Exposure to ZnO-NPs enhanced gut-associated microbial activity in Eisenia fetida

With advent of the nanotechnology era, the environmental risk has continuously been receiving engineered nanomaterials, as well as their derivatives. Our current understanding of the potential impact of nanomaterials and their effect on soil organism is limited. The present study fills the gap between effect of manufactured nanomaterials (NPs) and their available natural scavengers. In the study, earthworm Eisenia fetida (EW), which occupies 60 to 80% of the total biomass and well known for its contribution to cellulolytic degradation of organic wastes, was exposed to ZnO-NPs. Findings suggests that E. fetida can survive even at high exposure of ZnO-NPs (10 mg/kg) and can exhibit increase in bio-accumulation of Zn content in its body tissue with decreased NPs. Exposure of 35 and 10 nm ≥3.5 mg/kg sized NPs showed an increase in cellulase activity by 38 to 41%. This increase in cellulolytic activity in EWs’ gut may also be helpful in the bioconversion of lignocelluloses waste. Eighteen strains of cellulose hydrolytic bacteria capable of producing cellulase were obtained from the guts of EWs exposed to ZnO-NPs. The results of biochemical and 16SrRNA gene sequence examinations showed that six strains belongs to Bacillus sp.; five strains belongs to the sublines of Bacillus and others belongs to the Pseudomonas sp. The study advocates the application of ZnO-NPs enhance gut-associated microbial activity.


INTRODUCTION
Manufactured nanoparticles have a wide range of application having unique preparation as compared with their bulk counterparts (Nel et al., 2006).Nano-forms of metals, metal oxides, carbon-based materials and biopolymers are being used in several applications.Zinc oxide nanoparticles (ZnO-NPs) are one of the most abundantly used nanomaterials in cosmetics and sunscreens as they efficiently absorb ultraviolet (UV) light and also do not scatter visible light.This makes ZnO-NPs more transparent, aesthetically compared to their bulk counterpart (Schilling et al., 2010).They are also being used in the food industry as additives and packaging due to their antimicrobial properties (Gerloff et al., 2009;Jin et al., 2009).
They are also being explored for their potential use as fungicides in agriculture (He et al., 2010), as anticancer drugs and in biomedical imaging applications (Rasmussen et al., 2010;John et al., 2010).The increased production and use of ZnO-NPs enhances the probability of exposure in occupational and environmental settings.They may be introduced to the environment through wastewater from industrial sites or domestic sewage from showering or swimming.NPs can be transported to soil via sewage sludge used for land application.Therefore, terrestrial ecosystems are expected to be an ultimate sink for a larger portion of NPs.This raises concerns about their ecological effects, entry into food webs, and ultimately, human exposure from the consumption of contaminated agricultural products.Hence, it is of great interest to understand the effect of NPs on soil organisms.
In this study, earthworm Eisenia fetida was exposed to ZnO-NPs because it occupies 60 to 80% of the total biomass and is well known for its contribution to lingocelluloses' decomposition of organic wastes.Earthworms (EWs) influence decomposition indirectly by affecting microbial population structure and dynamics.The gut of some species of earthworm poses cellulolytic activity (Siturzenbaun, 2009).However, it has long been recognized that most earthworms and other animals living in the soil do not produce their own endogenous cellulase; instead they depend on the cellulase from their resident gut microorganism (Domínguez et al., 2005).However, endogenous cellulase genes have been recently reported from earthworm Pheretima hilgendor (Nozaki et al., 2009).Despite these newly discovered abilities, earthworm cannot assimilate lignocellulose by means of endocellulase alone, since efficient lignocellulose degradation requires synergetic action of a suite of other enzymes, including exocellulase, hemicellulase (xylanase) and lignin peroxidase (Lynd et al., 2002).According to Brown and Doube (2004), a synergistic earthworm-microbial digestive system (dual-digestive system) is indispensible for the digestion and utilization of lingocellulose by earthworms.Several studies have demonstrated that earthworm gut contain aerobic microorganism in abundance (Dash et al., 1986;Karsten and Drake, 1995).Moreover, some aerobes have been shown to proliferate during passage through the earthworm gut, reaching densities greater than those in the soil (Fischer et al., 1995;Kristuek et al., 2007;Parthasarathi et al., 2007); considering the dual-digestive system described and the abundance of aerobes in earthworm gut, we hypothesized that some species of cellulolytic aerobes can survive when exposed to ZnO-NPs and may also contribute to lignocelluloses digestion in gut.
Our current understanding of the potential impact of nanoparticles on cellulolytic activity of earthworms is limited.In earlier studies, we reported that the application of 100 nm and 50 nm ZnO-NPs showed no significant DNA damage on E. fetida and recorded their coelomocyte potential ability to uptake ZnO-NPs from soil ecosystem (Gupta et al., 2014).The question of the impact of ZnO-NPs on earthworm-gut associated microbiota needs to be answered.The study suggests, ZnO-NPs enhanced the activity of cellulolytic bacteria in the gut of E. fetida.

