Poster on microtube bacterial encapsulation

Mr. Chaitanyakumar Desitti1, Dr. Sheldon Tarre1, Dr. Uta Cheruti1, Prof. Eyal Zussman2,

Mr. Ron Avraham2 and Prof. Michal Green1

1Civil and Environmental Engineering, Technion, 2 Mechanical Engineering, Technion

Abstract

Atrazine

Atrazine cycle

Use of herbicides

Effect on frogs

Change in gender

Campaign against Atrazine

Contact Info

Chaitanyakumar Desitti [email protected]

Immobilization of microbial cells has been found

to provide for the stability of enzymatic activity,

protect the cells, sustain specific bacterial population

for extended periods, prevent biomass loss and

minimize effluent post- treatment in bioreactors.

Pseudomonas sp. ADP , which is known as a fast

degrading atrazine bacterium, has been successfully

encapsulated in electro-spun core-shell hollow

polymeric microfibers (microtubes).

The long term objective of this research project is

the utilization of these microtubes in a bio-reactor

for atrazine removal from polluted ground water. In

preliminary experiments, long-term atrazine

degradation was studied using P. ADP bacterium in

microtubes with no addition of external carbon

source (non-growth condition), in consecutive

batches under semi-sterile conditions.

P.ADP is known to use atrazine only as nitrogen

source and not as a carbon source. The results

demonstrated that electrospun microtubes inoculated

with P. ADP can biodegrade atrazine for more than

two years without the addition of external carbon

source. The biodegradation of atrazine was

accompanied by a release of ammonium indicating

atrazine degradation.

Analysis of the microbial community by

polymerase chain reaction followed by denaturing

gradient gel electrophoresis (PCR-DGGE) showed a

shift towards several other microbial species. This

shift in population can probably explain the above

results of long term atrazine degradation without

external carbon source.

SHELL

CORE

Electrospinning setup Bacteria in fibers Fibers on plastic carrier SEM of microtube Microtubes in batch

Bioremediation is eco-friendly and cleaner

technology for micro pollutant removal in polluted

water. However, effective bioremediation requires

large concentrations of active bacteria that can be

difficult to maintain under in-situ or ex-situ conditions

and contribute to the contamination of the product

water.

Encapsulation of bacteria is an efficient method to

maintain a specific bacterial population. Electrospun

nanofibers having high porosity, nano-scale interstitial

space, and large surface-to-volume ratio are attractive

for environmental engineering applications. The co-

spun nanotube technology has been applied for pure

enzyme encapsulation (Dror et al., 2008) and bacterial

cell encapsulation (Klein et al., 2009; Klein et al.,

2012).

Atrazine is used as an agricultural herbicide in

many parts of the world to control a variety of weeds.

However, recent studies have shown that atrazine

causes sexual abnormalities in frogs (Hayes et al.,

2002), reduced testosterone production in rats

(Trentacoste et al., 2001) and elevated levels of

prostate cancer in workers at an atrazine

manufacturing factory (Sass et al., 2003). Small

amounts of atrazine residues are frequently detected in

surface and well water samples (Thurman et al., 1992).

Pseudomonas sp. ADP can use atrazine only as a

nitrogen source but not as a carbon

source (Mandelbaum et al., 1995).

Introduction

Results & Discussion

Long-term consecutive batch experiments for atrazine degradation by P. ADP

in microtubes under non growth condition

Atrazine removal and ammonium production by encapsulated Pseudomonas sp. ADP cells

in consecutive batches under non-growth conditions.

Band-4

Band-1

PCR based DGGE on enriched

culture lane-1 and micro tube

bacterial population on lane-2.

Enriched culture

Pseudomonas sp. strain ADP DSM 11735 (AM088478.1)

Band-3

Band-2

Variovorax paradoxus (NBRC 15149)

Band-4

Microbacterium testaceum strain DSM 20166 (NR026163)

Band-1

Chitinophagaceae bacterium EM 4 (JQ717375.1) 100

100

100

100

100

90

0.05

Phylogenetic tree based on 16S r DNA V3-V5 sequences

representing the respective DGGE bands. Bootstrap analysis based

on 1000 replicates. Scale indicates 5% sequence divergence.

