Bacterial Bioflocculant for Multifunctional Features

By S. Sivaramakrishnan and R.T.V. Vimala

Bacterial Bioflocculant for Multifunctional Features highlights research findings on the production and characterization of self-assembling bioflocculant from bacterial consortium (encompassing Bacillus subtilis, Enterococcus faecalis and Proteus mirabilis). The book describes the various high-throughput techniques for characterization of wastewater at microbiological and molecular level. Sections cover pharmaceutical compounds, macromolecular compounds and other contaminants, the biotoxicity exhibited cellular and nuclear abnormalities in the zebra fish, and high-throughput techniques used for evaluating the flocculating efficiency of the bacterial bioflocculant to remove the contaminants in different other applications.

Bacterial Bioflocculant for Multifunctional Features will help users undertake further advanced research in bacterial bioflocculant for bioremediation technology and environmental prospectives. In addition, it will also inspire readers to understand bioflocculation and its functions.

• Offers alternative, less expensive biotechnologies where wastewater can be reused
• Focuses on the multipotent bacterial bioflocculant which plays a decisive role in bioremediation
• Discusses techniques for microbially decontaminating polluted wastewater to increase re-usability

• Language: English
• Publisher: Academic Press
• Release date: Jan 31, 2022
• ISBN: 9780323913843

S. Sivaramakrishnan

Dr. S. Sivaramakrishnan is engaged in research (more than 20 years) and teaching. His specialization is in Agricultural Biotechnology in relation to biopesticides and Nanobiotechnology towards biomedical applications. He has produced 16 Ph.D.s and guiding M.Sc., M.Phil. and Ph. D. and M.Tech. students for their research work. He has won young scientists award four times. He is a member of the editorial board of European Journal of Nematology and is a reviewer for nearly ten journals. He has scientific publications in national and international reputed journals. He has been the principal investigator of major DBT, DST and UGC projects. He has international collaborations, signed MoU between USA, Korea, Iran and Turkey. He has organized international conferences and national awareness programme

Book preview

Preface

Current research on environmental issues has focused on a number of problems that have taken center stage in sustainability science and environmental engineering since the turn of the century. One of the most intensively studied concerns was the issue of water conservation measures, as well as the worrisome rise in pollutants and environmental health.

A significant aim of the authors is to look for new water reutilization alternatives that take advantage of limitations on the use of chemical flocculants and the issue of limited water availability. Pricing wastewater treatment so high deters environmental planners from using it. As a result, there is a lack of appropriate wastewater treatment technologies for metropolitan environments.

Aiming to meet the need for new advances in the use of bacterial bioflocculants as alternatives to harmful inorganic and synthetic flocculants, which are a crucial component in wastewater treatment, this book was prepared to provide an up-to-date look at bacterial bioflocculants. A multipotent bacterial bioflocculant isolated from municipal wastewater microbial alliance was used by the authors to develop a bioflocculant that has many functions in the environment and the bioremediation process, in addition to its properties as reducing agents, nanosynthesis, insecticides, and biomedicines.

Agricultural waste materials such as peanut hull and wheat bran were used as a low-cost nutritional growth medium for bioflocculant producing microorganisms to increase the bioflocculant yield. This bioflocculant, which is composed primarily of polysaccharide, showed excellent flocculation capabilities. The mechanism behind flocculation should be better understood in order to comprehend the impact of variables on its activity. The molecular characterization of overall harmful pollutants in municipal wastewater was analyzed using high-throughput techniquesto assess the environmental risk of wastewater. After the water was treated with the bioflocculant, the water was substantially decontaminated. This research presents a hopeful possibility for the future of wastewater treatment and other biotechnological processes, using the bacterial bioflocculant as an alternative to traditional biochemical treatment methods.

The book is designed to offer the core concepts that will be required to remove contaminants from wastewater and reuse it. The results presented in this book will significantly improve the readers’ understanding of wastewater complexity, bioflocculant processes and its multifunctional features, and various high-throughput studies. This book is exceedingly advantageous for anyone concerned with water quality and management.

Dr. S. Sivaramakrishnan

Dr. R.T.V. Vimala

Chapter 1

Introduction

Abstract

The current focus on organic flocculants is owing to their flocculation behavior and biodegradability, resulting in an environment free of challenges. Extracellular polysaccharides are produced by the cellular growth of microorganisms, such as bacteria, fungi, yeast, and algae, and are known as bioflocculants. Also, bioflocculants have been commercialized on a big scale because of their easy manufacturing methodology, low production cost, and the degraded characteristic. The broad spectrum of toxins in wastewater necessitates development of bioflocculating systems for successful wastewater treatment and reuse. Further, due to their different functional groups and topologies, bioflocculant is both a reducing and stabilizing agent. Therefore, bioflocculant is beneficial for generating nanoparticles for biomedical applications. The focus of this book is on municipal wastewater bacteria, and the biocidal property of the self-assembling unique bacterial bioflocculant in the treatment of wastewater.