Test compound
ZnO-NPs (100 nm, 50 nm, 35 nm, 10 nm) were purchased from Sigma Aldrich chemical (St.Louis, MO, USA).Size of particles was measured in 20 µl particle suspension from the test medium on 400 mesh carbon-coated copper grid and was observed in TEM (40-100 kv) at the Sophisticated Analytical Instrumentation Facility, Department of Anatomy, All India Institute of Medical Science, Delhi, India.

Test organism and method of exposure
Exposure of ZnO-NPs on E. fetida followed the published organisation for Economic Co-operation and Development (OECD) guideline (2004).Twenty-clitellate adult E. fetida weighing 0.30±0.12g in three replicate exposure chamber, containing 1 kg dry mass of artificial soil medium were chosen for the test experiment.The soil medium consists of 70% quartz sand, 10% peat moss and 20% kaolin.The pH was adjusted with the addition of a small amount of crushed limestone.Various doses (He et al., 2010) of NPs (0.5-10.0 µg/kg) were added to the dry soil and mixed by homogenizer for 5 min.The moisture content was maintained for 60%.After 40 days of exposure, earthworms were washed with autoclaved tap water; their body surface were sterilized by a brief rinse with 70% ethanol and immediately anesthetized on crushed ice.

Estimation of Zn content in earthworms' tissue
Three EWs of each group were weighed and digested with 3 ml of concentrated nitric acid for 24 h.After digestion, the acid was evaporated; then the residue was dissolved in 4% nitric acid.Zinc concentration was quantified with Inductively coupled plasma atomic emission spectroscopy (ICP-AES) at The Energy Research Institute, New Delhi, India.

Enzymatic activities
Cellulase activity was estimated by determining the released reducing sugars after the incubation of samples (5 g fresh weight) with carboxymethyl cellulase (CMC), sodium salt (0.7%) for 24 h at 50°C in a 690 nm microplate reader (Schinner and Von Mersi, 1990).

Isolation of cellulolytic bacteria
The complete intestine was dissected out and homogenized in autoclaved distilled water, containing 0.5 mm glass beads with vortex mixing for 5 min.The resulting suspension was serially diluted with water and used as inoculum.Cellulose-hydrolytic bacteria were isolated using Bushnell Hass medium (BHM) amended with carboxymethyl cellulase (CMC) as the sole carbon source.The CMC-amended with BHM medium consist of (g/l); CMC, 10; MgSo 4 .7H 2 O, 0.2; K 2 HPo 4, 1; KH 2 Po 4, 1; NH 4 NO 3 1; Fecl 3 .6H 2 0, 0.05 and CaCl 2 , 0.02.After enrichment in CMC amended medium, the inoculums (0.1 ml; successively diluted to 10-5 times) were repeatedly streaked on BHM agar plates containing the amended CMC.After one week of incubation, the plates were stained by Congo red to observe cellulolytic activity of isolated strains.The cellulase activity of each culture was determined by measuring the zone of clearing on agar plate.The individual colony having significant clear zone was selected and transferred to a fresh CMC-amended BMH medium; then the inoculum was serially diluted 10-5 times and streaked over BMH agar plates repeatedly, and the bacteria were re-isolated.Through several such processes, eighteen pure bacterial cultures were obtained, morphologically observed in light microscope and biochemically characterized using Garrity et al. (1985) method.

16S rRNA gene sequencing and phylogenetic analysis of isolates
Amplification and sequence analysis of the 16S rRNA gene was performed as described by Chen et al. (2001).The sequences of isolates were compared with other available sequences in the gene bank.The multiple sequence alignment including the eighteen cellulose-degrading strains and their close relatives were obtained using National Center for Biotechnology Information (NCBI) platform.The phylogenetic reconstruction was inferred using the neighbor-joining method UPGMA, maximum-likelihood and Fitch-Margoliash methods BioEdit software in the Bio Edit program.A phylogenetic tree was drawn using the TREE VIEW program.The sequence identities were calculated using NCBI software.

Nucleotide sequence accession numbers
The 16S rRNA gene sequences of the bacterial isolates have been deposited in NCBI nucleotides sequence databases.Obtained accession numbers of the isolates are shown in Table 1.

