2010:006
M A S T E R ' S T H E S I S
Immobilisation of arsenic in anaerobic environment
Carlos Calvo López
Luleå University of Technology C/D Master thesis Chemical Technology
Department of Civil and Environmental Engineering Division of Waste Science and Technology
2010:006 - ISSN: 1402-1781 - ISRN: LTU-C/DUPP--10/006--SE
2 ACKNOWLEDGEMENTS
I would like to express my gratitude to my supervisor Jurate Kumpiene, who gave me the opportunity to carry out my master thesis at LTU, for her continuous help and being always so kind to me.
I am also grateful to Markku Pelkonen who helped me a lot in of the master thesis and spent a lot of time realizing analysis.
I am also grateful to Ulla-Britt Uvemo and Desiree Nordmark for being so kind to me and helping me a lot in the laboratory carrying out analysis.
The study was financially supported by the European Union Structural Funds Objective 2, North Sweden Soil Remediation Center.
3 SUMMARY
Nowadays, the main method applied to remediate As contaminated soil is excavation and landfilling. Soil treatments normally are implemented under aerobic conditions.
This study was conducted to investigate the possibility of stabilizing As in reducing conditions by adsorption to and co-precipitation with Fe-sulphides or formation of amorphous As-S phases. The sulphide formation is considered to result from the activity of sulphate reducing bacteria (SRB) present in sludge as a part of their metabolism. The bacteria produce sulphide by reducing sulphate and oxidizing organic compounds.
A total of 36 bottles were prepared, 18 of them are considered as sample bottles and did not contain sludge or Na-lactate (organic source) together with soil. The bottles amended with sludge contained 4.5 g/l of sulphate and 3.5 g/l of Na-lactate.
The results showed that the concentration of dissolved As was dramatically higher in the bottles amended with sludge, probably due to insufficient SRB activity or the necessity of having a longer-term experiment.
4 TABLE OF CONTENTS
ACKNOWLEDGEMENTS ... 2
SUMMARY ... 3
INTRODUCTION ... 7
MATERIAL AND METHODS ... 9
1.1 Soil characterization ... 9
1.2 Soil amendments ... 9
1.3 Literature study ... 10
1.4 Statistical analysis ... 10
1.5 pH and electrical conductivity ... 10
1.6 Redox potential ... 10
1.7 Element analysis ... 10
1.8 Sulphate analysis ... 10
1.9 COD and Fe+2/Fe+3 ... 11
1.10 Sampling and measurements ... 11
1.11 Conditions of the experiment ... 11
1.12 Design of the experiment ... 12
1.13 Selection of sludges ... 12
1.14 Preparation of bottles ... 12
RESULTS ... 14
2.1 Visual inspection of bottles ... 14
2.2 Analysis of data ... 15
2.2.1 As concentration overview ... 15
2.2.2 Comparison between control bottles ... 18
5
2.2.3 Comparison between bottles amended with sludge ... 19
2.3 Other significant parameters ... 20
2.3.1 Sulphate ... 20
2.3.2 pH ... 20
2.3.3 Redox potential ... 21
2.3.4 COD ... 21
2.3.5 Calcium ... 22
2.3.6 Iron ... 23
2.4 Correlations and statistical analysis ... 23
2.4.1 Bottles containing soil and water ... 23
2.4.2 Bottles containing soil, water and sulphate ... 24
2.4.3 Bottles containing soil, water, sulphate, Na-lactate and Borregard sludge 25 2.4.4 Bottles containing soil, water, sulphate, Na-lactate and Domsjo sludge 25 2.4.5 Evidence of microbial activity in the bottles ... 25
DISCUSSION ... 27
3.1 Method discussion ... 27
3.2 Arsenic immobilization ... 27
CONCLUSIONS ... 29
FUTURE WORKS ... 30
PRE-STUDY 1 ... 31
6.1 Objective ... 31
6.2 Theoretical basis ... 31
6.3 Material and Methods ... 31
6.4 Results and discussion ... 31
6.5 Conclusions ... 32
PRE-STUDY 2 ... 33
7.1 Objective ... 33
7.2 Theoretical basis ... 33
7.3 Material and Methods ... 33
7.4 Results and discussion ... 34
6
7.4.1 Results ... 34
7.4.2 Discussion ... 36
7.5 Conclusions ... 37
7.6 Selection of the final sludges ... 37
LITERATURE REVIEW ... 38
8.1 Introduction ... 38
8.2 Toxicity of arsenic ... 38
8.2.1 Introduction ... 38
8.2.2 Legal limits and Lethal Dose of arsenic ... 39
8.2.3 Humans and animals ... 39
8.2.4 Plants ... 40
8.3 Factors affecting arsenic mobility ... 40
8.3.1 General conditions ... 40
8.3.2 Arsenic stabilization ... 41
8.3.3 Reduction of As(V) to As(III) ... 42
8.3.4 Oxidation of As(III) to As(V) ... 42
8.3.5 Role of Sulphate-Reducing Bacteria (SRB) in As mobility ... 43
8.4 Sulphate-Reducing Bacteria ... 43
8.4.1 Introduction ... 43
8.4.2 SRB competitors ... 44
8.4.3 SRB competitors ... 45
8.5 Hypothesis ... 46
REFERENCES ... 47
APPENDICES ... 50
7 INTRODUCTION
Natural and anthropogenic As enrichment in soil occurs around the world. As enrichments causes an important health issue in several countries.