Keywords

Bioflocculant; Consortia; Wastewater; BOD; COD; Bioassays; Nanoparticles; Bioremediation

Biological approaches have garnered a tremendous role in the advancement of technology worldwide, with empirical and innovative capacities that impart an edge over conventional chemical methods. Recently, the production of organic flocculants has gained a great deal of interest owing to its unique flocculation behavior and biodegradability to build a hassle–free healthy environment. The use of microorganisms for biodegradation of toxic substances has piqued interest, and most of the emphasis has been on isolating robust microbes from nature, which has a broad variety of potential applications in different bioremediation procedures and can be referred to as a green technique. Bioflocculants are environmentally sustainable biological macromolecules that are beneficial because they are safe for living species and free from secondary pollution threats. Their usage has been regarded as a possible solution to aquatic life toxicity and environmental degradation. One of the main users of flocculants is the water and wastewater treatment sector. Bioflocculants have been extensively utilized in wastewater treatment, steroid oestrogen removal, pharmaceutical protein precipitation, heavy metal adsorption, color removal, cell removal, drinking water purification, food production, fermentation, and biomass recovery (Drakou et al., 2018; Sathiyanarayanan et al., 2013; Wang et al., 2007; Zhong et al., 2014). They can easily remove organic/inorganic pollutants and nutrients even in very minute concentration. They potentiate huge remarkable applications in the scientific and biotechnological fields as they have higher efficiency compared to conventional flocculants. Bioflocculants are found as capsular, slime, loosely bound, and tightly bound compounds according to the nature of their association with the cells or the method of extraction.

On another hand, with the growing population and the advancement of science and technology, more water is consumed in daily life and production, and more wastewater is generated. River water is the largest global supply of freshwater for domestic, irrigation, and industrial usages. In any developing and developed country, water conservation is one of the key issues. Industrial and municipal wastewaters are the biggest pollutants of natural water resources. Particularly, municipal wastewater is the main contributor to diverse water pollution problems. Wastewaters from these sources are rich in various contaminants at various concentrations. Municipal water which contains both domestic and industrial wastewater may differ from place to place depending upon the type of industries and industrial establishment. It is a significant source of environmental degradation from domestic product, pharmaceuticals, and eutrophication from nutrient overloading. Therefore, wastewater contains a wide range of contaminants such as suspended particles, dyes, toxic organic chemicals, heavy metals, petroleum hydrocarbons, chlorinated hydrocarbons, various acids, alkalis, and nutrient content like nitrogen and phosphorus, and other chemicals which greatly change the physicochemical properties of water (Wijaya and Soedjono, 2018). All these contaminants are quite harmful or even fatally toxic and cause gastroenteritis, liver damage, nervous system impairment, skin irritation, and liver cancer in aquatic populations. The removal of these chemicals and particles is biggest challenge for effective wastewater treatment and its disposal. Polluted water is not only affecting the freshwater segment but also contaminates the groundwater supplies. Organic pollutants have been detected in groundwater and causing health problems around the world.

Water pollution caused by organic and inorganic pollutants has become a major concern today. The unused effluents from the process are discharged as wastewater that contains turbidity, color, high levels of toxic chemicals, biochemical oxygen demand (BOD), and chemical oxygen demand (COD). If these discharges are not properly treated and released into the sea, river, or lakes, they have a negative impact on the environment. Therefore, it becomes a difficult but necessary task to treat and remove the pollutants present in water bodies.

Scarcity of water resources has led to the development of techniques for successful wastewater treatments and its reutilization. Physical and chemical processes are extensively used in the treatments, but due to certain drawbacks, new methods are being investigated. Inorganic flocculants (polyaluminum chloride and ferric chloride) and organic flocculants (polyacrylamide and polyethylenimine) employed in wastewater treatment and other biotechnological applications create a menace to aquatic organisms and human beings. Using chemically synthesized compounds results in a numerous health problems, including Alzheimer’s, genotoxic disorders, and carcinogenicity. Another crucial aspect is the expense of chemical flocculants that are not affordable by many developing countries. The setbacks due to chemical flocculants necessitated the discovery of biodegradable flocculants that are ecofriendly and can be used to replace the biotechnological applications of synthetic flocculants (Agunbiade et al., 2017; Guo et al., 2018; Wan et al., 2013).