Microbial community analysis Genomic DNA was extracted by using FastDNA Spin Kit for Soil, PCR was

performed amplifying the variable region V3-V5 of the bacterial 16S rDNA, using the

primers 341F and 907R PCR, DNA extracts were subjected to the PCR-DGGE analysis.

The DGGE profile of enriched culture bacteria showed one band whereas the microtube

DGGE consisted of four dominate bands (band 1-4). All the bands were sequenced and tree

was developed by using Neighbor-Joining method with support of MEGA. Enriched culture

was identified as the pseudomonas strain ADP (DSM 11735 (AM088478.1).

In microtube DGGE, band-1 is closely related to Chitinophagaceae bacterium, Band-2

has Variovorax paradoxus; Band -3 shows Pseudomonas sp. strain ADP, Band-4 was

identified as Microbacterium testaceum.

References

Dror, Y., Kuhn, J., Avrahami, R., Zussman, E., 2008. Encapsulation of enzymes in biodegradable tubular structures. Macromolecules 41,

4187–4192.

Hayes, T. B., Collins A., Lee M., Mendoza M., Noriega N., Stuart, A. A., Vonk, A., 2002. Hermaphroditic, demasculinized frogs after

exposure to the herbicide atrazine at low ecologically relevant doses. Science 99, 5476– 5480.

Klein, S., Kuhn, J., Avrahami, R. , Tarre, S., Beliavski, M., Green, M., and Zussman, E., 2009. Encapsulation of Bacterial Cells in

Electrospun Microtubes. Bio.macro.mol. 10, 1751–1756.

Klein, S., Avrahami, R., Zussman, E., Beliavski, M., Tarre, S, and Green, M., 2012. Encapsulation of Pseudomonas sp. ADP cells in

electrospun microtubes for atrazine bioremediation. J. Ind. Microbiol. Biotechnol 39(11), 1605–1613.

Mandelbaum, R. T., Allan, D. L., Wackett, L. P., 1995. Isolation and characterization of a Pseudomonas sp. that mineralizes the S-tri-azine

herbicide atrazine. Appl Environ Microb 61, 1451–1457.

Sass, J., and Brandt-Rauf, P. W., 2003. Cancer incidence among triazine herbicide manufacturing workers. J Occup Environ Med 45, 343–344.

Thurman, E. M., Goolsby, D. A., Meyer, M. T., Mills, M. S., Pomes, M. I., Kolpin, D. W., 1992. A reconnaissance study of herbicides and

their metabolites in surface water on the midwestern United States using immunoassay and gas chromatography/mass spectrometry. Environ

Sci Technol 26, 2440–2447.

Trentacoste, S. V., Friedmann, A. S., Youker, R. T., Breckenridge, C. B., Zirkin, B. R., 2001. Atrazine effects on testosterone levels and

androgen-dependent reproductive organs in peripubertal male rats. J Androl 22, 1142-1148.

Lane-1 Lane-2

Enriched

culture

Microtube

bacterial

population

Band-2

Band-3

Conclusions: Analysis of the microbial community showed a shift from pure culture of

P.ADP to towards several other microbial species. This shift in population can probably

explain the above results of long term atrazine degradation without external carbon source.

Phase-3 shown in below graph

Phases Number

of

batches/

days

Initial

atrazine

conc.

(ppm)

Atrazine

biodegraded

(ppm)

Percentage of

atrazine

biodegraded

Ammonium

theoretical

(ppm)

Ammonium

measured

(ppm)

Percentage

of

ammonium

recovered

Phase-1 18/40 20±2 15±5 75.0 4.9±1.7 2.5±1 51.5

Phase-2 46/149 20±2 18±3 90.0 5.8±0.7 4.5±1

77.4