Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology Application

Abstract

This Chapter discusses the entrapment, growing and biofilm formation by an environmental bacterium immobilized in polyethyleneoxide cryogel to be applied in environmental biotechnology. The KCM-R5 bacterium was isolated from the heavy metal-polluted environment near a large Pb-Zn smelter, also producing precious metals in Bulgaria. Molecular-genetic analysis revealed affiliation with Pseudomonas rhodesiae. The strain is capable of growing in high concentrations of phenol and different phenol derivatives. Polyethylene oxide was found to be friendly and nontoxic to bacteria polymer enabling bacteria easy to penetrate in it and fast to grow. KCM-R5 biofilms were grown for 30 days in batch culture with phenol (300-1000 mg L−1) dissolved in the mineral medium. The bacterium was able to involve phenol in its metabolism and use it as a single carbon supplier. The results obtained in the study showed 98% phenol biodegradation using the biotech installation described. The proposed PEO cryogel-P. rhodesiae KCM-R5 bacterium biotech biofilter can be used for environmental biotechnology application in industrial wastewater detoxification.

1. Introduction

In recent years, the growing amount of polymer-encapsulated bacteria and engineered bacterial biofilms have enhanced both wastewater management and biodegradation of industrial pollutants. Amidst the aromatic substances, monocyclic phenol and its nitro- and chlorophenol derivatives represent one of the most harmful environmental pollutants. Phenol (Figure 1) is a by-product of benzene production and widely exploited in the chemical industry.

Figure 1.

Structural formula of phenol.

The continuous application of phenol and its derivatives such as ortho-nitrophenol (о-NP), 2,4- dinitrophenol (2,4-dNP), 2,5-dinitrophenol (2,5-dNP), penthachlorophenol (PCP) and 2,4- dichlorophenoxyacetic acid (2,4-D) (Figure 2) in the chemical, agricultural, woodworking and oil processing industries has resulted in their persistent presence in the environment.

Figure 2.

Structural formulas of nitro- and chlorophenol derivatives.

Worldwide, high concentrations of phenol and phenol derivatives were detected in industrial wastewaters, which further flow into rivers, seas and oceans. Bisphenol A (BPA) as phenol derivative is amid he most prominent plasticizers and is omnipresent in surface and ground water. This toxic substance is detected in many aquatic organisms. Mathieu-Denoncourt et al. [1] reported that BPA was the most toxic (96 h LC50s) to aquatic invertebrates (0.96-2.70 mg/L) and less toxic to fish (6.8-17.9 mg/L). It plays toxic effect on amphibians being more noxious to embryos than to juveniles. It plays neuro-toxic and reproductive effect reported by Santoro et al. [2].

Phenol is harmful to human causing blood pressure increase, leukemia, skin necrosis and pores creation, damages of the phospholipid bilayer, heart arrhythmia, tight junctions disruption, liver and kidney injury, earlier child birth and gastro-intestinal perforations [3, 4]. Phenol did not demonstrate a carcinogenic effect (Figure 3) [4].

Figure 3.

Harmful effect of phenol on human organs.

It is estimated that the median lethal dose of phenol in humans is 14-214 mg kg⁻¹or 1-15 g [4].

Among bacteria, bacterial species like Pseudomonas [5, 6, 7], Bacillus [8] and Geobacter [9] are capable of effective phenol biodegradation. Besides bacteria, fungi of the genera Aspergillus [10], Trichosporon can also successfully degrade phenol. Most authors describe mainly the degradation by free planktonic cells, but data about degradation by encapsulated bacteria are scarce. Both natural or synthetic polymers can be used as bacteria carriers. Biodegradation of phenol is accomplished via ortho- or metha cleavage of the aromatic ring. First step is conversion of phenol to catechol by attachment of additional hydroxyl group (Figure 4).

Figure 4.

Mechanism of phenol degradation [11].

Further the catechol is degraded either via the metha-mechanism, a process catalyzed by the enzyme catechol 2,3-dioxigenase or via the ortho-mechanism using catechol 1,2-dioxigenase [11].

