TUTCRIS - Tampereen teknillinen yliopisto


Digestate valorization for bioremediation of petroleum hydrocarbons contaminated soils



KustantajaTampere University
TilaJulkaistu - 11 joulukuuta 2019
OKM-julkaisutyyppiG5 Artikkeliväitöskirja


NimiTampere University Dissertations


Petroleum contaminated soils constitute an environmental issue which may be solved with the help of bioremediation. Soil bioaugmentation with petroleum degrading microorganisms is an efficient clean-up strategy. Currently scientific interest is focused on searching new sources of microorganisms able to degrade hydrocarbons which serve as species pools for enrichments during inoculum preparation. Bioaugmentation strategy are especially important in soils with
low level of organic matter and low microbial counts (e.g. after intensive chemical treatments). No studies were performed up to date while considering the potential of organic fertilizers and amendments as a microbial seeding source for bioremediation. In this thesis, for the first time, digestate as an example of organic amendment, was studied in terms of indigenous microbial
community which can be involved in degradation of linear hydrocarbons. Digestate is an organic by-product of biogas production via anaerobic digestion processes and has a great potential as soil fertilizer due to concentrated nutrients and low content of easily biodegradable compounds (which could be used by bacteria as a preferential carbon source over hydrocarbons). However, the potential of microbial community of digestate was never studied in terms of petroleum hydrocarbons (PHCs) degradation.

In this thesis, digestate was examined as microbial seeding for bioremediation of weathered petroleum hydrocarbon contaminated soils. The goals were : I) to check the presence of alkanes degrading bacteria in digestate enrichments and compare alkanes degradation potential with enrichments from petroleum contaminated soils, ii) verify the effect of digestate application on soil microbial community and microbial activity, iii) study the presence of functional genes
responsible for alkanes degradation (alkB genes) in digestate and amended soils.

During the first experiment, 7 microbial enrichments were developed from various digestates (including composted digestate), a petroleum contaminated soil and from mix of soil with digestate. After 3 weeks of incubation the highest diesel fuel removal was observed for enrichments originating from composted digestate and from the petroleum contaminated soil (78 and 77 % diesel fuel removal, respectively). Enrichments obtained from digestate mixed with soil displayed lower performance than single source enrichments. In all enrichments,
presence of alkB genes was promoted during the incubation. The experiment revealed the presence of alkB genes in bacteria from digestate and confirmed their ability to degrade diesel fuel.

In a second experiment, 6 different treatments were performed in microcosm using two industrial petroleum contaminated soils having different textures: a clay rich soil and a sandy soil. After 30 days of incubation, the highest petroleum hydrocarbons removal was observed in microcosms containing: digestate together with bulking agent (17.8 % and 12.7 % higher than control in clay rich soil and sandy soil, respectively) or; digestate together with immobilized bacteria (13.4 % and 9 % higher than control in clay rich soil and sandy soil, respectively). Distinct microbial groups were formed in amended and non-amended soils. Genera containing species able to degrade hydrocarbons like Acinetobacter and Mycobacterium were abundant in digestate and soil amended with digestate. The study proved that digestate contains high concentration of alkB genes, significantly higher than contaminated soils. Application of digestate significantly increased the level of alkB genes in soils which remained high during the treatment.

In a third experiment, a contaminated soil was incubated with digestate and bulking agent (used to increase porosity of soil mixture and facilitate air transfer) in bioreactors with active aeration. Initial alkB concentration was 1.5 % in contaminated soil and 4.5 % in digestate. During incubation of soil with digestate, alkB percentage increased up to 11.5 % and after additional
inoculation with immobilized bacteria this value increased up to 60 % (alkB percentage for treatment with mineral nutrients reached 0.4 %). Addition of digestate positively affected soil respiration and bacterial density, which was concomitant with enhanced hydrocarbons degradation. Incubation of soil with digestate for 2 months resulted in 74 % of hydrocarbons removal, while extra addition of immobilized bacteria increased this value to 95 %. Digestate
increased soil bacterial density and diversity of hydrocarbons degrading taxa. The experiment clearly revealed the advantage of digestate over mineral fertilizer due to soil enrichment in TPH degrading taxa and thus a more efficient bioremediation.

This thesis for the first time analysed the potential of indigenous bacteria from organic nutrient source in bioremediation. The obtained results proved that digestate is a good source of bacteria caring alkB genes curtail in alkanes metabolism. Moreover, observed population of bacterial caring alkB genes was significantly greater in digestate comparing to contaminated soils. Application of digestate allowed to increase microbial activity and maintain high content of
alkB genes in the soil which enhanced PHCs degradation.

Experiments performed during the thesis are contributing for better understanding of bioremediation process with the use of organic amendment as a nutrient source. Presented advantages of digestate over mineral fertilizers were evaluated and confirmed. This thesis for the first time proposes organic amendment, like digestate to be considered not only as a nutrient source but also as a valuable source of microorganisms for soil bioaugmentation/biostimulation. Developed experimental treatments are a good starting point for further assessment of digestate during field scale treatments, however detailed risk assessment analysis including effect of potential pathogens contained in digestate on human health and studies analysing the effect of digestate leachates on groundwater quality need to be performed.

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