The positive fate of biochar addition to soil in the degradation of PHBV

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Remediation and Control Technologies

The positive fate of biochar addition to soil in the degradation of PHBV-silver nanoparticles composites Suely Patrícia Costa Gonçalves, Mathias Strauss, and Diego Stéfani Teodoro Martinez Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01524 • Publication Date (Web): 24 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Environmental Science & Technology

The positive fate of biochar addition to soil in the degradation of PHBV-silver nanoparticle composites

Suely P.C. Gonçalves, Mathias Strauss and Diego Stéfani T. Martinez

Brazilian Nanotechnology National Laboratory (LNNano), Brazilian Center for Research in Energy and Materials (CNPEM), Zip Code 13083-970, Campinas, São Paulo, Brazil.

*Corresponding author Dr. Diego S. T. Martinez Brazilian Nanotechnology National Laboratory (LNNano), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, São Paulo, Brazil. P.O. Box 6192, Zip Code 13083-970. E-mail: [email protected]

1 ACS Paragon Plus Environment


Abstract The environmental contamination of soils by polymeric and nanomaterials is an increasing global concern. Polymeric composites containing silver nanoparticles (AgNP) are collectively one of the most important products of nanotechnology due to their remarkable antimicrobial activity. Biochars are a promising resource for environmental technologies for remediation of soils considering their high inorganic and organic pollutant adsorption capacity and microbial soil consortium stimulation. In this work, we report, for the first time the use of biochar material as a tool to accelerate the degradation of polyhydroxybutyrate-co-valerate (PHBV) and PHBV composites containing AgNP in a tropical soil system, under laboratory conditions. This positive effect is associated with microbial community improvement, which increased the degradation rate of the polymeric materials, as confirmed by integrated techniques for advanced materials characterization. The addition of 5 to 10% of sugarcane bagasse biochar into soil has increased the degradation of these polymeric materials from 2 to 3 times after 30 days of soil incubation. However, the presence of silver nanoparticles in the PHBV significantly reduced the degradability potential of this nanocomposite by the soil microbial community. These results provide evidence that AgNP or Ag+ ions caused a decline in the total number of bacteria and fungi, which diminished the polymer degradation rate in soil. Finally, this work highlights the great potential of biochar resources for application in soil remediation technologies, such as polymeric (nano)material biodegradation.

Keywords: environmental nanotechnology, remediation, nanoparticles, biotransformation, nanoecotoxicity. 2 ACS Paragon Plus Environment

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1. Introduction Polymeric materials have great scientific-technological relevance in food packaging, engineering materials and biomedicine. (1,2) The promises offered by functional nanomaterials have huge potential to develop polymeric nanocomposites with distinguished technological applications. For example, the incorporation of silver nanoparticles (AgNP) in polymers has been explored to produce antimicrobial nanocomposites for biomedical and food packaging industries. (3,4,5) The massive needs by our technological centric society for polymeric composites pose environmental challenges on the safe and sustainable disposal of these materials in the environment. (6) The terrestrial ecosystem is the main destination of such kind of materials, and most of the petroleum-based polymers are long lasting materials when discharged in soil. To overcome this problematic, the development and use of the biodegradable biobased polymeric materials is a promising alternative to reduce the wastes in the environment. (7) The biodegradation and biotransformation processes primarily depend on the polymer type and exposure conditions. An important issue is the detailed understanding of the polymers degradation mechanisms, and how silver nanoparticles contribute to these processes. (8) Polyhydroxybutyrate-co-valerate (PHBV) has attracted attention due to its biodegradability and biocompatibility. (9) Such kinds of biopolymers are produced by a wide variety of microorganisms and plants. (10) Although PHBV is considered as ecofriendly material, there are few reports on the total life cycle of biopolymer nanocomposites (11)