The current chapter discusses the variety of natural and synthetic polymers used for bacterial entrapment; the content, development and structure of bacterial biofilms, and encapsulation of the xenobiotic degrading bacterium Pseudomonas rhodesiae KCM-R5 in PEO cryogels, creating a biofilter, bacterial biofilm formation and phenol degradation by said polymer-bacterium biofilter.

2. Natural and synthetic polymers used for entrapment of bacteria

Natural polymers most commonly used are: (i) Alginate: alginate, alginate/soy protein isolate (SPI), algnate/cashew gum, (ii) Cellulose derivatives: cellulose acetate, ethyl cellulose, cellulose fibers, (iii) Chitosan: chitosan, the binary system beta-cyclodextrin modified chitosan, chitosan/synthetic poly(ethylene oxide), (iv) Starch and maltodextrin: gum acacia/maltodextrin, Arabic gum/maltodextrin/starch, (v) Whey protein, (vi) Fibroin: fibroin/poly-caprolactone, (vii) Gelatine. The main advantages of natural polymers are their biocompatibility and nontoxicity to living cells and biological structures, e.g. essential oils [12].

Synthetic polymers used for bacterial encapsulation are polyvinylchloride, polylactic acid, polycaprolactone, polycaprolactone/hydroxiapatite composites, poly(methil methacrilate), poly(vinyliden fluoride), poly(ethileneoxide), poly(ethylene brassilate-co-squaric acid) [12, 13, 14]. A limited number of studies have been reported for phenol degradation by bacterial biofilms formed by immobilized bacteria. Immoblilization of bacteria was conducted in polyacrylamide [15], polyurethane [16], polyamide [17], polyacrylonitrile [18, 19] or polyvinyl alcohol [20].

In the last 20 years, different organic carriers for bacterial immobilization were investigated [15, 16]. Among synthetic polymers, poly(ethylene oxide) hydrogels are excellent candidates because they are nontoxic and biocompatible materials which meet all of the requirements for strength, absorbency, flexibility and adhesiveness [17]. Hydrogels of poly(ethylene oxide) have been synthesized in situ by applying a facile optimized protocol, which will be further described.

3. Structure and development of bacterial biofilms

Biofilms are an excellent strategy for bacterial survival in a sessile way and 40-80% of bacteria on earth can form biofilms [21, 22, 23, 24, 25, 26]. The first to observe under a microscope microbes living on the surfaces of teeth was the Dutch merchant Antony van Leuwehoek. He can also be considered the first discoverer of bacterial biofilms. The invention of the electronic microscope in the 1930-ies provided an insight into the structure and organization of biofilms. Biofilms colonize different surfaces like plant and animal tissues, medical devices, potable water pipes, and natural lakes and rivers. In the early 1970-ies, the ambiguous role of disinfectants in the disruption of bacterial biofilms was proved, a finding published by [24]. The authors discussed bacterial resistance to chlorine, one of the most widely used disinfectants, due to bacterial biofilms.

Bacterial biofilms are complex living communities composed of a wide range of components and molecules such as bacterial cells, their polysaccharides, proteins, lipids, DNA and RNA. The external DNA (eDNA) in particular plays an important role in the early phase of biofilm arrangement [27].

Several factors can influence biofilm generation [28, 29]. The main factors are related to the bacterial surface and its charge. Hydrophobicity is a main factor influencing the adsorption and change in the surface tension of bacteria. Biofilm formation involves all flagellar and non-flagellar bacterial structures - fimbriae, pilli and flagella [30]. Investigations on the structure of fimbriae show that they contain predominantly residues of hydrophobic amino acids, such as valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and cystein [23]. Fimbirae also contain adhesion molecules [29] which attach to substrates and thus bacteria can deliver nutrients for their metabolism. Temperature and substrate availability also impact biofilm formation.

It is important to note that bacterial adhesion [29] and biofilm formation increase on rough surfaces compared to smooth surfaces. The larger surface area and the weaker shear forces facilitate biofilm formation. As Donlan [29] described, the physicochemical properties of the surface is of great importance in biofilm build-up. Bacteria attach more easily to hydrophobic, nonpolar, rough surfaces like Teflon or plastics than to hydrophilic surfaces like glass or steel [31, 32, 33]. Bacterial biofilms develop on tooth enamel in the oral cavity. The pellicle contains albumin, lipids, glicoproteins, gingvinal fissure liquid, lysozime and bacteria dwelling in the oral cavity. Mittelman [34] discussed in his publication that the host produces complex bacterial biofilms as saliva, respiratory secretion, tears, urine and blood, which strongly influence bacterial attachment. The development of bacterial biofilm is shown in Figure 5.