Statistical analysis
Two-way analysis of variance (ANOVA) was performed using the Statistical Package for the Social Science (SPSS) 10.5 software.The objective of the statistical analysis was to determine any significant differences among the parameters analyzed in different treatments during the determination of uptake of ZnO-NPs and their potential use as a biotransformation agent.

Survivability of earthworms exposed to ZnO-NPs
The survivability of EWs was observed (Table 2) after exposure of a wide range (0.5 to 10 mg/kg) of ZnO-NPs (100, 50, 35 and 10 nm).It was observed that E. fetida could survive even at high concentration (10 mg/kg).Commercially, ≤0.5 mg/kg ZnO-NPs are used for different purposes including nanofertilizer for the release of Zn in soil ecosystem.Thus, the highest concentration was considered to be 10 mg/kg.However, there was sporadic mortality in some treatments, which were neither concentration-dependent nor statistically-significant.

Zn content in EW's tissue
After 28 days of exposure, the Zn content in body tissue was measured (Table 3); the result exhibits increase in bioaccumulation with decrease in size of NPs.The highest Zn content (36.18±3.17µg/kg) was recorded at the exposure of 10 nm.The results suggest that EWs can uptake and accumulate 20 to 36 µg/g Zn in their body tissue, depending on the dose and size of ZnO-NPs.Gardea-Torresdey et al. (2005,) observed that Au +1 and Au +3 can also be consumed by EWs from the soil and can subsequently be reduced to metal within the tissues.To our knowledge, this is the first evidence to prove that this process also account for the reduction of ZnO to Zn by EWs.

Increase in cellulolytic activity
The cellulolytic activity of EWs' gut increased with the decrease in size of NPs (Table 4).Although, no significant variations were observed in cellulolytic activity as compared to the control for 100 and 50 nm exposures at ≤7.5 mg/kg.In contrast, the exposure of 35 and 10 nm ≥3.5 mg/kg sized NPs, showed increase in enzymatic activity by 38 to 41%.Increase in cellulolytic activity in EWs may be helpful in bioconversion of lignocelluloses waste.Degradation of cellulose is a slow process limited by several factors involving cellulases (Sinsabaugh and Linkins, 1988).Decomposition of lignocellulosic residues is directly mediated by extracellular enzymes (Sinsabaugh et al., 1992).Therefore, analysis of the dynamics involved in the increase of cellulolytic activity with the exposure of ZnO-NPs may clarify the mechanism relating to the rate of decomposition with substrate quality and nutrient availability as reported by Sinsabaugh and Linkins (1993).Observations are contrary to Hu et al. (2010) who reported that NPs adversely affects cellulase activity in E. fetida.

Phylogenetic analysis of cellulolytic bacterial isolates
Eighteen strains of cellulose hydrolytic bacteria (Table1), capable of producing cellulase, were obtained from the gut of EWs exposed to ZnO-NPs.All strains grew well at 35°C on CMC-amended BHM medium under aerobic conditions.Colonies on CMC agar plates are circular, smooth, creamy yellow circles within 3 days (Figure 1).Microscopic examination showed that the isolated strains were in straight rods with Gram-positive reaction.The 16S rRNA gene were also used to characterize cellulolytic isolates (Figure 1).Based on the sequence identity of 16S rRNA gene strains, bs4 and bs5 showed the highest similarity towards the Bacillus subtilis type strain.Strain b1 exhibits close resemblance (99.8%) with Bacillus licheniformis.Strains bs2, bs 11 and bs19 represent different sublines of Bacillus.Moreover, bs8, bs9, bs10, bs20 and bs25 showed  knowledge.The bio-chemical properties of isolated cellulose hydrolytic bacteria of EWs' gut exposed to ZnO-NPs are shown in Table 5.The 16S rRNA gene phylo-genetic tree using the neighbor-joining method was constructed for the eighteen strains as indicated in Table 1.According to the results of the biochemical and the 16SrRNA gene sequence examinations, the six strains (bs1, bs2, bs4, bs5, bs11 and bs19) belongs to Bacillus sp.; another