Reductive dissolution of Fe-S hydroxides and minerals in anaerobic environments, releasing Fe(II) and As, is due to the activity of FeRB (Fe-Reducing Bacteria) when adding organic matter and also due to low redox potential.
Iron is known as one of the principal sorbents of As in several medias and sulfide (S-2) for its high affinity for As to form amorphous minerals (As-S phases) and Fe-As-S (arsenian pyrite) in presence of iron.
The aim of this thesis was to test if As can be immobilized in contaminated soil under reducing conditions that will occur in a landfill.
The sludge is considered to be the source of sulfate reducing bacteria (SRB) that in the presence of organic matter can reduce sulfate to sulfide and immobilise As.
8
9 MATERIAL AND METHODS
1.1 Soil characterization
The soil used in the experiment was collected from Solgårdarna in Boden, northern Sweden, and this soil contained high concentration of As. Table 1 shows element concentrations in soil.
Table 1: Total element concentrations in the soil (mg/kg dw) Element mg/kg
Al 16520.2
As 1861.5
Ca 9768.5
Cr 419.8
Cu 294.1
Fe 35893.5
K 10017.3
Mg 5039.6
Mn 1359.6
Na 206.1
P 857.0
S 373.1
Zn 479.2
1.2 Soil amendments
Several sludges, sulphate (explain where the sulphate comes from – pure chemical, waste, or other) and lactate (give the full name of the chemical and its producer) were chosen as amendments to the soil. The sludges used came from:
-Udebbo:sludge from the Uddebo wastewater treatment plant (WWTP), the sludge available is the final residue without dewatering.
-Kaldnes : it is derivated from a side-product of alcohol production.
-Borregard : activated sludge from paper mills.
-Domsjo : activated sludge from paper mills.
10 1.3 Literature study
A literature study was made in order to understand mechanisms that release and make arsenic stable under reducing conditions. This literature study was used designing the pre-study and the main experiment.
1.4 Statistical analysis
A general statistics package MINITAB® 14 was used for statistical analysis of the results. The confidence level used was 95 %. The principal tools used were linear regression and test of confidence.
1.5 pH and electrical conductivity
pH was measured with a pH electrode (KCl 3 M) connected to pH-Meter WTW pH 330. Electrical conductivity was measured with a standard conductivity cell (CDM 210 MeterLab®).
1.6 Redox potential
Redox potential was measured with a Pt-electrode with 3 M KCl reference system, connected into PHM 95 MeterLab®. All the redox potential measurements were done immediately after taking the samples.
1.7 Element analysis
Element analysis was carried out with inductively couple plasma optical emission spectroscopy (ICP-OES), model 2000V PerkinElmer, at Lueå University of Technology. All samples were filtered using 0.45 μm syringe filters and acidified with concentrated HNO3 before analyses.
1.8 Sulphate analysis
Sulphate analysis was carried out using Quattro Bran+Luebbe. All sulphate samples were frozen before analysis.
11 1.9 COD and Fe+2/Fe+3
Chemical oxygen demand (COD) and iron Fe+2/Fe+3 were determined using Spectometer NOVA 60 Merck®. The methods used were:
-COD: Methods 14895 and 14541 -Iron(Fe+2/Fe+3): 14896
These methods are explained in Analytical Procedure Appendices of Spectometer Nova 60 available in Merck-chemicals website.
