WO2003059543A1

WO2003059543A1 - Process for the biological degradation of chloro-organic compounds in groundwater

Info

Publication number: WO2003059543A1
Application number: PCT/EP2003/000150
Authority: WO
Inventors: Pasquale Sacceddu; Enrico Davini; Andrea Robertiello
Original assignee: Enitecnologie S.P.A.
Priority date: 2002-01-18
Filing date: 2003-01-08
Publication date: 2003-07-24
Also published as: ITMI20020086A1; GB2398563A; GB2398563B; DE10392217T5; AU2003235713A1; ITMI20020086A0; GB0414674D0

Abstract

A process is described for the decontamination in situ of groundwater polluted by chloro-organic compounds, using whey as electron donor and a source of carbon, nitrogen, phosphorous and other substances useful for the growth of autochthon and/or allochthon micro-organisms capable of using chloro-organic compounds as final electron acceptors. Whey is also used for ensuring that the negative redox potential necessary for the development of dehalo-reducing micro-organisms, is reached in the groundwater.

Description

PROCESS FOR THE BIOLOGICAL DEGRADATION OF CHLORO-ORGANIC COMPOUNDS IN GROUNDWATER

The present invention relates to a process for the de-

10 contamination in situ of groundwater polluted by chloro- organic compounds, which uses whey as electron donor and a source of carbon, nitrogen, phosphorous and other substances useful for the growth of autochthon and/or al- lochthon micro-organisms capable . of using chloro-organic

L5 compounds as final electron acceptors.

These compounds form a group of contaminants frequently found in sediments, in waste-water, in soil, in gaseous effluents and in groundwater. A high number of chlorinated products are included in the American Environ-

20 mental Protection Agency lists as priority pollutants due to their potential danger for the environment .

In particular, synthesis aliphatic hydrocarbons, such as tetrachloroethane (PCE) , trichloroethene (TCE) and other correlated molecules, are widely applied in the field of

.5 electronic components, as degreasers of metallic parts, in the manufacturing of plastic materials, as non-flammable solvents .

Due to their widespread use and high chemical stability, they represent a major environmental problem as they can cause persistent contamination in the soil, sediments and underground water in areas of industrial sites .

Many halogenated hydrocarbons have toxic effects, often also of a mutagenous and cancerous nature.

In particular, among aliphatic chlorinated products PCE is the contaminant which is most frequently found in groundwater. Its capacity of penetrating between the layers of the earth with a low permeability and collecting in sacs as a dense liquid, immiscible with water (dense-non- aqueous-phase-liquid, DNAPLs) , characterizes it as a per- sistent source of pollution in groundwater.

The treatment of underground water contaminated by these products is normally effected with the "pump-and- treat" technique, which consists in intercepting the groundwater, by the creation of water extraction wells, and subsequent treatment of the liquid above ground, normally by means of volatilization and/or adsorption of the products on activated carbon.

Alternatively, in the last few years, a new technique based on the installation of specific trenches (barriers) of earth filled with adsorbing or reactive permeable mate- rials, is being developed. Permeable reactive barriers based on zerovalent Iron are particularly used in the case of chloro-organic compounds .

As the duration of the treatment can last for long pe- riods of time, even tens of years, both systems create problems relating to the running (necessity for electric energy for the pumping systems, periodic replacement of the adsorbing or reactive material, etc.) and also the costs (energy, disposal of the exhausted adsorbing material, etc. ) .

Studies carried out in laboratories and on site, have demonstrated that PCE can be "co-metabolized" by a variety of aerobic micro-organisms possessing oxygenase, which are capable of dehalogenating it, i.e. transforming it into compounds having a lower number of chlorine atoms, but which cannot use it as growth substrate.

It seems, in fact, that PCE cannot be metabolized directly by aerobic micro-organisms as it is a chemical species which is completely resistant to biodegradation in the presence of oxygen.

During the last ten years, experiments carried out with anaerobic microbial flora of the natural environment, have shown that PCE, under low redox potential conditions, is progressively dechlorinated to TCE, dichloroethene (DCE) isomers, vinyl-chloride (VC) and finally to ethylene. The latter can, in fact, be degraded by means of a conventional oxidative process. [Tandoi, V. Di Pinto A.C. et al . , 2000. Chlorinated ethenes removal by sequential reductive dehalogenation and aerobic oxidation processes. Convention on "Remediation of contaminated sites - New frontiers" Milan, 10 November 2000] .

The transformation of PCE in anaerobiosis is a reduction reaction which therefore requires substances which act as electron donors . The reaction is probably catalyzed by the presence of vitamins (B₁₂) , cofactors (F₄₃₀) and by the mediation of cations of transition metals (prosthetic groups of enzymatic complexes) .