DISCUSSION
To degrade lignocelluloses in ZnO-NPs exposed earthworms, the presence of cellulolytic aerobes in the earthworm's gut is significant.Since, the gut is free of oxygen and anaerobic bacteria, which comprise of a major population in the gut micro-biota (Karsten and Drake, 1995;Wust et al., 2011).Aerobic soil microorganisms, including cellulolytic ones, associated with lignocelluloses are ingested by an earthworm and then introduced into the anterior digestive tract, where the zinc rich oxic conditions allow their growth and produce enzymes for lignocelluloses digestion.Subsequently, the microorganisms are exposed to the anoxic conditions of the gizzard and intestine, resulting to the activation of aerobic microorganisms with continuous activity of their enzymes, including those of saccharification lingocelluloses during gut passage.The resulting degradation products (glucose, xylose and their oligosaccharides) are thus consumed by the earthworm and, also used by anaerobes as a fermentation substrate (Fujii et al., 2012).The grown biomass of anaerobes is digested and consumed by earthworms as a source of essential amino acids and fatty acids (Sampedro et al., 2006).Glycosidase activity should remain even if the growth of enzyme-producing microorganisms is halted by anaerobiosis, because these enzymes are resistant to proteolytic inactivation and are able to continue the saccrification for several hours, which corresponds to the food transit time of earthworms (Dash et al., 1986;Lynd et al., 2002).The growth of cellulose hydrolysis bacteria as well as other aerobes is halted by anaerobiosis, casting these microorganisms to become a minor population in the gut.However, some are resistant to digestion and finally excreted as a part of the cast, because earthworm digestive fluid contains microbial activity that is selective for certain species, but neutral or stimulating for the growth of others (Byzov et al., 2007;Khomyakov et al., 2007).
ZnO nanoparticles have a broad spectrum of antibacterial activities.An inhibitory effect of ZnO nanoparticles on B. subtilis, Staphylococcus aureus, S. epidermidis, Streptococcus pyogenes and Enterococcus faecalis has been reported (Xie et al., 2011;Vani et al., 2011;Wang et al., 2012).Contrary to the earlier findings, our results showed that ZnO nanoparticles favors the bacterial activity in EWs'gut.In the case of the behavior of NPs particles in EWs, two central questions arise: Do particles remain in their nanoparticulate state in gut of EWs or do they congregate to coarse agglomerates/ aggregates?; Do substances such as humic acid that exist in the Ews'gut influence agglomeration behavior?
In this study, we assumed that the particle looses their nanoparticulate state and agglomerate in the gut of EWs due to the presence of humic acid.The release of engineered nanoparticles into the soil ecosystem leads to the contact of these particles with microbiota of EWs'gut; this contact does not affect EWs'microbiota.
The growth of microbiota due to nanoparticle-specific characteristics of metal oxide nanoparticles depends on the dispersion and stabilization of these particles.Our earlier findings, suggests that ZnO particles do not generated significant hydrogen peroxide in the gut of EW (Gupta et al., 2014).This indicates that ZnO-NPs do not remain in the nanoparticulate state of EWs'gut; it aggregates in the gut of the EWs and confirms their biotransformation ability.Therefore, microbiota of EWs' gut is not adversely affected by nanoparticles.

Conclusion
ZnO-NPs are among the most commonly utilized group of nanomaterials and have a wide range of application.As a well-known phytocatalyst, ZnO-NPs have received much attention in the degradation and complete mineralization of environmental pollutants.Finding suggests that the cellulase activity in earthworm gut increased with the decrease in size of ZnO-NPs.Therefore, the analysis of the dynamics involved in the increase of cellulolytic activity with exposure of ZnO-NPs may clarify the mechanisms relating to the rate of decomposition with substrate quality and nutrient availability.The isolated and identified eighteen efficient cellulolytic bacteria have the ability to hydrolyze different cellulosic substrates in ZnO-NPs spiked vermireactors.However, further investigations are required to understand the mechanism that proliferate growth of cellulose-hydrolytic bacteria by ZnO-NPs.

Figure 1 .Figure 2 .
Figure 1.Phylogenetic tree of cellulose hydrolytic isolates of gut of E. fetida constructed using the Robust Phylogenetic Analysis software (LIRMM).Trees for bacterial isolates are based on the partial 16r DNA sequences.

Table 1 .
Accession numbers of bacterial isolates obtained by submission of 16S rRNA sequence to NCBI.

Table 2 .
Percentage of mortality of E. fetida in ZnO-NPs fortified vermireactors.

Table 4 .
Cellulolytic Activity (µg/g/h) in gut of E. fetida after the exposure of various ZnO-NPs.
All values are mean and standard deviation of three replicates.*Significant(p<.01) close similarity (99.8%) with type strain Pseudomonas aeruginosa.The highest similarity of bs12 and bs14 was found close to Paenibacillus alvei.PR7 and PC7 show 99.9% similarity with Paenibacillus dendritiformis.Strain bs13 represents different sublines of Paenibacillus and bs21 belongs to Enterobacter.Strain bs6 shows close similarity (99.9%) with Brevibacillus limnophilus; and is reported for the first time in EWs'gut to the best of our

Table 5 .
Biochemical properties of eighteen bacterial isolates from EWs' gut exposed to ZnO -NPs fortified vermireactor.