Uso de Leersia hexandra (Poaceae) en la fitorremediación de suelos contaminados con petróleo fresco e intemperizado / Use of Leersia hexandra (Poaceae) for soil phytoremediation in soils contaminated with fresh and weathered oil

ResumenLa industria petrolera ha generado derrames crónicos de petróleo y su acumulación en Gleysoles en zonas anegadas en el estado de Tabasco, en el sureste de México. El anegamiento es un factor que limita el uso de tecnologías de remediación por el alto costo y los bajos niveles de degradación del petróleo, sin embargo, Leersia hexandra Sw. es un pasto que crece en estas zonas contaminadas con petróleo intemperizado. El objetivo del estudio fue evaluar la densidad de bacterias, producción de biomasa vegetal y fitorremediación de L.hexandra en suelo contaminado con petróleos fresco e intemperizado, bajo condiciones experimentales de anegamiento. Se realizaron dos experimentos (E1 y E2) en un túnel de plástico. El E1 se basó en ocho dosis: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 y 180 000 mg kg-1 base seca (b.s.) de hidrocarburos totales de petróleo fresco (HTPF), y en el E2 se evaluaron cinco dosis: 14 173, 28 400, 50 598, 75 492 y 112 142 mg kg-1 b. s. de hidrocarburos totales de petróleo intemperizado (HTPI), con ocho repeticiones en cada experimento, además se utilizó un testigo con 2 607 mg kg-1 b. s. de HTP de origen biogénico. Las variables evaluadas a los tres y seis meses fueron 1) densidad microbiana de las bacterias fijadoras de nitrógeno de vida libre totales (BFN), del grupo Azospirillum (AZP) y Azotobacter (AZT), por cuenta viable en placa seriada; 2) producción de materia seca total (MS), se cuantificó por el peso seco por gravimetría, y 3) el porcentaje de descontaminación de los hidrocarburos (DSC) por extracción en equipo soxhlet. En suelos con HTPF, la población de BFN, AZP y AZT se estimuló hasta cinco veces más que el tratamiento testigo a los tres y seis meses; sin embargo, concentraciones de 150 000 y 180 000 mg kg-1 b. s. inhibieron entre un 70 y 89 % la densidad bacteriana. A su vez, en suelos con PI, la inhibición se registró hasta en un 90 %, a excepción del tratamiento con 14 173 mg kg-1 b. s., el cual estimuló las BNF y AZT en 2 y 0.10 veces más que testigo, respectivamente. La producción de MS fue continua en los experimentos hasta los seis meses, con valores de 63 y 89 g en PF y PI, respectivamente; sin diferencias significativas con el testigo (p ≤ 0.05). El DSC alcanzó valores del 66 % al 87 % en HTPF como HTPI a los seis meses, respectivamente. Estos resultados demuestran la habilidad del L. hexandra para desarrollar una rizósfera con alta densidad de BFN, producir biomasa vegetal y fitorremediar Gleysoles con petróleo fresco e intemperizado en ambientes tropicales inundados.

Abstract:The oil industry has generated chronic oil spills and their accumulation in wetlands of the state of Tabasco, in Southeastern Mexico. Waterlogging is a factor that limits the use of remediation technologies because of its high cost and low levels of oil degradation. However, Leersia hexandra is a grass that grows in these contaminated areas with weathered oil. The aim of the study was to evaluate the bacteria density, plant biomass production and phytoremediation of L. hexandra in contaminated soil. For this, two experiments in plastic tunnel were performed with fresh (E1) and weathered petroleum (E2) under waterlogging experimental conditions. The E1 was based on eight doses: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 and 180 000 mg.kg-1 dry basis (d. b.) of total petroleum hydrocarbons fresh (TPH-F), and the E2, that evaluated five doses: 14 173, 28 400, 50 598, 75 492 and 112 142 mg. kg-1 d. b. of total petroleum hydrocarbons weathered (TPH-W); a control treatment with 2 607 mg.kg-1 d. b. was used. Each experiment, with eight replicates per treatment, evaluated after three and six months: a) microbial density of total free-living nitrogen-fixing bacteria (NFB) of Azospirillum (AZP) and Azotobacter group (AZT), for viable count in serial plate; b) dry matter production (DMP), quantified gravimetrically as dry weight of L. hexandra; and c) the decontamination percentage of hydrocarbons (PDH) by Soxhlet extraction. In soil with TPH-F, the NFB, AZP y AZT populations were stimulated five times more than the control both at the three and six months; however, concentrations of 150 000 and 180 000 mg.kg-1 d. b. inhibited the bacterial density between 70 and 89 %. Likewise, in soil with TPH-W, the FNB, AZP and AZT inhibitions were 90 %, with the exception of the 14 173 mg.kg-1 d. b. treatment, which stimulated the NFB and AZT in 2 and 0.10 times more than the control, respectively. The DMP was continued at the six months in the experiments, with values of 63 and 89 g in fresh and weathered petroleum, respectively; had no significant differences with the control (p≤0.05). The PDH reached values of 66 to 87 % both TPH-F and TPH-W at six months, respectively. These results demonstrated the ability the L. hexandra rhizosphere to stimulate the high NFB density, vegetal biomass production and phytoremediation of contaminated soils (with fresh and weathered petroleum), in a tropical waterlogging environment. Rev. Biol. Trop. 65 (1): 21-30. Epub 2017 March 01.

