National Technological

University
Regional Resistencia Research Group on Environmental
Faculty and Chemical Issues

Biofilm formation on agroforestry waste

supports by bacterial and native consortium
to optimize furfural’s biodegradation
Alejandro Farías, Enid Utgés, Antonella Temporale y Eliana Zalazar

INTRODUCTION
Furfural - an heterocyclic aldehyde - is obtained from quebracho colorado
(Schinopsis balansae) sawdust, produced by a tannery industry in the
province of Chaco, on the northeast of Argentina. Even being a recalcitrant
and toxic substance for various living organisms [1], it can be biodegraded by
native bacteria [2, 3], with a previously adaptation [4]. These microorganisms
transform organics and natural contaminants into harmless compounds. Their
ability to adhere to a surface or support by creating biofilms - polymeric
matrix -, gives them high resistance and the capacity of survival in diverse
environments [5, 6].
OBJECTIVE
To evaluate the capacity of Microbacterium sp., Bacillus licheniformis and
Brevundimonas sp. by themselves or as a consortium to develope biofilms
above four supports taken from agroforestry waste (rice husk, pine
sawdust, vegetable sponge and quebracho chips); and determine the
optimum support to incorporate in a fluidized bed reactor to remove the
furfural.
MATERIALS AND METHODS
Microorganisms
RESULTS
Figure 2. Washed and dried supports: (a) Rice husks, (b) Pine sawdust,
(c) Vegetable sponge, (d) Quebracho chips.
A great development of individual, double and triple bacterial colonies was
observed. However, formation of biofilms by consortia was highly more
noticeable, comparing to individual colonies.
No statistically significant differences were detected in gravimetric method,
with very high standard deviations.
SEM images shows:
Quebracho chips (Fig. 3.a) consortium biofilm completely covers their
vegetal structure.
Vegetable sponge (Fig. 3. b), the biofilm practically covers the entire
surface, although some biomass devoid areas are perceived.
Pine sawdust (Fig. 3.c) has cellular aggregates dispersed all over its surface
and an incipient biofilm development could be assumed.
Rice husks (Fig. 3.d), isolated bacterial clusters and some scattered cells
were noticed.

Figure 3. Consortium SEM images: (a) Quebracho chips x2000,

(b) Vegetable sponge x2000, (c) Pine sawdust x4500, (d) Rice husk x4500.

Figure 1. Bacterial growth in plates with semi-solid nutritional media.

(a) Brevundimonas sp., (b) Microbacterium sp., (c) Bacillus licheniformis. CONCLUSIONS
Every tested bacteria is a biofilm developer.
Support conditioning Gravimetric method is inconclusive by itself to affirm or rule out the biofilm
formation.
Distilled water at Based on results obtained by SEM, quebracho chips and vegetable sponge
80°C Cooling and
Classification could be considered the optimal supports because the consortia developed
Dried at 60°C weighing
great stability biofilms on their surfaces.
Washing Cooling into a desiccator
Tyler Series Rice husks and pine sawdust produce a lesser extention of biofilm.
1 ± 10-4 g weighed of each
sieves and drying support Higher biofilm development could be associated with the roughness.
Autoclaved in glass vials Based on this results, bacterial immobilization using quebracho chips and
vegetable sponge as supports, in a fluidized bed system, would increase the
Consortium Enrichment in culture media chances of furfural removal in concentrations higher than those tested with
planktonic bacteria.
Pre-enrichment in a
meat peptone
Isolation solution
Enrichment mineral
Incubation REFERENCES
Sludge bacteria medium M9 At 30°C on a orbital
of tannery shaker, at 200 rpm, 1. Ran, H., Zhang, J., Gao, Q., Lin, Z., and Bao, J., Analysis of biodegradation performance of
industry Activation for 24 h furfural and 5- hydroxymethylfurfural by Amorphotheca resinae ZN1, Biotechnol. Biofuels, Vol. 7,
No. 1, pp. 1-12, 2014.
2. López, M., Nichols, N., Dien, B., Moreno, J. and Bothast, R., Isolation of microorganisms for
biological detoxification of lignocellulosic hydrolysates, Appl. Microbiol. Biotechnol., Vol. 64, No 1,
Inmobilization on supports and biofilms formation pp. 125-131, 2004.
3. Zheng, D., Bao, J., Lu, J., and Gao, C., Isolation and characterization of a furfural-degrading
bacterium Bacillus cereus sp. strain DS1. Current microbiology, 2015, Vol. 70, No 2, pp. 199-205.
Individual strain (A, B or C) Incubation, 4. Farías, A. R., Utgés, E. E., Tenev, M. D., Hervot, E. I., and Utgés, E. M., Baccaro, J., Mlot, Z.,
Inmobilization Double consortia (A+B, B+C, A+C) filtration and Ensayo de crecimiento bacteriano en un efluente industrial recalcitrante, PROIMCA-PRODECA
Triple consortia (A+B+C) Congress, La Rioja, Argentina, August 11-13, 2015 pp. 1-14.
Supports in mineral washing
5. Rabin, N., Zheng, Y., Opoku-Temeng, C., Du, Y., Bonsu, E., and Sintim, H., Biofilm formation
medium M9 Bacterial Gravimetric
mechanisms and targets for developing antibiofilm agents, Future Medicinal Chemistry, 2015, Vol.
Bacterial concentration determination
standardized at 1.2
inoculation Biofilm confirmation 7, No 4, pp. 493-512.
with scanning electron 6. Koo, H., Allan, R. N., Howlin, R. P., Stoodley, P., & Hall-Stoodley, L., Targeting microbial
DO610
microscopy, SEM biofilms: current and prospective therapeutic strategies. Nature Reviews Microbiology, 2017, Vol.
15, No 12, pp. 740.