Figure 5.

Bacterial biofilm development.

As shown in Figure 5, the stages of bacterial biofilm development include the crucial initial steps of finding, interacting with, and adhering of planktonic bacteria to a surface [35, 36]. Once irreversibly attached to a surface, bacteria form microcolonies. Biofilm matures and when it has completely matured it is affected by shear forces and undergoes rupture resulting in free planktonic cells. The liberated planktonic cells fall on new surfaces and colonize them, forming new biofilms [22, 37].

Both pH and the high amount of nutrients increase the concentration of ferric, sodium and calcium cations. These cations affect the adhesion of Pseudomonas fluorescens reducing the chemical forces between the negatively charged bacterial cells and the glass surface [29]. Several studies reported that mycolic acid-containing bacteria like Mycobacterium [38], Corynebacterium [39] and Nocardia [40] attach more intensively than non-mycolic ones. The longer chain length of mycolic acid correlates with high and rapid bacterial adhesion. Silva and de Ataujo [41] discussed the inhibitory role of lectins on biofilm formation. Lectins are proteins which bind to carbohydrates and polysaccharides of the outer membrane of bacteria. Lectins are ubiquitous in nature and can be found in large amounts in cereals and legumes.

Depending on the affinity of motile and nonmotile bacteria to adhesion, motile bacteria are capable of more active attachment. Nonmotile bacteria are slower in forming biofilms. The flagella of motile bacteria are crucial for the early stages of biofilm formation [30, 42].

Pseudomonas aeruginosa, one of human opportunistic pathogens was used as model organism to study bacterial biofilm formation cells [43]. Authors show that three extracellular polysaccharides (EPS) - alginate, Psl, and Pel are mainly responsible for the biofilm formation. EPS can represent between 50% and 90% of the total organic carbon in the biofilm [44]. EPS composed of polysaccharides are neutral biopolymers [45]. When EPS contain uronic acids such as D-glucoronic or D-galacturonic acid, they contribute to their anionic nature. The anionic property is important for the association with calcium and magnesium bivalent cations, which cross-link and provide greater strength to the bacterial biofilm. EPS can be either hydrophobic or hydrophilic but are generally highly hydrated due to water accumulation via the hydrogen bonding. This is the reason why natural biofilms can hardly be desiccated. In addition to divalent cations, EPS can bind to metal ions, proteins, DNA or lipids. Some EPS can even bind to humic acids [46].

The main extracellular polymeric substances which bacteria produces when exposed to phenol are PN (exopolymeric protein) and PS (lower polysaccharides) as described by Gao et al. [47]. During the biotransformation of heavy metals synthesize as EPS both homopolysaccharides and heteropolysaccharides [48]. Among the homopolysaccharides are identified dextrane, mutane, alternant, reuteran, gurdlan, levan and inulin. Gupta et al. reported the most abundant amidst heteropolysaccharides - alginate, xanthan, hyaluronan and sphingans [48]. P. aeruginosa responds to chlorine-based disinfectants by synthesis of alginate-based EPS as described by Xue et al. [49].

Undoubtedly, the architecture of each bacterial biofilm is unique. They can be mono-, double or multi-layer thick. When consisting of several layers, a network of many water channels can be observed inside the biofilm. According to the bacterial diversity, biofilms can consist of one bacterial strain but most often they contain mixed bacterial cultures. Different bacteria form thicker or thinner biofilms. Sometimes, when the biofilm is formed in the human body, it can also include nonbacterial compartments like erythrocytes or fibrin. Such types of biofilms form on heart valves. Bacterial biofilms formed on urinary catheters are known to consist of bacteria capable of urease-catalyzed degradation of urea, resulting in the release of ammonia. Ammonia induces precipitation of the calcium and magnesium inside the biofilm, leading to encrustation and catheter blockage [50].