Biologic processes are evolving as potential alternate management methods, including bioflocculants for wastewater. Bioflocculant refers to the extracellular polysaccharides exuded by microorganisms, such as bacteria, fungi, yeast, and algae as a product of their cellular proliferation. They are made up of composite multichain polymeric substances with repeated pieces of sugar units, glycoprotein, uronic acids, and nucleic acids. They have defined molecular chain length and composition and can flocculate even tiny particles. Even though substantial development has been found in the laboratory, large-scale bioflocculant production faces economic challenges due to the high cost of production and poor yield. The main component needed for a microbial medium for bioflocculant production includes carbon, nitrogen, sulfur, and phosphorus, and these usually add up to the cost of production. Nitrogen sources such as yeast extract, peptone, and carbon sources, such as glucose, fructose, and sucrose and other conventional media sources are exorbitant components as they are commercially produced from valuable and comparatively expensive products (Mohammed and Dagang, 2019). This contributes to an extra cost of media formulation for microbial bioflocculant synthesis and restricts the market potentials of this commodity.

In the quest for replacing costly commercial nutrient sources, cheap recyclable sources from wastewater, animal waste constituents, and industrial wastes received considerable scientific explorations for their bioflocculant yielding capacity, especially when fermented with appropriate microorganisms under suitable culture conditions. Most of the wastes contain high BOD and COD concentrations. They are made up of biodegradable carbohydrates, proteins, and lipids which can serve the same purpose as the exorbitant conventional media. In addition to utilizing these wastes for bioflocculant production, the environmental problems in the context of contamination, diseases, and depletion of waste of essential bioresources, such as proteins, enzymes, and lipids caused by their improper disposal have also been combatted (Bukhari et al., 2020; Lasekan et al., 2013; Wang et al., 2007). Nonetheless, the use of these wastes for the development of bioflocculants depends mainly on the isolation or use of already isolated bioflocculant producing microbial strains with capability in fermenting the cost-effective substrates, optimization of the substrate concentration, and culture conditions. This book focuses on the utilization of municipal wastewater as a cost-effective alternative source for the processing of bioflocculants.

Microbial species in wastewater consortia have proven to improve bioflocculant when compared to pure strains. This is owed to the fact that microbial species coexist in ecological niches in nature and form convoluted relationships which include symbiosis and synergism. The production of bioflocculant from renewable sources has made the goal of commercialization much closer to reality. Therefore, mass level cultivation with easy production methods, low production cost, less requirement of coreagents, and the property of degradation are the reason for their commercialization on an industrial scale as compared to chemical flocculants. In addition, chemical data alone do not allow the evaluation of toxic effects. Toxicity bioassays are necessary to integrate the biological effects of all compounds present and other factors such as bioavailability, toxicants interaction, and others. Besides, bioflocculant acts as both reducing and stabilizing agent due to their properties and structures with many functional groups; which consequently make bioflocculant suitable and advantageous to produce nanoparticles (NPs) destined to be applied in the biomedical sciences (Araujo et al., 2018; Zhu et al., 2004). Some of these bioflocculant-producing microorganisms have been isolated from sludge, soil, wastewaters, rivers, dams, alkaline lake, and marine intertidal sludge. However, there is paucity of information regarding bacterial bioflocculant from the municipal wastewater consortium and their role in flocculation. Hence, there is need to explore the diversity of bacteria for bioflocculation and validate their possible industrial applications, wastewater treatment, and synthesis of NPs. On this note, this book discusses the bacteria that have been isolated and screened from the municipal wastewater and the need to explore the self-assembling novel bacterial bioflocculant, its potential to treat the wastewater, its multifunctional features toward bioremediation and environmental prospective.

References

Agunbiade, M.O., Van Heerden, E., Pohl, C.H., Ashafa, A.T., 2017. Flocculating performance of a bioflocculant produced by Arthrobacterhumicola in sewage waste water treatment. BMC Biotechnol 17 (1), 1–9.

Araujo, I.M., Silva, R.R., Pacheco, G., Lustri, W.R., Tercjak, A., Gutierrez, J., Júnior, J.R., Azevedo, F.H., Figuêredo, G.S., Vega, M.L., 2018. Hydrothermal synthesis of bacterial cellulose–copper oxide nanocomposites and evaluation of their antimicrobial activity. Carbohydrate Pol 179, 341–349.

Bukhari, N.A., Loh, S.K., Nasrin, A.B., Jahim, J.M., 2020. Enzymatic hydrolysate of palm oil mill effluent as potential substrate for bioflocculant BM-8 production. Waste Biomass Valorization 11 (1), 17–29.

Drakou, E.-M., Amorim, C.L., Castro, P.M., Panagiotou, F., Vyrides, I., 2018. Wastewater valorization by pure bacterial cultures to extracellular polymeric substances (EPS) with high emulsifying potential and flocculation activities. Waste Biomass Valorization 9 (12), 2557–2564.

Guo, J., Chen, C., Jiang, S., Zhou, Y., 2018. Feasibility and mechanism of combined conditioning with coagulant and flocculant to enhance sludge dewatering. ACS Sustain. Chem. Eng. 6 (8), 10758–10765.