Under particular reducing conditions, such as those associated with negative redox potential values, in the presence of a suitable substrate which acts as electron donor, PCE can therefore be drastically degraded to ethylene (ETH) [M.H.A. van Ee ert and G. Schraa. 2001. The potential of anaerobic bacteria to degrade chlorinated compounds . Water Sci. Technol. 41(8); 49-56]. The degradation steps are summarized in figure 1.

The reducing demolition of PCE has been prevalently demonstrated with the use of mixed microbial mediums in consortium: at present, only one case is known, of complete dehalogenation in a pure medium, effected by a single bac- terial isolate classified as Dehalococcoides ethenogenes. Various electron donors are cited in literature, such as methanol, ethanol, pentanol, glycerol, glucose, formi- ate, piruvate, lactate, propionate, molasses, toluene and H₂, for the biological dechlorination of organo-chlorinated compounds as final electron acceptors in a strict anaerobic environment, with mixed microbial mediums and in separate cases.

The use of all these substances is not only costly but also requires further additions to the system to be decon- taminated, of at least suitable nitrogen and phosphorous sources indispensable for the multiplication of the dehalo- reducing micro-organisms.

It has now been found that these drawbacks can be overcome by using whey, as a low-cost electron donor, for bio-remediation processes.

Whey, available on the market as a by-product of the dairy industry, is a component of cow-milk. More specifically what remains as by-product of whole milk after being subjected to caseation necrosis. The product obtained, con- taining about 75% of lactose with respect to the dry product, is almost completely free of fats and sodium chloride. The serum proteins (albumin, globulin, gamma globulin) are however maintained, together with some essential amino- acids (leucine, isoleucine, valine, threonine, tryptophan, lysine, phenylanaline) , inorganic components (sodium, po- tassium, magnesium, calcium, phosphorous, zinc, iron) and some vitamins (A, E, folic acid, biotin and pantothenic acid) . Whey can therefore contemporaneously form a carbon, nitrogen, phosphorous source and also a source of other growth factors for micro-organisms.

Former patents in the name of the Applicant (EP- 692458, Italy 1,270,605, Italy 1,296,940, MI2001A 001258) claim a biological system which uses a specific mixed anaerobic flora, based on lactobacilli and sulfate-reducing bacteria; this mixed flora is capable of catalyzing chemical reactions in various matrixes (sediments, earth, water) to inertize inorganic compounds (metals) or to transform and degrade organic compounds (chloro-aromatics, chlorinated pesticides, dioxins) . In accordance with this, an objective of the present invention relates to a process for the decontamination in situ of groundwater polluted by chloro-organic compounds, using whey as electron donor, a source of carbon, nitrogen, phosphorous and other substances used for the growth of au- tochthon and/or allochthon micro-organisms capable of using chloro-organic compounds as final electron acceptors.

The process, which can be used for all chloro-organic contaminants, is particularly effective in removing chloro- aliphatic products such as tetrachloroethene (PCE) , tri- chloroethene (TCE) , dichloroethene (DCE) and vinyl chloride (VC) .

In particular, vinyl chloride, a cancerous compound for human beings, is considered as being a difficult contaminant to eliminate from groundwater as it is not suffi- ciently withheld by the activated carbon. The spontaneous biological degradation mechanisms, moreover, tend to stop the dechlorination of PCE to VC and/or DCE.

The whey is normally used in a quantity equal to 5-20 grams (as dry substance) per gram of COD corresponding to the chlorinated contaminants contained in the water to be treated.

When necessary, further additions of whey are effected to ensure that the negative redox potential necessary for the development of dehalo-reducing micro-organisms, is reached in the groundwater.

The process can be applied using both micro-organisms present in the groundwater (autochthons) according to the bio-enhancing procedure, and also a suitable inoculum of micro-organisms selected for the purpose (allochthons) ac- cording to the bio-augmentation procedure.

The process, object of the present invention, can be conveniently effected by pre-arranging a series of inlet points, upstream of or inside the polluted area, normally positioned with respect to the groundwater flow, in which the whey is added in the quantities specified above. The following examples are illustrative but do limit the scope of the invention described. EXAMPLE 1

Preparation of the graft medium of dehalo-reducing micro- organisms

A sample of groundwater chronically polluted by chlorinated compounds was removed from a well at a depth of 6 πt, poured into a sterile dark glass bottle which was filled up to the neck and immediately transferred into the labora- tory.

The water was analyzed to determine :

- the content of aliphatic chlorinated products via solid phase micro-extraction (SPME) and gaschromatography (GC) ;

- the total anaerobic microbial charge (via dilutions in series and MPN (Most Probable Number) using a Wilkins-

Chalgren Anaerobe Broth - OXOID-CM 643 B medium; and the presence of nitrogenated substances (Total Kjel- dahl Nitrogen) and phosphates (via colorimetry) .

