AU2016101966A4 - A process for the bioremediation of hydrocarbons in contaminated soil or sediment - Google Patents

Abstract

A PROCESS FOR THE BIOREMEDIATION OF HYDROCARBONS IN CONTAMINATED SOIL OR SEDIMENT The present invention relates to methods of bioremediation of soil and sludge contaminated with hydrocarbons using microorganisms, nutrients and biosurfactant. More particularly, the invention related to an EcoPiling system for biodegrading hydrocarbon in soil or sediment with cultured microorganisms, necessery nutrients for microorganisms growing, and biosurfactant which increases touch area between microorganisms and hydrocarbons.

Description

A PROCESS FOR THE BIOREMEDIATION OF HYDROCARBONS IN CONTAMINATED SOIL OR SEDIMENT 2016101966 09 Nov 2016

FIELD

[0001] The present invention relates to remediation of soil and sediment contaminated with petroleum hydrocarbons (TPHs) and polycyclic aromatic hydrocarbons (PAHs). More particularly, the invention relates to a soil remediation process using micro-organisms and plant-based surfactants.

BACKGROUND OF THE INVENTION

[0002] Bioremediation may be defined as the use of micro-organisms, plants or enzymes to degrade contaminants from environment. Biopiling, also know as bioheaps, biocells or biomounds, is an ex situ bioremediation technology that has been extensively used for remediating a wide range of petrochemical contaminants in soils and sediments. Biopiling involves the heaping of contaminated soild/dried sediments into piles and stimulating the biodegrading activity of aerobic microbial populations by creating optimum or near optimum growth conditions within the pile (Jorgensen et al. 2000). This includes the introduction of oxygen through aeration, adjusting pH and moistures levels, and addition of nutrients (nitrogen and phosphorus). As a consequence of these optimum growth conditions, the enhanced microbial activity results in the rapid degradation of the bioavailable organic pollutants (Gomez 2013). The effectiveness of biopiling has been successfully demonstrate at laboratory and field scale for a mumber of different classes of hydrocarbons (Rojas et al. 2007, Xu et al. 2014).

[0003] There are many limitations regarding biopiling technologies. Ongoing operation and maintenance activities are needed for the biopiling technology which results high energy consumption and cost inefficiences. Also, the biopiling can be a 2016101966 09 Nov 2016 very slow process. Therefore, there is need to develop a functional synergetic remediaiton process which is relevantly fast, cost effective and energy efficient to remediate hydrocarbons contaminated soil or sediment.

SUMMARY

[0004] The purpose of this invention is to add cultured aboriginal micororganisms, plant-based surfactant and nutrients to biopiling system to achieve a more efficient remediation system. According to the present invention, there is provided a cost-effective and eco-friendly Ecopiling system for remediating hydrocarbons impacted soil or sediment. The EcoPiling system comprises of: 1) Tailor design site-specific microbial consortum comprising pollutant degrading bacteria. The consortium are encapsulated in (alginate, agarose, polyvinyl alcohol (PVA) , acrylamide (ACAM), polyethylene glycol); the pollutant degrading vacteria in this invention are preferably isolated from the same soil which is to be decontaminated and are non-genetically modified; the members of the consortium in this invention belong to species Pseudomonas, Rhodococcus, Ralsonia, Alcaligenes, Streptomyces, Aeromonas Rhizobia, Burkholderia, Agrobacterium, Achromobacter, Micrococcus, Bacillus, Actinomyces, Cladosporium, Staphylococcus, Acinetobacter, Xanthomonas, Sphingomonas, Arthrobacter, Flavobacterium, Corynebacterium, Brevibacterium, Nocardia; 2) Biourfactants and nutrients. Biourfactants can be any plant-based surfactants or surfactants synthesised by living cells, such as Emulsan, Sophorolipids and Rhamnolipid.

[0005] Pretreatment of the soil. 1) mix the contaminated soil with cultured bacteria, biosurfactants, nutrients and water; 2) design an EcoPile with its length direction perpendicular to the prevailing wind, trapezoidal in shape with a 2:1 slope from base to top, lm-3m in height, and 3m-100m in length. 2016101966 09 Nov 2016 [0006] Constrution of the EcoPile. 1) place perforated pipes at approximately lm intervals laying laterally across the length of the EcoPile over the impermeable membrane; 2) place a base layer of contaminated soil with a layer of perforated pipes at lm intervals and a layer of tailor-designed encapsulated microbial consortium; 3) raise the height of the EcoPile in consecutive 50-500cm layers comprising contaminated soil, perforated pipes and encapsulated microbial consortium to a height of lm-3m.

DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 presents a side sectional view of an EcoPile according to the present invention. The EcoPile. 1. Phyto-cap layer; 2. Clean soil layer; 3 Perforated pipe; 4. Alginate encapsulated bacterial consortium; 5. Heavy duty liner; 6. Leachate collection channel.

[0008] Figure 3 presents the average pH and % moisture leaves from EcoPile soils of one of the embodiment over a two-year period.

[0009] Figure 4 presents the average soluble nitrate and phosphate levels within EcoPile soils from one of the embodiment over the two-year period.

[0010] Figure 5 presents TVC and TPH degrader count within Ecopile soils from one of the embodiment over the two-year period.

[0011] Figure 6 presents the levels of Soxhlet extractable fats and oil from Ecopile soils of one of the embodiment over a two-year period.

[0012] Figure 7 presents GC profiles of Soxhlet extractions from the EcoPile soil of one of the embodiment. TPH standard, Chromatogram 1; GC profile of EcoPile soil in 2016101966 09 Nov 2016 3 months, Chromatogram 2; GC profile of EcoPile soil in 24 months Cheomatogram 3.

[0013] Figure 8 presents the GC profiles of EcoPiles soil from one of the embodiment. 0 Day, Cheomatogram 1; 56 days, Chromatogram 2.

EXAMPLE

EcoPiling system to remediate TPH contaminated soil [0014] Petroleum impacted soil were excavated from former food manufacturing site which had four pockets of mineral oil contaminated subsoil (2 m - 4 m below the surface). In total, 4,870 m of soil was excavated from these contaminated pockets and the analysis showed the TPH level of 1613 ppm. The contaminated soil was stockpiled in an unused car park (cement) on the site. Nutrients were mixed into the soil in the form of a nitrogen: phosphorus (25:4) fertiliser at a rate of 5 g/m3. -5

Biosurfactants were mixed into the soil at a rate of 5 g/m . The soil was also augmented with a consortia TPH degrading bacteria that had been isolated from the contaminated soil from the same site.

[0015] The consortia were isolated by incubating 10 g of TPH contaminated soil in 500 ml minimal media (Abraham et al. 2002) at 30 °C, 100 rpm for 2 weeks and sub-culturing in the same media supplemented with diesel oil every 2 weeks for 3 months. The consortia were encapsulated in an alginate bead carrier matrix. Typical bacterial numbers in these beads range from 108 - 109 CFU per bead. The alginate beads were applied at a rate of 37 g/m3 (3 x 106 CFU/g soil). The EcoPiles were constructed so that they were perpendicular to the prevailing wind. The base layer of soil (0.5 m) was placed over a heavy-duty polythene liner and 50 mm perforated pipe was placed at approximately 1 m centres, laid laterally across the pile to allow for passive ventilation. The EcoPlie was then raised in consecutie 0.5 m layers, 2016101966 09 Nov 2016 comprising TPH contaminated soil and perforated piping to a height of 2 m. The Ecopiles were constructed to be trapezoidal in shape with a 2:1 slope from base to top. Finally, each EcoPile was capped with uncontaminated topsoil (5 cm deep) and seeded with a 30/30/40 clover, rye grass (Lolium multiflorum) and meadow grass seed mix.

[0016] Nine EcoPiles were constructed, typically 8 m in base width, 4 m in top width, 2 m in height and of varous different lengths (20 m - 75 m) with exact dimension. Monitoring of important physico-chemcial parameters was carried out at regular intervals throughout the trial. Two years after construction, the TPH levels in the petroleum impacted EcoPile were below detectable limits. SAMPLING: [0017] Soil samples were taken from EcoPile 1,3,4,6 and 8, every 3-4 months over a two-year period. Nine individual samples were taken from each Ecopile and mixed to form 1 composited sample per EcoPile. These composite samples were analysed in triplicate for pH, moisture, nitrate, phosphate, total aerobic bactterial counts, total petroleum hydrocarbon degrader counts and TPH levels. TVC was estimated using standard plate count methods while TPH degraders were estimated using a modified most probable number method (Johnson et al. 2002) substituting 10 μΐ of diesel oil for PAHs. Soluble nitrates and posphates were analysed using ion chromatography. Total oil/fat contents were determined by Soxhlet extraction on 10 g of soil using 300 ml hexaneracetone (1:1) solvent and refluxing for 24 h. Excess solvent was removed by distillation and the fat/oil content was measured grvimetrically. Soil samples were sent to an independent testing laboratory for TPH analysis.

