BIOFILMS IN BIOREMEDIATION: CURRENT RESEARCH AND EMERGING TECHNOLOGIES

• Chapter 1
Engineering Successful Bioremediation
Michael Harbottle
Bioremediation has become a well-established tool in the armoury of engineers wishing to address the problems of contaminated land, water or waste. Successful delivery of bioremediation requires a combination of expertise from such diverse fields as civil engineering, soil science and environmental microbiology; whilst the contribution from engineering may appear to be straightforward, a detailed understanding of often complex ground conditions (physical, chemical and biological) is needed to avoid failure. In this chapter the common techniques for the implementation of bioremediation are described, alongside an introduction to the effects of real materials and environments on the behaviour of contaminants and microorganisms, particularly those of natural heterogeneity in geological materials, and the potentially detrimental effects these may have on achieving a successful outcome within a reasonable period of time. The chapter concludes with a brief summary of potential enhancements for the practical implementation of bioremediation currently at the research stage.
• Chapter 2
The Biofilm Concept from a Bioremediation Perspective
Benjamin Horemans, Pieter Albers and Dirk Springael
Biofilms have been extensively studied since they were identified as the primary growth mode of microbial life. In the clinical world and food processing industries, biofilms are considered a threat to human health, but biofilms also have beneficial properties as they are deployed in waste recycling and water treatment. Biofilm-based bioremediation systems such as biofilters, aerobic and anaerobic granular sludge reactors and rotating disk contactors are widely used nowadays. As with most microbial-based technologies, the creation of a robust and reliable biofilm-based remediation technology remains challenging. For this reason, an in-depth understanding of biofilm formation and of the specific processes that occur inside biofilms, is mandatory to improve bioremediation. In this chapter, we discuss the nature and role of biofilms from a bioremediation perspective. We first address the basics of biofilm formation and how this should be considered when using biofilms for bioremediation. The main stages in the biofilm life cycle, i.e., attachment, aggregation, biofilm maturation and dispersal are discussed. Afterwards, we address the role of biofilm properties such as the associated matrix of extracellular polymeric substances and spatial heterogeneity, and elaborate on important processes within the biofilm such as microbial interactions and gene exchange. Many of these properties and processes are unique for biofilms and provide creative opportunities to improve biofilm-based bioremediation.
• Chapter 3
Biofilm Survival Strategies in Polluted Environments
Marc A. Demeter, Joseph A. Lemire, Raymond J. Turner and Joe J. Harrison
Biofilm communities have the remarkable ability to withstand mechanical and toxic chemical perturbations that wipe out their planktonic cell counterparts. Many features of the biofilm mode-of-life protect microbial cells. These include an extracellular matrix, changes in cellular biochemistry, and social interactions that facilitate the exchange of metabolites, signal molecules and genetic material between cells. Here we provide a primer on the fundamental mechanical, physiological and ecological mechanisms that protect biofilm cells from antimicrobial substances and fluctuating shear forces. Biofilm research is not only advancing our understanding of these survival strategies, but also leading to the design of biofilm-based enrichment techniques that may harness new metabolic power for bioremediation.
• Chapter 4
Tactic Responses of Bacteria to Pollutants: Implications for the Degradation Efficiency of Microbial Biofilms
Diana L. Vullo
In nature, microorganisms are mostly found attached to surfaces forming biofilm communities. The study of these biofilms is primarily focused on basic research to better understand their multicellular way of life, and negative implications for clinical cases and industrial processes. However, there is good reason to increase our knowledge regarding how to stimulate biofilm formation by bacteria, and particularly to exploit these biofilms for their efficient bioremediation of the wide spectrum of the environmental pollutants. Biofilm establishment and maintenance relies on a complex interaction of different mechanisms since bacterial movement and attachment is mediated by swimming, swarming and twitching motility, quorum sensing mechanisms, biosurfactant secretion and the presence of the chemotactic responses. These chemotactic responses include reactions to inorganic species and xenobiotics that are commonly present in polluted aquatic or soil environments as a result of industrial processes. Microorganisms that display positive chemotactic responses are able to swim towards an adsorbed chemical and, following biofilm formation, can increase pollutant bioavailability by surfactant synthesis, further impacting the rate and extent of pollutant degradation or transformation. The manipulation of biofilm condition and chemotactic response may be managed in ex situ bioreactors to improve the bioremediation efficacy of a wide range of pollutants, and particularly for the treatment of metal contaminated media.