Lasekan, A., Bakar, F.A., Hashim, D., 2013. Potential of chicken by-products as sources of useful biological resources. Waste Manag. 33 (3), 552–565.

Mohammed, J.N., Dagang, W.R.Z.W., 2019. Role of cationization in bioflocculant efficiency: a review. Environ. Process. 6 (2), 355–376.

Sathiyanarayanan, G., Kiran, G.S., Selvin, J., 2013. Synthesis of silver nanoparticles by polysaccharide bioflocculant produced from marine Bacillus subtilis MSBN17. Colloids Surf. B 102, 13–20.

Wan, C., Zhao, X.-Q., Guo, S.-L., Alam, M.A., Bai, F.-W., 2013. Bioflocculant production from Solibacillussilvestris W01 and its application in cost-effective harvest of marine microalga Nannochloropsisoceanica by flocculation. Bioresource Technol. 135, 207–212.

Wang, S.-G., Gong, W.-X., Liu, X.-W., Tian, L., Yue, Q.-Y., Gao, B.-Y., 2007. Production of a novel bioflocculant by culture of Klebsiellamobilis using dairy wastewater. Biochem. Eng. J. 36 (2), 81–86.

Wijaya, I., Soedjono, E., 2018. Physicochemical characteristic of municipal wastewater in tropical area: case study of Surabaya City, Indonesia, IOP Conference Series: Earth and Environmental Science, IOP Publishing, 012018.

Zhong, C., Xu, A., Chen, L., Yang, X., Yang, B., Hong, W., Mao, K., Wang, B., Zhou, J., 2014. Production of a bioflocculant from chromotropic acid waste water and its application in steroid estrogen removal. Colloids Surf. B 122, 729–737.

Zhu, Y.-b., Feng, M., Yang, J.-x., Ma, F., Wu, B., LI, S.-g., Huang, J.-l., 2004. Screening of complex bioflocculant producing bacteria and their flocculating mechanism. J. Harbin Inst. Technol. 36 (6), 759–762.

Chapter 2

Biosynthesis of bioflocculant from bacterial consortium of municipal wastewater and its characterization

Abstract

Bioflocculant has been emphasized in various research initiatives due to its unique features. It is a nontoxic biopolymer molecule produced by microorganisms or their metabolites and is made up of numerous biomolecules, such as polysaccharide, protein, DNA, sugar, polyamino acids, and lipids. This novel bacterial bioflocculant biosynthesised from a bacterial consortium of B. subtilis, P. mirabilis, and E. faecalis of municipal wastewater was proven to be efficient in flocculation. The produced bioflocculant has been characterised by FTIR, XPS, 3D-EEM, MALDITOF-MS, etc. FTIR analysis shows that composition of bioflocculant was made up of majorly protein and polysaccharides. XPS and 3D-EEM were also used to corroborate it. Further, FE-SEM observations revealed an irregularly formed bioflocculant structure. Maximum flocculating activity was influenced by the factors such as inoculum size (1.5%), carbon (sucrose), and nitrogen (peptone) sources, metal ions (Ca²+), initial pH of the medium (7), time (72 h), and dosage [1.5 mL (9mg)/100 mL]. Moreover, peanut hull extract and wheat bran extract were employed as fermentation mediums in order to commercialize the product at an economical point of view, with response surface methodology being statistically utilized. The highest yield of 7.5g/L bioflocculant was obtained, with 92% flocculating activity. As a result, the bioflocculant produced might be regarded a potential, low-cost, and environmentally friendly wastewater treatment method.

Keywords

Response surface methodology; Bioflocculant; Fermentation medium; Optimization; Flocculating activity; Characteristics

Chapter Outline

2.1 Introduction 8

2.2 Types of flocculants 9

2.2.1 Inorganic flocculants 12

2.2.2 Organic flocculants 13

2.2.3 Natural flocculants 13

2.3 Bacterial bioflocculant 16

2.4 Stages of bioflocculant production 17

2.5 Isolation of bioflocculant producing bacteria 17

2.6 Identification of effective bacterial strains 19

2.7 Composition of bacterial strains 19

2.8 Construction of bacterial consortia 21

2.9 Factors influencing bioflocculant production and flocculating activity 23

2.9.1 Effect of inoculum size 24

2.9.2 Effect of carbon and nitrogen sources and c/n ratio 26

2.9.3 Effect of metal ions 28

2.9.4 Effect of initial pH 28

2.9.5 Effect of time 30

2.9.6 Effect of dosage 32

2.9.7 Effect of temperature 33

2.10 Low cost nutritional growth medium 34

2.11 Optimization of culture medium for bacterial bioflocculant production 36

2.12 Purification of bioflocculant 39

2.13 Composition of the bacterial bioflocculant 40

2.14 Mechanism of bioflocculation and coagulation 41

2.15 Determination of flocculating activity