When it comes to AgNP loaded polymers there is great concern about the possible detrimental environmental health effects due to acute or chronic exposure to them, especially when applied to commercial and large-scale products. (8) The migration of Ag species from food storage polymeric containers to the environment have been reported in the literature (8,12,13), but there still not enough data on the risks to the consumers and ecotoxicological adverse effects considering the exposure to these products during its life cycle. (14,15) Silver nanoparticles can exert their antibacterial activity by three different mechanisms: i) release of Ag+ ions, which can bind to and thiol groups (SH), destabilizing the proteins structures and cell membrane enzymes of the bacteria cell membrane; ii) adsorption of AgNP on the bacterial cell membrane, which can lead to pore formation, extravasation of the cytoplasmic content and consequently to cell death; and iii) production of reactive oxygen species (ROS), free oxygenated radicals derived from oxygen, which can also cause DNA structure destabilization and proteins denaturation. (16) The biological effects of AgNP in calcareous soils with two different textures and salinity levels showed that the effects on soil enzyme activities and microbial respiration strongly depend on the Ag dose and type of soil. (17) It is critical to evaluate the interactions and transformations of biopolymers and nanocomposites in the soil environment towards responsible and sustainable actions. Common approaches to improve degradation of biopolymer composites in soil mostly lie on changes of polymers chemical composition/structure or by engineering bioplasticdegrading microorganisms/enzymes consortia (11). In this work, we are focusing to evaluate a new approach for biopolymers nanocomposites degradation in soil exploring promissing advantages of biochar. In this sense, it is an innovative and sustainable strategy


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towards biochar use on environmental remediation technologies concerning soils contaminated with plastics and emerging nanocomposites.

Biochar has been applied to soil improvement and remediation for agricultural and environmental applications by its incorporation into soils, but had never before been used to improve the biodegradation of polymeric materials(18, 19,20). Biochar is the solid product of the thermochemical conversion process of any kind of organic matter at oxygen-limited conditions at temperatures above 400 oC known as pyrolysis. The production of biochars from agricultural residues meets the needs for their technological employment in high materials consumption applications. One of the most expressive and intensive agroindustry, which could be explored in this sense, is the sucro-energetic industry which produces sugar, ethanol-based biofuels and electricity from sugarcane. (21) The processing of 1 ton of sugarcane in these industries generates about 200-300 kg of sugarcane bagasse residues (22), which could be used as an abundant and renewable resource for biochars production. In Brazil for example, which is the biggest sugarcane producer in the world, about 700 Mt of sugarcane are processed per year (23) that result in close to 200 Mt of bagasse. Biochars can adsorb and immobilize inorganic and organic pollutants from contaminated soils. (24) This ability arises from ion exchange, electrostatic attraction and surface complexation between the polluting entities and the organic functional groups or mineral fractions of the biochars. (25) In agriculture, the incorporation of biochars into soil has remarkable beneficial effects such as fertility and water retention enhancement, and soil physical structure and aeration improvement, which may lead to an increase of the crops productivity. (26, 27) Furthermore, when added to soils, biochars are known to increase the



abundance and to change the dynamics of the bacteria and fungi communities, and influence their enzymatic activities. (28, 29) The effects of the addition of biochars into soils are associated to the properties their selves, which vary according to the raw material characteristics (e.g. source, particles size, ashes content, humidity) and the pyrolytic processing conditions (e.g. reactor geometry, temperature, reaction time, heating rate). Additionally, the soil properties (e.g., texture, nutrients and organic matter composition, pH) and climatic factors (e.g. temperature and humidity) also act on those effects. (30) The soil microbial community and their enzymatic activities, that include biogeochemical processes, are impacted by the physicochemical properties of biochar. (31) The addition of biochars to soils improve the soil aeration, water content and pH conditions for the well development of the microbiota, their pores and surface act as a shelter for microorganisms, they may supply essential nutrients, interrupt the communication between microbial cells, and increase the soil sorption and degradation capacity of toxic substances for these organisms. (32) In this work, we studied the influence of biochar addition to soil in the microbial consortium improvement and the degradation rate of the PHBV composites. We report for the first time, to best our knowledge, the use of sugarcane bagasse biochar as a renewable material to accelerate the biodegradation of polyhydroxybutyrate-co-valerate (PHBV) and PHBV composites containing AgNP in a tropical soil. Our findings suggest that biochar can be applied as an innovative environmental remediation technology for plastics and nanocomposites.