• Chapter 5
Whole-cell Biosensors for Monitoring Bioremediation
Audrey S. Commault and Richard J. Weld
Whole-cell biosensors are sensing devices that typically use biofilm-dwelling microorganisms to detect specific physical or chemical aspects of environmental samples. Microbial detection produces a signal that is transformed into user accessible data which can be as simple as colour change on a paper strip or as complex as quantitative digital display. Whole-cells can be embedded on a transducer or used separately as part of a multi-step assay format. Both unmodified and genetically modified cells have been used in this way. Metabolic reporters are used to detect toxicity that inhibits cell metabolism while catabolic reporters can be used to detect specific contaminants. Biosensors can provide data on the bioavailability of contaminants and are very relevant to monitoring bioremediation. Several commercially available sensors have been developed and some of these have been widely tested and demonstrated to be effective at measuring environmental contaminants.
• Chapter 6
Modern Methods in Microscopy for the Assessment of Biofilms and Bioremediation
Guneratna Kuttuva Rajarao
Imaging techniques have an important and well-accepted role in many fields including bioremediation. New advanced methods are enhancing quantitative in situ analyses of the structure, composition, processes and dynamics of complex biofilm microbial communities that perform key roles in pollutant degradation and immobilization. Biofilm matrices consist of water or solvent (97%) bound to microbial cells and the physical properties are determined by the solutes present in it. Biofilm systems are suitable for bioremediation due to their enhanced persistence, complexity, mechanisms to increase pollutant availability for degradation and also because of enhanced by gene transfer among the associated organisms. Several microscopy-based techniques have been developed to better understand the role of microbial communities and associated features such as quorum sensing for community structure and functioning. However, there are impediments to characterizing biofilms in situ. This chapter gives an overview of microscopic methods for biofilm bacterial community analysis and mainly focuses on recent advancements in microscopic methods for the assessment of extracellular polymeric substances (EPS) that form a key component of the biofilm matrix.
• Chapter 7
Molecular Methods for the Assessment of Microbial Biofilms in Bioremediation
Gavin Lear
Advances in molecular methods are providing new insights into the diversity and organisation of biofilm microbial communities as well as the genetic basis of biofilm-mediated contaminant degradation. In the first part of this chapter, we consider the various methods available to inform on the taxonomic and genetic composition of microbial biofilms and to visualise the location of microorganisms genes within complex biofilm communities. Methods directed towards the analysis of functional subsets of these communities, including contaminant degraders are also described. The advantages and disadvantages of a variety of contemporary DNA fingerprinting and sequencing methodologies are each considered. In the second part of this chapter, case studies focusing on the use of molecular techniques in bioremediation assessments are presented. We highlight how the labelling of bacterial taxa with gene specific probes can reveal the complex structural organisation of natural biofilms as well as the impact of pollutants on their composition and organisation. Finally, we detail how the DNA of contaminant-degrading bacteria can be isolated to identify even low abundances of pollutant degrading taxa within diverse and active biofilm communities. The research opportunities provided by modern molecular methods continue to rapidly advance our understanding, and application of, biofilms in bioremediation.
• Chapter 8
Biofilm-mediated Degradation of PAHs and Pesticides
M. Pazos, L. Ferreira, E. Rosales and M.A. Sanromán
Environmental pollution caused by emerging organic compounds, such as polycyclic aromatic hydrocarbons (PAHs) and pesticides has attracted considerable attention in recent years. PAHs constitute a class of organic substances consisting of two or more fused benzene rings and exhibiting recalcitrance and strong mutagenic/carcinogenic properties. On the other hand, pesticides are used for controlling, repelling, preventing or eradicating pests, and include not only a wide range of chemical compounds but also, antimicrobial or disinfectant agents. Their composition often poses a threat for humans and the environment. As a result of the complex structure of both types of pollutants and their recalcitrant and persistent properties, the remediation of these compounds by conventional methods is often difficult. To solve this problem alternative treatment processes have been studied. Among them, biological treatment by biofilms has been found to be suitable for their remediation because of their high microbial biomass and ability to immobilise pollutants.