[Use of Leersia hexandra (Poaceae) for soil phytoremediation in soils contaminated with fresh and weathered oil]. / Uso de Leersia hecandra (Poaceae) en la fitorremediación de suelos contaminados con petróleo fresco e intemperizado.

The oil industry has generated chronic oil spills and their accumulation in wetlands of the state of Tabasco, in Southeastern Mexico. Waterlogging is a factor that limits the use of remediation technologies because of its high cost and low levels of oil degradation. However, Leersia hexandra is a grass that grows in these contaminated areas with weathered oil. The aim of the study was to evaluate the bacteria density, plant biomass production and phytoremediation of L. hexandra in contaminated soil. For this, two experiments in plastic tunnel were performed with fresh (E1) and weathered petroleum (E2) under waterlogging experimental conditions. The E1 was based on eight doses: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 and 180 000 mg.kg-1 dry basis (d. b.) of total petroleum hydrocarbons fresh (TPH-F), and the E2, that evaluated five doses: 14 173, 28 400, 50 598, 75 492 and 112 142 mg. kg-1 d. b. of total petroleum hydrocarbons weathered (TPH-W); a control treatment with 2 607 mg.kg-1 d. b. was used. Each experiment, with eight replicates per treatment, evaluated after three and six months: a) microbial density of total free-living nitrogen-fixing bacteria (NFB) of Azospirillum (AZP) and Azotobacter group (AZT), for viable count in serial plate; b) dry matter production (DMP), quantified gravimetrically as dry weight of L. hexandra; and c) the decontamination percentage of hydrocarbons (PDH) by Soxhlet extraction. In soil with TPH-F, the NFB, AZP y AZT populations were stimulated five times more than the control both at the three and six months; however, concentrations of 150 000 and 180 000 mg.kg-1 d. b. inhibited the bacterial density between 70 and 89 %. Likewise, in soil with TPH-W, the FNB, AZP and AZT inhibitions were 90 %, with the exception of the 14 173 mg.kg-1 d. b. treatment, which stimulated the NFB and AZT in 2 and 0.10 times more than the control, respectively. The DMP was continued at the six months in the experiments, with values of 63 and 89 g in fresh and weathered petroleum, respectively; had no significant differences with the control (p≤0.05). The PDH reached values of 66 to 87 % both TPH-F and TPH-W at six months, respectively. These results demonstrated the ability the L. hexandra rhizosphere to stimulate the high NFB density, vegetal biomass production and phytoremediation of contaminated soils (with fresh and weathered petroleum), in a tropical waterlogging environment.

Associative diazotrophic bacteria in grass roots and soils from heavy metal contaminated sites

This work aimed to evaluate density of associative diazotrophic bacteria populations in soil and grass root samples from heavy metal contaminated sites, and to characterize isolates from these populations, both, phenotypically (Zinc, Cadmium and NaCl tolerance in vitro, and protein profiles) and genotypically (16S rDNA sequencing), as compared to type strains of known diazotrophic species. Densities were evaluated by using NFb, Fam and JNFb media, commonly used for enrichment cultures of diazotrophic bacteria. Bacterial densities found in soil and grass root samples from contaminated sites were similar to those reported for agricultural soils. Azospirillum spp. isolates from contaminated sites and type strains from non-contaminated sites varied substantially in their in vitro tolerance to Zn+2 and Cd+2, being Cd+2 more toxic than Zn+2. Among the most tolerant isolates (UFLA 1S, 1R, S181, S34 and S22), some (1R, S34 and S22) were more tolerant to heavy metals than rhizobia from tropical and temperate soils. The majority of the isolates tolerant to heavy metals were also tolerant to salt stress as indicated by their ability to grow in solid medium supplemented with 30 g L-1 NaCl. Five isolates exhibited high dissimilarity in protein profiles, and the 16S rDNA sequence analysis of two of them revealed new sequences for Azospirillum.