Bacterial biofilms are perfect structures for plasmid DNA horizontal transfer, which occurs more easily between cells in biofilms than between planktonic cells because of the tighter cell-to-cell contact [51]. Quorum sensing also plays an important role in attachment or detachment of the biofilm [52].

4. Industrial area where the environmental bacterium KCM-R5 was isolated

KCM-R5 is an environmental bacterial isolate collected from a Pb-Zn smelter area and successfully entrapped in a synthetic polymer – poly(ethylene oxide) hydrogels (PEO) [53]. PEO hydrogels are macroporous polymers with high molecular weight and appropriate for bacterial immobilization due to their biocompatibility, strength and adhesiveness [54]. Additionally, they demonstrate nontoxicity, flexibility and durability. Initially, PEO hydrogels were obtained in situ by γ-irradiation of aqueous solutions [55], and two decades later, via methods based on chemical crosslinking [56]. UV crosslinking at cryogenic temperatures contributes to an important feature of the PEO hydrogels, namely the formation of macroporous structure. This macroporous structure is highly compatible with bacteria and enable their easy penetration, movement, hence, biofilm generation inside the hydrogels. The second main advantage of poly(ethylene oxide) hydrogel synthesis under cryogenic conditions than at room temperature is the extraordinarily high yield of gel fraction and better crosslinking [55, 56].

The environmetal bacterial isolate KCM-R5 was isolated from a soil sample collected at the industrial area of KCM Pb-Zn smelter (plant for production of non-ferrous metals), located in Central Bulgaria, near the town of Plovdiv. This plant is the biggest smelter on the Balkan Peninsula and producer of Pb, Zn, Au, Ag and Pt and their alloys since 1962. At approximately 1 km away is the pesticide factory AGRIA Ltd., founded in 1932. Both plants have been polluting the environment with heavy metals and hydrocarbons for years. Recently, the new wastewater treatment plant operating at KCM has reduced the outflow of polluted water. The produce of both plants is sold on the local market but is mainly exported worldwide. After the isolation, the bacterium was successfully cultivated in nutrient broth and nutrient agar and on selective media containing various heavy metals and 2,4-Dichlorophenoxyacetic acid (2,4-D).

5. Molecular-genetic analysis of KCM R5 bacterial isolate

A molecular-genetic analysis of the bacterial DNA was conducted aiming the identification of the bacterium. 16S rDNA of the KCM R5 strain was amplified, restricted with the frequently cutting endonuclases MspI, HaeIII and RsaI (New England BioLabs, UK) and sequenced. PCR amplification was performed using the primers 8F (forward) (5’-AGAGTTTGATCCTGGCTCAG-3′) and 1513R (reverse) (5’-GGTTACCTTGTTACGACTT-3′). This primer pair is preferable because it generates the longest amplicon ofapproximately 1400 bp. The amplification protocol consisted of one cycle of initial denaturation at 95°C for 3 min, 35 cycles of DNA denaturation at 94°C for 90 sec, primer annealing at 55°C for 40 sec, and primer extension at 72°C for 1.5 min, ending with a final extension step at 72°C for 20 min. Sequencing was accomplished with an automated sequencer 310 ABI-PRISM (Applied Biosystems, USA). The sequences obtained were analyzed using BLAST program and the bioinformatic analysis showed that the 16S rDNA sequence of KCM-R5 is affiliated with Pseudomonas rhodesiae with 99.9% identity. The 16S rDNA sequence of the strain was submitted to the Gene Bank-EMBL Database under the accession number AJ 830707. Figure 6 presents the dendrogram of the strain P. rhodesiae KCM-R5 (Gamma- Proteobacteria) with its closely related relatives.

Figure 6.

Dendrogram of the strain Pseudomonas rhodesiae KCM R5.

Members of the genus Pseudomonas are heterotrophs, rod-shaped, psychrotrophic and motile. According to Gram staining, pseudomonads are Gram-negative. Gram staining of the bacterial isolate KCM R5 shown in Figure 7 demonstrated that it is a Gram-negative bacterium.

Figure 7.

Gram staining of Pseudomonas rhodesiae KCM R5.