• Chapter 9
Detoxification of Hexavalent Chromium from Industrial Wastewater using a Bacterial Biofilm System
Zainul Akmar Zakaria, Wan Azlina Ahmad, Wan Haslinda Wan Ahmad and Sindhu Mathew
The incomplete removal of chromium from industrial wastewaters, particularly Cr (VI) species is of importance due to its persistence, stability and high solubility. Chemical reduction followed by precipitation is the most common Cr (VI) treatment technique used in the industry. Such an approach has disadvantages as large volumes of sludge are generated, obnoxious gases may be released and the process can be associated with high operating costs. Microorganisms such as bacteria have long been reported to have the ability to reduce Cr (VI). The use of Cr (VI) resistant-reducing bacterial biofilm offers an interesting solution to treat Cr (VI) containing industrial wastewater as well as the bioremediation of Cr (VI) polluted areas. Nevertheless, promising results obtained from laboratory scale experiments do not necessarily equate to success under large scale operating conditions. This is because important operational parameters are normally controlled in the laboratory and huge natural fluctuations in the wastewater are avoided. Hence it is necessary to assess the robustness of the system at a larger scale where it can be subjected to fluctuations naturally occurring in wastewater, and as typically encountered in factory compounds or contaminated areas.
• Chapter 10
Hydrocarbonoclastic Biofilms
Dina M. Al-Mailem and Samir S. Radwan
Biofilms harboring hydrocarbonoclastic (hydrocarbon-degrading) bacteria occur naturally, and are widely distributed in aquatic, terrestrial and even atmospheric environments. They seem to play a role in the 'self-cleaning' of environments contaminated with oil, oil vapor and oil derivatives. Such biofilms may develop on inanimate substrates such as gravel particles, small stones and waste metal, wood and plastic pieces. They may also be associated with animate biotic substrates, e.g. biofouling materials, macroalgae, cyanobacterial mats, the roots (rhizospheres) and leaves (phyllospheres, phylloplanes) of higher plants, as well as with fish and other aquatic animals. Hydrocarbonoclastic bacteria also live naturally in association with phototrophic picoplankton in surface waters and mud flats. Biofilms comprising hydrocarbonoclastic bacteria can be established artificially on inert substrates such as gravel particles, or on glass and plastic plates. These man-made biofilms are promising tools for the bioremediation of oil-contaminated materials in bioreactors. In biofilms, hydrocarbonoclastic bacteria enjoy various benefits which facilitate and enhance their oil-biodegradation activity, especially when coexisting with phototrophic and diazotrophic microorganisms which enrich the biofilm with oxygen and nitrogenous compounds. This principally occurs because oxygen and nitrogen are limiting factors to the mineralization of hydrocarbons by bacteria.
• Chapter 11
Use of Biofilm Permeable Reactive Barriers for the In Situ Remediation of Mobile Contaminants
Youngwoo Young Seo
Bioremediation has been used for the destruction of organic chemicals in soils, groundwater and industrial wastewaters and can be categorized into both in-situ and ex-situ remediation strategies. Among in-situ bioremediation technologies, the use of engineered permeable reactive barriers (PRBs) within contaminated aquifers has grown as a way to prevent the migration of mobile pollutants from contaminated plumes with minimal maintenance costs. During the past decade, PRBs have become an important in-situ remediation option for contaminated groundwater. However, the influence of biofilm formation on the performance of PRBs is not well understood yet. This chapter reviews the influence of biofilm formation on the performance of PRBs for the continued removal of mobile contaminants. First, the general application of biofilm-mediated processes for the remediation of soil and groundwater bioremediation is discussed. Second, the use of PRBs incorporating biofilm grown on different supporting media is reviewed. Third, the influence of biofilm formation on the long term performances of PRBs is evaluated. Finally, emerging research trends and needs of relevance to the use of PRBs in remediation are considered.