1.10 Sampling and measurements
Sampling was every two weeks. During the second and sixth week the volume taken was 5 ml. The fourth week the volume taken was 10 ml and the last week the volume taken was 20 ml. Each quantity corresponds to the minimum quantity necessary to make the appropriate analysis.
All samples were taken using needles 0.6 mm x 25 mm and filtered through a 0,45μm membrane. Redox potential, pH and conductivity were measured immediately after taking the samples in this order and the remaining sample volume was frozen. COD, sulphate and ICP analysis were made using thawed samples.
Gas production was measured using a glass syringe.
1.11 Conditions of the experiment
The conditions of the experiment are chosen according to the literature review. The main identified factors that affect As mobility are the following:
-Redox conditions -pH
-Trace element content -Sulphate concentration
-Dissolved organic carbon (DOC)
The conditions and their justification are explained in the literature review.
12 1.12 Design of the experiment
The final experiment design is made according to the above listed main factors.
However, redox potential plays an important role in As mobility but it is not chosen as a initial factor due to the difficulty in its adjustment.
The experiment was performed using four types of sets, each one with 3 initial pH values and 3 replicates per each pH (n=3), and two extra sets. The first two types of sets are considered as controls. The entire experiment was as follows:
Soil + water (Initial pH 5, 7, 9). 9 bottles total.
Soil + water + sulphate (initial pH 5, 7, 9). 9 bottles total
Soil + water + sulphate + Borregard sludge (initial pH 5, 7, 9). 9 bottles total
Soil + water + sulphate + Domsjo sludge (initial pH 5, 7, 9). 9 bottles total
Extra bottle with Borregard sludge and 20 mg/l Fe+2by adding FeCl2 no pH adjustment. 1 bottle.
Extra bottle with Borregard sludge in a room at 4 ºC, no pH adjustment. 1 bottle.
The conditions of the experiments were the following:
Liquid/Solid ratio (L/S) = 2
Temperature = 30 ºC
Concentration of sludge = 5 g/l. (g of dry sludge per l)
All bottles were vacuumed and flushed with N2. 1.13 Selection of sludges
The selection of sludges was made according to Pre-studies 1 and 2. In these studies, the availability and activity of SRB in the sludges and soil were tested. Domsjo and Borregard were the selected sludges for the final experiment. The justification of the selection of the sludges is explained in Pre-study 2 (Selection of sludges and conditions).
1.14 Preparation of bottles
All the sets were prepared by putting soil into 250 ml bottles (give the volume of the bottles) and adding distilled water as shown in table 1. The homogenized sludges were added to the soil-water mixtures. Next the sludge was added into the bottles, achieving a sludge solids concentration of 5g/l(g of dry sludge per l). Table 1 shows all the quantities used in the experiment.
13 Table 1: Material used in the experiment
Per bottle
Number of bottles
Total
quantity Unit
Soil 71.35 38 2711.3 g
Na-lactate 0.49 20 9.8 g
CaSO4 0.54 40 21.66 g
Sludge Borregaard 38.22 11 420.38 ml
Sludge Domsjo 37.38 9 336.45 ml
Distilled water 3371.87 ml
FeCl3 2.4 1 2.4 mg
Glass bottles 1 38 38 -
HCl (1 M) few ml
NaOH (1 M) few ml
Then Na-lactate and sulphate were added to the bottles and mixed. As soon as it was shaken for at least 30 min, pH was adjusted by adding some ml of HCl 1 M or NaOH 1 M, depending on the final pH (5, 7, 9) for each bottle and then some ml of distilled water were added in each bottle achieving 20 ml extra (distilled water + pH adjustment) per bottle.pH was measured when mixing due to lack of equilibrium when measuring statically. The bottles were shaken only during pH adjustment.
As soon as pH was adjusted (desired pH ± 0.5), all bottles were closed tightly, vacuumed, filled up with N2 and stored in a dark room at 30 ºC excepted the bottle stored in the 4 ºC room for eight weeks.
14 RESULTS
2.1 Visual inspection of bottles
All bottles were inspected visually. The bottles that had sludge had a structure similar to the one in figure 1 (except Domsjo.pH=5. Sample 1 and Borregard.pH=5. Sample 2) and the ones that did not have sludge in figure 1.
Figure 1: The bottle is divided into three parts: water ( transparent yellow), bio-film layer (black) and soil respectively (brown).