Ubiquitous in nature, the size of the bacteria of genus Pseudomonas varies between 1 and 5 micrometers in length and 0.5-1.0 micrometers in width. Bacterial flagella and pilli are important for the adhesion process. Pseudomonads are known to produce a vast amount of extracellular polysaccharides (EPS) [57]. They are able to produce biofilms even on smooth stainless steel surfaces, multiplying alone in the biofilm or co-existing with other bacterial species [58]. The biodegradation of phenol in wastewater by immobilized cells of Pseudomonas putida was described by [7, 59, 60].

When pseudomanads exist in mixed biofilms, they are more stable. In such biofilms P. aeruginosa or P. fluorescens synthesize a blue toxic substance called pyocianin (Figure 8) able to kill bacteria competing pseudomonads [27]. Norman et al. [61] demonstrated that pyocyanin influenced the functional diversity of a crude oil-degrading culture containing P. aeruginosa and affected the overall degradation of the crude oil.

Figure 8.

Structural formula of pyocyanin.

6. Heavy metal tolerance and growth of Pseudomonas rhodesiae KCM R5 on phenol and phenol derivatives as planktonic cells

The tolerance of planktonic cells of P. rhodesiae KCM-R5 to phenol and phenol derivatives was studied by cultivation of the strain on phenol, o-nitrophenol, pentachlorophenol, 2,4-dinitrophenol, 2,5-dinitrophenol and 2,4-dichlorophenoxiacetic acid (2.4-D) added to mineral media of Furukawa and Chakrabarty [62]. The medium contained per liter 5.6 g К₂HPO₄x3H₂O, 3.4 g КH₂PO₄, 2 g (NH₄)₂SO₄, 0.34 g MgCl₂x6H₂O, 0.001 g MnCl₂x4H₂O, 0.0006 g FeSO₄x7H₂O, 0.026 g CaCl₂x2H₂O and 0.002 g Na₂MoO₄x2H₂O. Phenol was applied at a concentration of 100 mg L⁻¹ while its five derivatives were added at a lower concentration of 20 mg L⁻¹ due to their higher toxicity and carcinogenicity, which may cause bacterial cells death. Xenobiotics were metabolized as a sole carbon source with no glucose or other carbohydrate addition. The investigation was performed for 144 h at 28°C. Figure 9 shows the growth of P. rhodesiae KCM R5. The strain demostrated the most intensive growth on 2,5 - dinitrophenol, 2.4-D, and pentachlorophenol (Figure 9).

Figure 9.

Growth of P. rhodesiae KCM-R5 on phenol and nitro- and chlorophenol derivatives as sole carbon sources.

7. Cryogels preparation

PEO cryogels necessary for bacteria entrapment were kindly supplied by Prof. Petar Petrov, DSc, Institute of Polymers, Bulgarian Academy of Sciences. Polyethylene oxide was dissolved in distilled water and polymerized by adding a photo initiator (4-benzoylbenzyl) trimethylammonium chloride. The obtained solution was poured into Teflon dishes forming layers 50 mm in diameter. The layers were further placed at -20°C for 2 h and irradiated with UV–VIS light for 2 min. PEO cryogels were extracted in distilled water for 7 days and freeze dried at -55°C, adopted from Doycheva et al. [55]; Petrov et al., [56], Satchanska et al., [63], Berillo et al., [54].

8. Entrapment of the bacteria into the PEO cryogels

The dried PEO cryogels were swelled by soaking without shaking in Furukawa and Chakrabarty medium for 24 h. The strain P. rhodesiae KCM R₅ was prepared for entrapment in the PEO cryogels by cultivation in Furukawa and Chakrabarty mineral medium with added 0.1% sterile glucose and 100 mg/L phenol until reaching OD 0.550. Then the bacterial culture was mixed with the pre-swollen PEO cryogels and shaken mildly at 100 rpm for 48 h. The resulting PEO-KCM R₅ unit consisting of cryogel and immobilized inside bacteria was gently placed inside the sterile Top Filter 45 mm, 500 ml system (Nalgene, Rochester, USA) and the locking rings were softly screwed up in order to avoid cutting of the cryogels, adopted from Satchanska et al., [63]; Donelli et al., [64] and Berillo et al., [65]. In the control swelled but empty (without immobilized bacteria inside) PEO cryogel was used.