2. Materials and Methods Chemicals 6 ACS Paragon Plus Environment

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The polymer used in this study was poly(hydroxybutyrate - co – valerate) (PHBV) HV 6.2% (FE 130 – Biocycle, Serrana/Brazil), d = 1.22 g cm-3; Mw~ 113.000 g mol-1. For the synthesis of the silver nanoparticles (AgNP) silver nitrate ( 90%, Allkimia, Brazil), polyvinylpyrrolidone (PVP, Mw ~ 40.000 g.mol-1, Sigma-Aldrich, USA), sodium hydroxide ( 98%, Quimex, Brazil) and ethyl alcohol ( 99.5% PA, Synth, Brazil) were used. The culture medium used for quantification of total microorganisms were Tryptic Soy Agar (Kasvi, Brazil), Rose Bengal chloramphenicol (Acumedia, England) and Plate count agar (Acumedia, England). The biochar was produced from milled sugarcane bagasse pellets in a fluidized bed pyrolysis reactor operating at 480 oC at the Bioware company (Brazil).

Soil Characterization Red clay latosol soil was collected in the locality 22º48'08"S 47 03'08" W from 0 to 15 cm depth profile at the CNPEM Campus in Campinas, São Paulo, Brazil. The soil was sieved with a 2 mm mesh sieve and its chemical and physical characteristics determined (Table S1-Support Information). The climate at the soil collection site is classified as humid temperate with dry winter and hot summer (Cwa), according to Koeppen climate classification.

Sample Preparation Silver nanoparticles were synthesized according to the following methodology (33): to 10 mL of ethanol 50 mg of PVP (polymeric stabilizer) were added and mixed for 5 min under magnetic stirring. Separately, a solution containing 4.5 mg of AgNO3 in 15 mL of ethanol



was prepared and then added to the PVP solution, followed by the addition of 5 mL of a 0.12 M NaOH ethanolic solution (reducing agent). The mixture was kept under magnetic stirring for 30 min. The polymer films were prepared by casting. For this, 0.2 g of PHBV (6.2% HV) were dissolved in 10 mL of chloroform ( 99.8%, JT Baker) under magnetic stirring at 50 °C for 30 min. For the preparation of PHBV-AgNP, 5% (v/v) of the AgNP colloid was added to the polymer solution (Ag/PHBV = 0.024%, wt%) and left under magnetic stirring for 20 min. Then, the polymer solutions were poured into Petri dishes (90 mm) and left on a flat surface for evaporation of the solvent at room temperature into a fume hood.

Antibacterial Test of PHBV-AgNP films Antimicrobial activity of the PHBV-AgNP films was determined following the ASTM standard (34), using the PHBV film as a reference and Escherichia coli (ATCC 8739) as inoculum. Briefly, E. coli culture was transferred to 100 mL sterile agar slurry to obtain 106 CFU mL-1 final concentration inoculum. Then, 1.0 mL of the inoculated agar slurry was pipetted on PHBV-AgNP film samples (n = 3) placed on sterile Petri plate. The samples were incubated for 24 h at 37 oC in the dark in an incubator. After incubation, the agar slurry was removed from the samples surfaces and transferred to test tubes containing saline solution. Serial dilutions were performed and spread on agar plates. Bacterial colonies were counted after 24 h of incubation. For comparison, PHBV films were used as reference material in the same conditions. Additionally, a control experiment was performed using only the agar slurry without contact to polymeric films.


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Soil-Biochar Experiment The polymer degradation experiments were carried out in soil columns to which 0, 1, 5 or 10% of sugarcane bagasse biochar (wt%) were added. After mixing, the soil-biochar mixtures were kept moistened (60% of the field capacity) and acclimated for 28 days in a acclimatized room under controlled temperature and humidity (28 (2) oC, 60% field capacity). Then, PHBV and PHBV-AgNP films (1 x 1 cm) were buried in the soil-biochar columns (100 g), in independent quadruplicates for each time lapse tested. The samples were kept for 7, 14, 21 and 30 days in the greenhouse at 28 (2) oC being moistened as needed. After each time, the samples were removed from the soil columns and cleaned. Appropriate rinsing of the composites surface is a critical issue on reliable composites films analysis. Aware of this, after removal from soil columns, the residual soil particles attached on the films surface were first removed with a soft brush, and then extensively rinsed with ultrapure water. After that, the samples were dried and stored in a desiccator at room temperature for further analysis. The mass of each sample was measured to determine its biodegradability. The results are presented as means ± standard deviations values in percentage of the samples initial mass. Statistical analysis was performed using ANOVA followed by Tukey's test with the significance level of p

The positive fate of biochar addition to soil in the degradation of PHBV

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