RESULTS

[0018] The hydrocarbon impacted soil was generally found to be granular in nature, fine to medium silty sandy (sand 84%, silt, 11% clay 5%) and contained 3.0-9.3% organic matter (mean=5.72%+2.17%). Analysis of the soil before EcoPiling by an external commercial laboratory samples showed TPH levels (C10-C40) of, on average, 2016101966 09 Nov 2016 0 1613 mg/kg. In total 1687 M of contaminated soil was excavated and stockpiled. The contaminated soil was amended with 20,888 kg of chemical fertiliser (24:5 N:P to achieve a 100:10:1 C:N:P ratio) and 190 kg of alginate beads containing the TPH degrading consortium. The soil was used to construct nine EcoPiles.

[0019] The high soil pH at the site (pH 7.9-8.7) was due to the presence of lime originating from the food manufacturing process. As the pH levels were just ouside of optimum range on pH adjustment of the soil was performed as the addition of the low pH fertiliser would potentially result in lowering the soil pH into the optimum range. The use of ammonium based fertilisers is known to result in a decrease in pH while urea based chemcial fertilisers will increase the PH of environmental media (Amebrant et al. 1990). The addition of the fertiliser did result in a slow decrease in soil pH over the two-year period. Although a leachate collection system was constructed, no leachate was generated from the EcoPiles even after heavy rain events.

[0020] The levels of soluble nitrate and phosphate were monitored over the two-year study period. Figure 4 shows that there was a rapid drop in soluble phosphate between the time of application and the second monitoring date (3 months later). Soluble phosphate was not detected in any of the soil samples taken subsequent monitoring dates. Soluble nitrate levels decreased rapidly over the course of the first twelve months dropping from 250 to mg/kg soil. Over the subsequent twelve months levels of soluble intrate continued to fall until they reached normal background levels.

[0021] Initial analysis of the soil before constructing the EcoPiles showed a low number of aerobic heterottrophic bacteria (1.3 x 104 CFU/g soil) and a lower number 2016101966 09 Nov 2016 •λ of aerobic oil degrading bacteria (4.2 x 10 CFU/g soil). This was attributed to the fact that the soil was subsurface material, composed mainly of sand. After inoculation with the TPH degrading consortia, nurient amendement and construction of the EcoPoles the TVC increased to an order of 108 CFU/g soil (Figure 5). This increase in bacteria population is likely to have been due to the mixing during EcoPile construction, the stimulating effect of the nutrients added and the inoculation with TPH degrading bacteria. As expected there were seasonal effects on the TVC bacterial population, with decrasing populations in during the Autumn/Winter months and increased populations in during Spring/Summer. Similar seasonal trends were observed with the TPH degrader counts where the populations increased in Spring/Summer and decreased in Autumn/Winter. The initial increase in TPH degrader counts may have been partially due to the slow release nature of the alginate bead delivery system that was used to inoculate the contaminated soil during construction.

[0022] Gravimetric analysis of Soxhlet extractable fats and oil was used to monitor the process of hydrocarbon degradation in the contaminated soil (Figure 6 EcoPile 1 had the highest level of extractable fats/oils at ~ 12,000 mg/kg soil. The soil used to construct this EcoPile originated from the most heavily hydrocarbon impacted location at the site. The remaining nine EcoPiles all resulted in similar quantities of extractable fat/oils ~5,000-7,000 mg/kg soil. After tweleve months the levels of Soxhlet extractable fat/oils were approximatedly 50% of those recorded at the start of the study. After a further twelve months Soxhlet extractable fat/oil were in the range of 1050-2500 mg/kg soil, representing a 62-81% decrease in extractable fats/oils. Analysis of the soils 24 months after the Ecopiling process had started by aexternal laboratory showed TPH levels (both aliphatic and aromatic C12-C40) of the EcoPiles were below detectable limits.