The soil in Figure 3 is considerably darker than in figure 2 which only contains soil and water.
Figure 2: Bottle containing only water and soil. It is possible to distinguish two different parts.
15 2.2 Analysis of data
2.2.1 As concentration overview
Figure 3, 4, 5, 6 shows arsenic concentration in each type of bottle during week 4 and week 8. It is clear that the addition of sludge and lactate to the soil mobilized As in soil.
Figure 3: Arsenic in system soil + water in pH's 5, 7 and 9
Figure 4: Arsenic in system soil + water+ sulphate in pH's 5, 7 and 9
16
Figure 5: Arsenic in system soil + water+ sulphate+ Borregard sludge in pH's 5, 7 and 9
Figure 6: Arsenic in system soil + water+ sulphate+ Domsjo sludge in pH's 5, 7 and 9
Arsenic concentration clearly increased in control bottles (water and sulphate) between week 4 and Jweek 8 (see figure 7, 8, 9).
Bottles amended with sludges showed big variance in the As concentration. The biggest variance is observed when pH was 5, this is probably due to the fact that two bottles of different experimental settings of pH=5 had a different visual structure, they do not contain bio-film layer.
17
Figure 7: Arsenic (mg/l) in the different experimental settings of pH=5
Figure 8: Arsenic (mg/l) in the different experimental settings of pH=7
18
Figure 9: Arsenic (mg/l) in the different experimental settings of pH=9
2.2.2 Comparison between control bottles
In week 8 it was not possible to affirm that the concentration of As in control bottles (type 1 and type 2) are different:
Paired T-Test and CI: W8 Type1; W8 Type2
Paired T for W8 Type1 – W8 Type2
N Mean StDev SE Mean W8 Type1 9 3,77000 1,63867 0,54622 W8 Type2 9 4,89444 1,48135 0,49378 Difference 9 -1,12444 2,24997 0,74999
95% CI for mean difference: (-2,85392; 0,60503)
T-Test of mean difference = 0 (vs not = 0): T-Value = -1,50 P-Value = 0,172
During week 4, there is not enough evidence to refuse the null hypothesis, so we can not affirm that arsenic concentrations during week 4 were different.
19 Paired T-Test and CI: Water W4; Sulphate 4
Paired T for Water W4 - Sulphate 4
N Mean StDev SE Mean Water W4 9 0,365222 0,167876 0,055959 Sulphate 4 9 0,283111 0,088267 0,029422 Difference 9 0,082111 0,128449 0,042816
95% CI for mean difference: (-0,016623; 0,180846)
T-Test of mean difference = 0 (vs not = 0): T-Value = 1,92 P-Value = 0,091
2.2.3 Comparison between bottles amended with sludge
Arsenic concentrations in bottles amended with sludge were normalized when applying logarithm to the values. It is not possible to affirm that As concentrations were different in Domsjo and Borregaard bottles during week 8.
Two-sample T for Ln W8
C1 N Mean StDev SE Mean Borregard 9 3,083 0,949 0,32 Domsjo 9 2,591 0,824 0,27
Difference = mu (Borregard) - mu (Domsjo) Estimate for difference: 0,492391
95% CI for difference: (-0,400523; 1,385304)
T-Test of difference = 0 (vs not =): T-Value = 1,18 P-Value = 0,258 DF = 15
However, it is possible to affirm that during week 4 (May) the concentrations were different. Borregard As concentration was higher (3.5 mg/l average) than Domsjo (2,5 mg/l average).
Two-sample T for Ln W4
C1 N Mean StDev SE Mean Borregard 9 3,457 0,938 0,31 Domsjo 9 2,544 0,816 0,27
Difference = mu (Borregard) - mu (Domsjo) Estimate for difference: 0,912871
95% CI for difference: (0,029364; 1,796378)
T-Test of difference = 0 (vs not =): T-Value = 2,20 P-Value = 0,044 DF = 15
20 2.3 Other significant parameters
2.3.1 Sulphate
Sulfate decreases dramatically in bottles amended with sludge, while in the bottles amended with only sulphate decrease softly (figure 10). This is probably due to precipitation of metal-sulphates and probably, the consumption of SO4-2 because of the presence of SRB.
Figure 10: Sulphate evolution during the experiment
2.3.2 pH
In bottles not amended with sludge all the pH tended to equilibrate at pH=5. In the bottles amended with sludges the pH was around neutral (figure 11).