Objetivou-se avaliar a densidade de populações de bactérias diazotróficas associativas em amostras de solos e de raízes de gramíneas oriundas de sítios contaminados com metais pesados, e caracterizar isolados destas populações através da análise fenotípica (tolerância aos metais pesados zinco e cádmio e à NaCl in vitro, perfis protéicos), e genotípica (seqüenciamento de 16S rDNA), comparados às estirpes tipo das mesmas espécies. As densidades foram avaliadas nos meios NFb, Fam e LGI, comumente utilizados para culturas de enriquecimento de populações de bactérias diazotróficas associativas. As densidades encontradas em amostras de solo e raiz de sítios contaminados foram semelhantes àquelas relatadas na literatura para solos agrícolas. Isolados de Azospirillum spp. de solos contaminados e estirpes tipo oriundas de solos não contaminados variaram substancialmente com relação à tolerância a Zn+2 e Cd+2, sendo que Cd+2 mais tóxico que Zn+2. Dentre os isolados mais tolerantes (UFLA 1S, 1R, S181, S34, e S22), alguns(1R, S34 e S22) foram mais tolerantes a metais pesados que rizóbios isolados de solos de áreas tropicais e temperadas. A maioria dos isolados mais tolerantes a metais pesados também foi tolerante ao estresse salino, o que foi indicado por seu crescimento em meio sólido suplementado com 30 g L-1 de NaCl in vitro. Cinco isolados apresentaram alta dissimilaridade em perfis protéicos e o seqüenciamento do gene 16S rDNA em dois deles revelou que apresentam novas seqüências de Azospirillum.

Development and standardization of cyst based liquid formulation of Azospirillum bioinoculant.

Azospirillum bioinoculant is well known for its high nitrogen fixing and plant growth promoting characters. The carrier based bioinoculants generally suffer from shorter shelf life, poor quality, high contamination and low field performance. Therefore, it is necessary to develop alternative new formulation of inoculants where cyst based inoculants can play significant role. The cyst based liquid formulation was developed by inoculating Azospirillum into the cyst inducing minimal salts medium (MSM). One hundred per cent conversion of vegetative cells into cyst cells was noticed in 96 h. The survival of cyst cells in the MSM was observed up to one year and two months and interestingly, the population level of 10(8) was maintained till the final observation. The cyst cells of Azospirillum accumulated poly-beta-hydroxybutyrate (PHB) granules and exhibited desiccation tolerance up to 20 days and temperature tolerance up to 40 degrees C. Thus the cyst based liquid formulation has twin advantage of longer shelf life and tolerance to harmful environmental conditions. Regeneration of cyst cells into vegetative cells in different media viz., tap water, sterile water, rice gruel and nutrient broth was studied. The changes started within 3 h and complete return of vegetative cells was observed at 24 h. Although all the media tested favoured regeneration, comparatively quicker regeneration was observed in nutrient broth and followed by rice gruel. Thus, cyst based liquid formulation of Azospirillum has all the survival advantages and can be used as a potential bioinoculant.

[Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum].

The oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum was studied under laboratory conditions. After screening, A. brasilense strain SR80 was chosen for further investigation. The strain was capable of degrading 56.5% of crude oil (added in a concentration of 1%) over 14 days in a medium containing malate as an additional source of carbon and energy. Studies of associative properties showed that the strain had positive chemotaxis to wheat root exudates, colonized wheat roots, and produced indolyl-3-acetic acid. The synthesis of indolyl-3-acetic acid was not inhibited by oil. Under hydroponic conditions, crude oil stimulated growth of A. brasilense SR80, which promoted development of the wheat root system in the presence of oil and enhanced the level of oil degradation by the plant-microbial association.

Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations.

The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp. L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2. Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants. Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings. The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd. Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly. The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A. lipoferum 137. Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots. The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements.