hours (4 to 8) as a response to substrate consumption; UPAEP 8, 12 (Figure 3 and 6) and *Arthrobacter* sp ATCC 31012 (figure 8) strains began at 20, 28 and 50 h. In contrast, *Bacillus subtilis* ATCC 21332 strain biosurfactant production occurred at the end of

Emulsification of Hydrocarbons Using Biosurfactant Producing Strains Isolated from Contaminated Soil in Puebla…

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31

UPAEP 6 strain showed the highest increase in biomass and biosurfactant production at 24 h. Maximum biomass production occurs at 44 h and with a maximum value of 5.3 g L-1. The maximum value of the biosurfactant production (80 %) at 40 h was high considering that crude oil is heavy with a density of 0.92-1.01 g mL-1 and an API gravity of 10.1-22.3 and viscosity can

On the other hand, UPAEP 9, UPAEP 10 and UPAEP 15 strains (Figures 4, 5 and 7) showed maximum biomass production at 28, 24, and 77 h with values of 3.6, 5.3 and 9.5 g L-1 respec‐ tively. Biosurfactant production started from the first couple of hours and up to 49 h by UPAEP 9 and UPAEP 15 strains reached emulsification of 58 and 69 %; and at 20 h UPAEP 10 strain

UPAEP 8 (Figure 3), UPAEP 12 (Figure 6) and *Arthrobacter* sp ATCC 31012 (Figure 8) strains showed slow biosurfactant production in contrast with the isolated strains. Biosurfactant production started only at 20, 28 and 50 h, and reached a maximum value of 65, 37 and 30 % respectively (at 40, 49 and 72 h). *Arthrobacter* sp ATCC 31012 showed slow growth, the highest biomass production was of 8.5 g L-1 at 55 h. Anyhow, UPAEP 12 strain showed a highest increase in biomass production between 28 and 46 h with a final value of 7 g L-1 (77 h) and

However, *Bacillus subtilis* ATCC 21332 strain (Figure 9) showed maximum biomass production in the first 10 h with 4.6 g L-1. Maximum biosurfactant production (27 %) is observed at the end

The *Candida petrophilum* ATCC 20226 strain (Figure 10) showed an important decrease in glucose up to 70 h (data not shown). Biosurfactant production began at 20 h. No relation to substrate consumption or to biomass production was observed. The maximum emulsification

The initial pH of the culture medium was 7.0 and lowers during the cellular growth of the studied isolates, therefore was adjusted with NaOH 0.1N to obtain a pH closer to neutrality (data not shown). Thus, the final pH of the culture medium ranged from 6.07 to 7.37 (Table 2). It is interesting to observe, that the drop in pH occurred just before the biosurfactant synthesis, possibly due to a prior synthesis of organic acids as precursors of biosurfactants by UPAEP 6, UPAEP 8, UPAEP 9, UPAEP 10 and UPAEP 15 strains. Yet, the pH was maintained between 6.5 and 6 with few changes during the entire kinetic by UPAEP 12 strain, and *Arthrobacter* sp ATCC 31012 showed only a small drop at 49 h. *Bacillus subtilis* ATCC 21332 and

UPAEP 8 strain showed maximum biomass production of 6.6 g L-1 at 24 h.

microbial growth (after of 76 h).

reach 10,000 cP [31] (Figure 2).

of the kinetic (70 h).

showed 70 % of biosurfactant production.

percentage obtained was 80 % after 70 h.

**Figure 1.** a) Emulsification of hydrocarbons. (b) Oil recovery.

## **5. Results**

## **5.1. Presumptive identification of isolated microorganisms**

Six microorganisms were isolated and identified according to their morphology. Table 1 shows the results of the presumptive tests for the identification of bacteria and yeasts by API galleries. The strains UPAEP 8 and UPAEP 15 were related to *Klebsiella pneumoniae* (99 and 97.6 % likelihood respectively). UPAEP 6 strain was closely related to *Klebsiella ornithinolytica* (99 %) and UPAEP 9 strain to *Klebsiella* sp (97 %). Whereas UPAEP 10 strain showed high likelihood (99 %) to *Serratia marcescens* and UPAEP 12 strain to *Candida inconspicua* (75 %).

## **5.2. Glucose consumption and biomass production**

The kinetic characteristics of the bacteria showed similar behavior regarding rapid growth, good adaptation to hydrocarbons and rapid glucose consumption.

All strains consumed glucose in a range of 92 to 100 %. However, the glucose consumption percentage of the commercial strains was lower than the isolates studied; with the exception of *Bacillus subtilis* ATCC 21332 which consumed 93.6 % of glucose (Table 2). Nevertheless, the glucose consumption was inversely proportional to the biomass production during the cell growth (data not shown). The emulsification index was directly proportional at production of biomass, except *Candida petrophilum* ATCC 20226 which showed no relation. Biosurfactant synthesis and biomass production by UPAEP 6, 9, 10, and 15 (Figures 2, 4,5 and 10) strains began during the first few hours (4 to 8) as a response to substrate consumption; UPAEP 8, 12 (Figure 3 and 6) and *Arthrobacter* sp ATCC 31012 (figure 8) strains began at 20, 28 and 50 h. In contrast, *Bacillus subtilis* ATCC 21332 strain biosurfactant production occurred at the end of microbial growth (after of 76 h).

**4.8. Biosurfactant recovery**

30 Biodegradation - Engineering and Technology

determine the yield.

**5. Results**

The purification of biosurfactant was performed according to a modified technique described in [30]. With the strains with highest percentage of emulsifier. The strains were previously grown in 500 mL of Lebac medium. The biosurfactant was then extracted from the bacteria with isopropanol-ethanol (3:1) analytical grade (Merck, México) in a separatory flask. It was centrifuged at 1200 rpm for 30 minutes (Solbat), and the supernatant was eliminated. The sample was then filtered using cellulose paper grade 101 (Millipore 2.5μ M). The precipitate obtained was dried for 24 h at 60 °C in an oven (FELISA) and stored in an Eppendorf vial to

(a) (b)

Six microorganisms were isolated and identified according to their morphology. Table 1 shows the results of the presumptive tests for the identification of bacteria and yeasts by API galleries. The strains UPAEP 8 and UPAEP 15 were related to *Klebsiella pneumoniae* (99 and 97.6 % likelihood respectively). UPAEP 6 strain was closely related to *Klebsiella ornithinolytica* (99 %) and UPAEP 9 strain to *Klebsiella* sp (97 %). Whereas UPAEP 10 strain showed high likelihood

The kinetic characteristics of the bacteria showed similar behavior regarding rapid growth,

All strains consumed glucose in a range of 92 to 100 %. However, the glucose consumption percentage of the commercial strains was lower than the isolates studied; with the exception

(99 %) to *Serratia marcescens* and UPAEP 12 strain to *Candida inconspicua* (75 %).

**Figure 1.** a) Emulsification of hydrocarbons. (b) Oil recovery.

**5.1. Presumptive identification of isolated microorganisms**

**5.2. Glucose consumption and biomass production**

good adaptation to hydrocarbons and rapid glucose consumption.

UPAEP 6 strain showed the highest increase in biomass and biosurfactant production at 24 h. Maximum biomass production occurs at 44 h and with a maximum value of 5.3 g L-1. The maximum value of the biosurfactant production (80 %) at 40 h was high considering that crude oil is heavy with a density of 0.92-1.01 g mL-1 and an API gravity of 10.1-22.3 and viscosity can reach 10,000 cP [31] (Figure 2).

On the other hand, UPAEP 9, UPAEP 10 and UPAEP 15 strains (Figures 4, 5 and 7) showed maximum biomass production at 28, 24, and 77 h with values of 3.6, 5.3 and 9.5 g L-1 respec‐ tively. Biosurfactant production started from the first couple of hours and up to 49 h by UPAEP 9 and UPAEP 15 strains reached emulsification of 58 and 69 %; and at 20 h UPAEP 10 strain showed 70 % of biosurfactant production.

UPAEP 8 (Figure 3), UPAEP 12 (Figure 6) and *Arthrobacter* sp ATCC 31012 (Figure 8) strains showed slow biosurfactant production in contrast with the isolated strains. Biosurfactant production started only at 20, 28 and 50 h, and reached a maximum value of 65, 37 and 30 % respectively (at 40, 49 and 72 h). *Arthrobacter* sp ATCC 31012 showed slow growth, the highest biomass production was of 8.5 g L-1 at 55 h. Anyhow, UPAEP 12 strain showed a highest increase in biomass production between 28 and 46 h with a final value of 7 g L-1 (77 h) and UPAEP 8 strain showed maximum biomass production of 6.6 g L-1 at 24 h.

However, *Bacillus subtilis* ATCC 21332 strain (Figure 9) showed maximum biomass production in the first 10 h with 4.6 g L-1. Maximum biosurfactant production (27 %) is observed at the end of the kinetic (70 h).

The *Candida petrophilum* ATCC 20226 strain (Figure 10) showed an important decrease in glucose up to 70 h (data not shown). Biosurfactant production began at 20 h. No relation to substrate consumption or to biomass production was observed. The maximum emulsification percentage obtained was 80 % after 70 h.