agriculture

1. Leather industry solid waste-a potential source for
the production of bioactive molecules to be used in
the Tertiary Treatment of domestic wastewater
By
FARIDHA BEGUM. I, K.RAMANI*
Biomolecules and Biocatalysis Lab
DEPARTMENT OF BIOTECHNOLOGY
SRM UNIVERSITY

2. • The conventional tertiary disinfecting process in domestic wastewater
treatment like chlorination, disinfectants and UV treatment are used
which are ineffective and leads to the development of drug resistant
strains.
• Rapid emergence in antibiotic-resistant strains of pathogens increases
the urgency for alternate strategies to control Multi Drug Resistant
Organisms (MDROs).
• Animal fleshing, a solid waste generated in large quantity in leather
industries possesses serious challenges in disposal. It has high protein
and lipid content but left unutilized.
INTRODUCTION

3. • Rumen fluid from slaughterhouses is another unutilized source that has
a rich consortium of microbial flora, effective in the waste degradation
used for anaerobic fermentation of animal fleshing for the production
of bioactive compound.
• This work focus on the efficient treatment of the MDROs and
pathogens present in the secondary biological treated domestic
wastewater using bioactive compounds derived from the microbial
fermentation of animal flesh.
• To date, there is no report on the use of bioactive compound from
animal flesh for the destruction/disinfection of MDROs and other
clinical pathogens in the secondary biological treated domestic
wastewater (SBTDW).
Introduction continuation….

4. METHODOLOGY
Anaerobic fermentation of animal flesh
Confirmation of animal flesh degradation using FT-IR, NMR, SEM
Extraction and characterization of crude bioactive compound
Antimicrobial activity of crude bioactive compound
Immobilization of crude bioactive compound onto Mesoporous activation carbon
Treatment of secondary biological treated domestic waste water

5. Day 3
RESULTS AND DISCUSSION
ANAEROBIC DEGRADATION OF ANIMAL FLESH (AF)
Complete degradation of animal Flesh was observed under anaerobic condition on 8th day.

6. AF DEGRADATION CONFIRMATION BY FT-IR
Figure 2 : FT-IR spectra of a) Untreated AF and
b) treated AF
Peak at 3128 cm-1 corresponds to hydroxyl group of carboxylic acids
Peaks at 2856-2834 cm-1 shows the CH3, CH2, CH stretching
The intensity of the peak is reduced at 1745 cm-1 which corresponds to ester
carbonyl functional group in the treated sample.
a b

7. NMR Unhydrolysed Hydrolysed
1H
13C
AF DEGRADATION CONFIRMATION BY NMR
In H1 NMR , methoxy
group is disappeared in
hydrolysed sample which
was present in 4.21 ppm in
the unhydrolysed sample.
In C13 NMR, the peak
appeared at unhydrolysed
sample in 172 ppm was
disappeared in hydrolysed
sample
Figure 3: NMR spectra of Unhydrolysed AF and Hydrolysed AF

8. AF DEGRADATION CONFIRMATION BY SEM
UNHYDROLYSED ANFL sample showed
prominent attachment of loose proteins
and clumpy clusters of bounded tissues
(a)
(b)
Fig. 4. Scanning Electron Micrograph of
unhydrolysed ANFL sample
Fig. 5. Scanning Electron Micrograph of
hydrolysed ANFL sample
Hydrolysed ANFL showed some clear
spaces, such spaces were occupied by
hydrolyzed peptides .
More breakage of tissue fiber was
seen.
In many places fiber-to-fiber
detachment were visualized.

9. Extraction and Characterization of crude bioactive compound
Extraction of crude bioactive compound
with different solvent systems –
Chloroform, Ethyl acetate, Methanol and
Butanol
Checking antibacterial activity of crude
bioactive compound
• Against pathogens
• Against MDROs
Characterization of crude bioactive
compounds
• TLC
• HPLC

10. Antibacterial activity of crude bioactive compound
S.No. Microbial
pathogens
Zone of
inhibition
(mm)
1. Escherichia coli 15
2. Staphylococcus
aureus
14
3. Klebsiella
pneumoniae
14
4. Proteus sp. 13
5. Salmonella sp. 12
6. Pseudomonas
aeruginosa
8
Proteus sp. P. aeruginosa
Salmonella sp.
E. coli S. aureus
K. pneumoniae
MRSA MBL
S.No. MDROs Zone of
inhibition
(mm)
1. MRSA 15
2. MBL 12
Against the microbial pathogens
Against multi drug resistant organisms (MDROs)
E. coli

11. Characterization of crude bioactive molecules
Fig.7. TLC results of crude bioactive compound
Fig.8. HPLC chromatogram of crude
bioactive compound

12. Comparative sequence analysis of 16S rRNA from Acinetobacter nosocomialis MPA1, Pseudomonas
aeruginosa MPA2, Acinetobacter bouvetii MPA3, Kitasatoprora putterlickiae MPA4 and representative strains
from GenBank using the neighbour-joining method
S4
S3
S1
S2
Results
Isolated MDRO strains
from SBTDW via
Membrane Filtration
S1: Acinetobacter nosocomialis MPA1
Accession No.: KX173286
S2: Pseudomonas aeruginosa MPA2
Accession No.: KX173287
S3: Acinetobacter bouvetii MPA3
Accession No.: KX173288
S3: Kitasatoprora putterlickiae MPA4
Accession No.: KX173289
Screening of multidrug resistant bacterial strains in secondary
biological treated domestic wastewater

13. Feasibility study:
TNTC 0 H TNTC 1 H TNTC 2 H 32CFU/ml 3 H
18 CFU/ml4H 10 CFU/ml 5H 4 CFU/ml 6 H 2CFU/ml 7 H
NIL 8H NIL 9 H NIL 10 H NIL 11 H
 Conclusion:
50 mg of crude bioactive compound showed
maximum destruction of bacterial strains in
short duration of the time.
TERTIARY TREATMENT OF SECONDARY BIOLOGICAL TREATED DOMESTIC
WASTEWATER(SBTDW) USING THE OBTAINED CRUDE BIOACTIVE COMPOUND:
• 25mg Crude Bioactive
compounds immobilized
onto mesoporous activated
carbon. This showed
maximum
destruction/disinfection of
bacterial cells present in 30
ml of SBTDW at 15th hour
Destruction of microbial cells as a function of time
Parameters optimisation:
Microbial screening of Real time waste
water treatment by 50mg of Crude
Destruction of bacterial cells as a function of time for the
varying concentrations of crude biomolecules.
• CFU/mL was calculated without dilutions.

14. (a). Untreated bacterial strains in SBTDW
Destruction of bacterial cells - SEM analysis
(b) Treated bacterial strains using crude bioactive
compounds.
Cells clumping with morphological changes as a result of
destruction of bacterial cells in treated SBTDW

15. Animal Flesh was degraded under anaerobic fermentation
Bioactive compound produced by Paracoccus pantotrophus utilizing
animal flesh showed antibacterial activity against clinical pathogens and
MDROs which was characterized by TLC and HPLC
Screening of multidrug resistant microorganisms (MDROs) in the
SBTDW
Production cost could be reduced by immobilizing the crude bioactive
compound onto mesoporous activated carbon (MAC) and reuse for the
number of cycles (to be studied).
The crude bioactive compound in the immobilized MAC showed
prominent disinfection effect against the bacterial strains presented in the
secondary biological treated wastewater.
The study proved that the crude bioactive compound from AF could be a
potential alternate for the conventional tertiary treatment process to
destruct the bacterial strains in the secondary biological treated domestic
wastewater.
CONCLUSIONS