• Chapter 12
Comparison of the Degradation Activity of Biofilm-associated Versus Planktonic Cells
Masaaki Morikawa and Kenji Washio
Biofilm formation is a common microbial strategy to construct and maintain a favorable niche in stressful natural environments. Consequently, the application of biofilms in bioproduction and bioremediation processes should be a natural and rational choice (Morikawa, 2006). In this chapter, we describe two examples that demonstrate the usefulness of biofilm-associated cells in bioremediation, and provide basic mechanisms that should advance environmental biotechnologies. First,Pseudoalteromonas strains were shown to be dominant biofilm forming bacteria in fish-farming fields and some produced extracellular proteases in a biofilm-dependent manner. The results suggest that Pseudoalteromonas bacteria living in the biofilm community, contribute in part to remove excess proteinaceous material from the sediment sludge and maintain good water quality in fish farms. Second, biofilm-associated cells of Pseudoalteromonas stutzeri T102 did not degrade naphthalene during initial hours of incubation but later they degraded it even faster than planktonic cells. This delayed but elevated degradation activity of the biofilm-associated cells could be attributed to 'super-activated cells' as they detach from biofilms. T102 biofilm-associated cells were capable of surviving for 10 weeks in natural petroleum-contaminated soils; by then T102 planktonic cells were mostly extinct. Naphthalene degradation activity in the soils that had been inoculated with T102 biofilms was indeed higher than that observed in soils inoculated with T102 planktonic cells. It would appear that biofilms may provide a constant stream of super-active contaminant degrading bacteria which could may be used in bioremediation applications.
• Chapter 13A
Using Microbial Biofilms to Enhance the Phytoremediation of Contaminants in Soil and Water. Part A: A Trial for Sustainable Phenol Degradation by Duckweed-colonizing Biofilms
Masaaki Morikawa, Fumiko Yamaga, Kazuya Suzuki, Koki Kurashina, Kyoko Miwa and Kenji Washio
Here, we demonstrate how conventional passive phytoremediation technologies can be transformed into active bioremediation technologies using growth-promoting, plant associated bacteria. Phenol-degrading bacteria were isolated from the surface of duckweed, Lemna aoukikusa, using an enrichment culture method. One of the isolates, Acinetobacter calcoaceticus P23, exhibited an excellent ability to degrade phenol and formed robust biofilms under laboratory conditions. P23 rapidly colonized the surface of sterilized duckweed and enhanced its growth. A long-term test using P23 colonised L. aoukikusa showed that the continuous removal of phenol could be attributed to a beneficial symbiotic interaction between duckweed and its surface bacterium. The rational reconstruction of multispecies microbial communities, including taxa with pollutant degrading and plant growth promoting activities, holds much promise for the development of innovative phytoremediation technologies.
• Chapter 13B
Using Microbial Biofilms to Enhance the Phytoremediation of Contaminants in Soil and Water. Part B: The Sustainable Biodegradation of Phenolic Endocrine-disrupting Chemicals by Bacteria in the Rhizosphere ofPhragmites australis
Tadashi Toyama and Kazuhiro Mori
Rhizoremediation, the degradation and removal of pollutants by exploiting the elevated microbial activity in the rhizosphere of plants, has gained much attention in recent years. In this second part of this chapter, we discuss the use of two rhizobacteria (Sphingobium fuliginis TIK1 and Sphingobium sp. IT4) colonizing the root of the common reed Phragmites australis, and the use of this association for the sustainable treatment of water polluted with phenolic endocrine-disrupting chemicals (EDCs). Strains TIK1 and IT4, isolated from Phragmites rhizospheres, degrade various phenolic EDCs (4-alkylphenols and bisphenols). The root exudates of Phragmites appeared to facilitate the degradation activity of these, using the exudates as a source of carbon and energy for cell growth. Additionally, the roots provide a large surface area for bacterial colonisation. Phragmites plants inoculated with these bacteria (i.e., both Phragmites-strain TIK1 and Phragmites-strain IT4 associations) were able to simultaneously and repeatedly remove various phenolic EDCs from polluted water. We conclude that the use of hydroponic systems using Phragmites-TIK1 and Phragmites-IT4 associations would be an effective strategy for the sustainable treatment of polluted waters contaminated by mixtures of phenolic EDCs.

The microbial bioremediation of contaminants is cost effective and reliable and a number of approaches are in widespread commercial use. Microbial bioremediation makes use of the metabolic activities of biofilm-dwelling microorganisms which are responsible for the majority of pollutant degradation in natural environments.
In this book, renowned scientists from around the world provide up-to-date and authoritative reviews of the latest scientific research that has contributed to our understanding of the vital importance of microbial biofilms for the biological remediation of contaminated environments. The results of a variety of key case studies are presented to highlight the broad range of treatment approaches and applications at our disposal. In addition, the authors discuss the future trends and likely growth areas in biofilm-related research.
This comprehensive volume is indispensable for anyone involved in bioremediation, biofilm research or environmental microbiology. It is also recommended as a reference work for all microbiology libraries.