The results of the tests effected showed the presence of PCE (12 ppb), TCE (8 ppb), DCE (traces), in the ground- water used.

The total anaerobic microbial charge proved to be equal to 4.0 x 10³ MPN/ml . Finally, the water proved to be free of nitrogenated and phosphorated substances. On the basis of the data collected, it was assumed that there were natural attenuation phenomena in the groundwater which could be attributed to autochthon microorganisms capable of dehalogenating the PCE.

Enrichment mediums starting from these micro-organisms were activated, in order to produce a standardized inoculum with which to effect further tests which could be completed within limited periods of time.

In this way, 12.5 ml of groundwater were used to inoculate, in 125 ml borosilicate glass flasks, 112.5 ml of a sterile culture medium whose composition (in bi-distilled water) is indicated in Table 1.

Table 1

(*) Solution of oligo-elements (Sec. Zeikus) FeCl₂ x 4H₂0 0.4 g/1; MnCl₂ x 4H₂0 0.1 g/1; CoCl₂ x 6H₂0 0.17 g/1; ZnCl₂ 0.1 g/1; CaCl₂ 0.02 g/1; H₃B0₃ 0.019 g/1.

The flasks prepared under perfect anaerobiosis conditions, operating in a controlled atmosphere of N₂/C0₂ (70:30), were statically incubated at room temperature (20- 25°C) .

A sample was periodically tested to register the progressive degradation of the PCE. When the total content of chlorinated compounds was reduced to below 2500 μg/1, 12.5 ml of culture broth were removed, under perfect anaerobiosis conditions, and used, in substitution of the original groundwater, for a new. subculture.

The enrichment of the dehalo-reducing microbial con- sortia was repeated 4 times, care being taken that the last subculture cycle was concluded with the complete disappearance of the chlorinated contaminants .

A graft culture of anaerobic micro-organisms was thus obtained, which proved to contain 1.0 x 10⁸ MPN/ml . EXAMPLE 2

Treatment test of contaminated groundwater in microcosms using molasses as carbon and electron source

A series of five 125 ml borosilicate glass flasks with hermetic sealing was prepared by pouring into each con- tainer, under perfect anaerobiosis conditions, 1.2 ml of graft culture produced as described in Example 1 and 123.8 ml of contaminated groundwater, having the composition described in Example 1, enriched with the components indicated in Table 2. Table 2

The micro-organisms thus prepared were incubated under staticity conditions at room temperature.

After 0 (blank), 23, 40, 59 and 100 days, each microcosm was analyzed to determine the redox potential and concentration of organo-chlorinated compounds.

The results of the analysis (expressed in ppb) are indicated in Table 3.

Table 3

EXAMPLE 3 Treatment test of contaminated groundwater in microcosms using whey as source of carbon, electrons, nitrogen and i- cro-elements .

A series of five 125 ml borosilicate glass flasks with hermetic sealing was prepared by pouring into each container, under perfect anaerobiosis conditions and operating in a controlled atmosphere of N₂/C0₂ (70:30), 1.2 ml of graft culture produced as described in Example 1 and 123.8 ml of contaminated groundwater, also described in Example 1, enriched with the components indicated in Table 4. Table 4

The microcosms thus prepared, in which phosphates were added with the sole purpose of buffering the pH, were incu- bated under staticity conditions at room temperature.

After 0 (blank), 23, 40, 59 and 100 days, each microcosms was analyzed to determine the redox potential and concentration of organo-chlorinated compounds.

The results of the analyses (expressed in ppb) are in- dicated in Table 5.

Table 5

From the results indicated in Tables 3 and 5, it can be observed that all the chloro-organic contaminants initially present in the groundwater have been completely de- graded .

Claims

WHAT WE CLAIM IS

1. A process for the decontamination in situ of groundwater polluted by chloro-organic compounds, using whey as electron donor and a source of carbon, nitrogen,

5 phosphorous and other substances useful for the growth of autochthon and/or allochthon micro-organisms capable of using chloro-organic compounds as final electron acceptors .

2. The process according to claim 1, wherein the whey is L0 used in quantities ranging from 5 to 20 grams, as dry substance, per gram of COD corresponding to the chlorinated contaminants contained in the water to be treated.

3. The process according to claim 1, wherein the chloro- L5 organic compounds are chloro-aliphatic compounds.

4. The process according to claim 1, effected by prearranging a series of inlet points upstream of or inside the polluted area, normally positioned with respect to the flow of the groundwater, in which the

!0 whey is added.

5. The process according to claim 1, wherein the whey is used to ensure that the negative redox potential necessary for the development of dehalo-reducing microorganisms, is reached.