9. Phenol biodegradation by PEO cryogel-P. rhodesiae KCM R5 biofilter and biofilm formation

The phenol biodegradation by the PEO cryogel-P. rhodesiae KCM R5 biofilm occurred via a sequencing batch process [66, 67]. The cycle of feeding via the upper container (phenol inflow) was 24 h and phenol concentrations was increased from 300 to 1000 mg L⁻¹. Volume of phenol inflow was 250 mL. The experiment was conducted 28°C, in triplicate. Every 24 hours 250 mL sterile medium that contained increasing phenol concentrations on the following scheme: 7 days with 300 mg L⁻¹, 5 days with 400 mg L⁻¹, 4 days with 600 mg L⁻¹ and 12 days with 1000 mg L⁻¹ phenol was poured into the upper funnel. The experiment lasted 28 days. No pressure was applied to the phenol-containing liquid and it run through the PEO cryogel-P. rhodesiae KCM R5 biofilm by only its gravity force, adopted by Satchanska et al., [63].

Inside the PEO cryogel-P. rhodesiae KCM R5 biofilm phenol degradation occurred and the solution of degraded phenol flowed out into the lower container (phenol outflow) [51, 52]. Phenol concentration in both phenol inflow and outflow was measured in succession at every 24 hours for a period of 28 days. Assessment of the phenol concentration in both inflow and outflow was carried out by colorimetric method using pyramidone. The protocol can be briefly described as follows: 0.125 ml phenol outflow liquid, 0.250 ml ammonium chloride buffer pH 9,3, 0.125 ml 3.5% pyramidone and 0.375 ml ammonium persulfate pH 7.0 were added to 12.375 ml distilled water to obtain 13 ml total volume. The reaction was incubated at room T ^oC for 45 min and its absorption was measured with a UV/VIS spectrophotometer at 540 nm. In the control, instead of the phenol outflow liquid 0.125 ml distilled water was added adopted by Satchanska et al., [63].

The phenol amount and biodegradation was calculated according to a standard curve and phenol biodegradation was calculated according the equation:

$$\mathrm{Efficiency}\mathrm{in}\left(\mathrm{X}\right)\mathrm{hour}\left(\%\right)=\mathit{Ci}–Cf/\mathit{Ci}\times 100$$E1

Data about phenol biodegradation [54, 55, 56, 57] by the PEOcryogel-P. rhodesiae KCM R₅ biofilm is presented in Figure 10.

Figure 10.

Phenol degradation by PEO cryogel-P. rhodesiae KCM R₅ biofilter.

After 28 days of biodegradation, the PEO-KCM R5 biofilter was disassembled and the cryogel with bacteria degrading phenol inside was taken out and subjected to Scanning Electron Microscopy analysis (SEM). The biofilter sample was covered with an Au microlayer and observed at JSM-5510 Scanning Electron Microscope (Jeol, Japan) in vacuum at 10000 V voltage and under different magnifications ranging from x500 to x20 000 (Figures 11 and 12).

Figure 11.

Macrostructure of swelled PEO cryogel without bacteria.

Figure 12.

Bacterial P. rhodesiae KCM R5 biofilm0 engeneered inside the biofilter.

10. Conclusions

Our molecular-genetic analysis showed that the environmental bacterium KCM-R5 is affiliated to Pseudomonas rhodesiae. The strain is tolerant to xenobiotics and can grow as planktonic cells on phenol and nitro- and chlorophenol derivatives as sole carbon sources. The constructed PEO cryogel-P. rhodesiae KCM R5 biofilm is capable of phenol degradation at a concentration of 1000 mg L⁻¹/24 h. Phenol biodegradation is due to the biofilm formed by P. rhodesiae KCM R₅ inside the PEOcryogel approved by observation using Scanning Electron Microscope. The so engineered PEO cryogel-P. rhodesiae KCM R5 biofilm can be used for environmental biotechnology application in industrial wastewater detoxification.

Acknowledgments

The author is grateful to Prof. P. Petrov, Institute of Polymers, Bulgarian Academy of Sciences, for supplying the PEO-cryogels and to Prof. Maria Angelova, Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, for the critical reading of the manuscript.

The author thanks RIDACOM Ltd., Bulgaria for the financial support in publishing the chapter.

Conflict of interest

The author declares no conflict of interest.