Month Total Petrolem Hydrocarbon ppm in EcoPile 0 1613 2016101966 09 Nov 2016 24

Non-detectable [0023] GC-FID profiles of the Soxhlet extracts showed that almost 100% of low boiling point hydrocarbons (< C20) had been removed after 24 months (Figure 7). These fractions tend to be the more water soluble and toxic components of mineral oils. Therefore, after twelve months the toxicity of the soil is likely to have been significantly reduced. The heavier fractions had been reduced by 67-80% after 24 months.

INDUSTRIAL APPLICABILITY

EcoPiling System to remediate crude oil and heavy metal containing sludge impacted soil [0024] Oil sludge impacted soil were excavated from an oil Field Company. The soil was contaminated with TPH and Cadmium. The level of TPH and cadmium was 30,000 ppm and 10 ppm respectively, analysed by an independent accredited laboratory. The TPH degrading consortia was isolated by incubating 10 g of TPH contaminated soil in 500 ml minimal media (Abraham et al. 2002) at 30 °C, 100 rpm for 2 weeks and sub-culturing in the same media supplemented with diesel oil every 2 weeks for 3 months. The consortia were encapsulated in an alginate bead carrier. Typical bacterial numbers in these beads range from 108-109 CFU per bead.

[0025] The alginate beads encapsulating the TPH consortium were applied at a rate of 1 kg/m3 on the entire surface of the constructed EcoPiles. The EcoPiles were constrcted as in Example 1. Three EcoPiles (A, B, C) and three control stockpiles (D, E, F) were constructed with dimensions shown in the following table. The layout of the treatment area was shown in Figure 10. After 60 days, the degradation rate of TPH was 33% with the EcoPiling treatment system and 5% with the controls. 2016101966 09 Nov 2016

EcoPile No. Length (m) Base width (m) Height (m) A 16 4 2 B 16 4 2 C 16 4 2 D 3.5 4 2 E 3.5 4 2 F 3.5 4 2

Claims (4)

1. An EcoPiling process for the remediation of hydrocarbons and/or sediment consisting essentially of: a. design site-specific microorganism consortium comprising pullutant degrading bacteria and biosurfactants. The consortium are encapsulated in material including, but not limited to, alginate, polyviyl alcohol (PVA), carylamide (ACAM) and polythylene glycol (PEG); The biosurfactants can be any surfacntants produced by microorganisms or plants. b. Pretreatment of soil: 1) excavate hydrocarbon or mixture of hydrocarbon from the contaminated pockets; 2) stockpile the contaminated soil on a site covered with impermeable membrane; 3) mix the contaminated soil/sediment with nutrient in the form of a nitrogen: phosphorus fertiliser, at a ratio of treated material and nutrient of 0.001-1% by weight; 4) mix the contaminated soil/sediment with the encapsulated consortium at a rate of 0.04-20 kg/m3; c. Construction of the EcoPile : 1) place perforated pipes at approximately 1 m intervals laying laterally across the length of the EcoPile over the impermeable membrane; 2) place a base layer of contaminated soil with a layer of perforated pipes at 0.2 m/m intervals; 3) place layer of tailor-designed encapsulated microbial consortium at a rate of 0.04-20 kg/m3; 4) raise the height of the EcoPile in consecutive 0.3 m - 0.5 m layers comprising contaminated soil/sediment, perforated pipes and encapsulated microbial consortium to a height of 1 m - 3 m; 5) cap the EcoPile with 0.01 m - 0.05 m layer of uncontaminated topsoil; d. Sowing of phytoremediation cap; 1) plant seeds or plants on the entire surface of the EcoPile;

2. The process as in Claim 1, wherein said bacterial consortium is preferable isolated from the same soil which is to be decontaminated and belong to species including Pseudomonas, Rhodococcus, Ralsonia, Alcaligenes, Streptomyces, Aeromonas Rhizobia, Burkholderia, Agrobacterium, Achromobacter, Micrococcus, Bacillus, Actinomyces, Cladosporium, Staphylococcus, Acinetobacter, Xanthomonas, Sphingomonas, Arthrobacter, Flavobacterium, Corynebacterium, Brevibacterium, Nocardia;

3. The process as in Claim 1, wherein said EcoPile has its length direciton perpendicular to the prevailing wind, trapezoidal in shape preferable with a 2:1 slope from base to top, 1 m - 3m in height, and 3m - 100m in length.

4. The process as in Claim 1, wherein said bio surfactants can be any surfacntants produced by microorganisms or plants.