Figure 11: pH trending in bottles amended with sludges
21 2.3.3 Redox potential
Redox potential is shown in figure 12. Bottles amended with sludge, lactate and sulphate show moderate reducing conditions and being more reducing in the bottles amended with Borregard. Control bottles achieved moderate oxidizing conditions.
Figure 12: Redox potential evolution during the experiment, for the bottles only amended with sulfate there are some negative values of redox which have been
discarded. This is due to a bad measurement by the electrode.
2.3.4 COD
The chemical oxygen demand (COD) development is shown in Figure 13. It is supposed that Fe-reduction consumed organic matter during the first two weeks and then the activity stopped. And between sixth and eighth week there was an evidence of activity in the bottles, specially in those amended with sludge from Domsjo, which is in agreement with preliminary experiment 2, which showed that Domsjo sludge was the one with microbial activity and consumed more organic matter.
22
Figure 13: Table and figure captures should contain full names - COD evolution during the experiment
2.3.5 Calcium
In order to have a better comprehension of the sulphate evolution it is necessary to know when the sulphate is being reduced and when it is being precipitated. The difference in calcium concentration can be due to the precipitation of Ca-sulphates like CaSO4 (Kps=4.93x10-5) or CaSO4 dihydrate (Kps=3.14x10-5) with relatively higher solubility. At basic pH, SO4-2
is the predominant ion, therefore the precipitation of salts is higher and concentration of calcium is lower. This is in agreement with the obtained results (Figure 14).
Figure 14: Concentration of calcium in solution.
23 2.3.6 Iron
In the bottles not amended with sludge iron increase from less than 1 mg/l to a 9 mg/l and 16 mg/l. In the bottles with soil amended with Borregaard sludge Fe concentration is reduced while in those amended with Domsjo sludge Fe is increased. The bottles only amended with sulfate had the highest concentration of iron in solution. (Figure 15).
Figure 15: Average iron concentrations (mg/l) per type of experimental sets.
2.4 Correlations and statistical analysis
In order to make a more accurate analysis of data, each type of bottle was surveyed separately, no pH study was made due to high variance in some values. In this section the regressions will be done with the increment (or decrement) in parameters studied between week 4 and week 8. This study will allow us to see the relation between As and other elements (Fe, Al, Ca, Cu, Zn, S and Sulphate) during arsenic release.
2.4.1 Bottles containing soil and water
There is a clear relation between the release of arsenic between week 4 and week 8 and other metals, specially Al and Fe. (Appendices Regression 1)
The metal that explains best the behaviour of arsenic is aluminium, that explains a 99%
of the arsenic behaviour. The p-value in the Analysis of Variance table (0.000) shows that the model estimated by the regression procedure is significant at a confidence level of 95%.
Regression Analysis: delta As versus Delta Al The regression equation is
delta As = 0,384 + 0,818 Delta Al
24 Predictor Coef SE Coef T P Constant 0,3839 0,1218 3,15 0,016 Delta Al 0,81752 0,02915 28,05 0,000
S = 0,170583 R-Sq = 99,1% R-Sq(adj) = 99,0%
Analysis of Variance
Source DF SS MS F P Regression 1 22,892 22,892 786,72 0,000 Residual Error 7 0,204 0,029
The p-values for the estimated coefficient of the constant and aluminium indicated that they are significantly related to arsenic behaviour at α-level=0.05.
The positive correlation indicates a simultaneous releasing of arsenic and aluminum.
This significant release of arsenic and other trace metals can be due to the significant change in redox conditions between week 4 and week 8. This change in redox potential possibly released some precipitated hydroxides or co-precipitates in the soil, p-value shows that at a CI=95% there is enough evidence to affirm that redox potential varied between week 4 and week 8.
Paired T for Redox W4 - Redox W8
N Mean StDev SE Mean Redox W4 9 322,822 18,833 6,278 Redox W8 9 254,522 18,988 6,329 Difference 9 68,3000 25,8302 8,6101
95% CI for mean difference: (48,4451; 88,1549)
T-Test of mean difference = 0 (vs not = 0): T-Value = 7,93 P-Value = 0,000
2.4.2 Bottles containing soil, water and sulphate
It is possible to affirm that Fe is the metal with a higher correlation with As, so it is possible to affirm that the simultaneous release of Fe and As. Iron explains 86% of arsenic behavior.
Regression Analysis: delta As versus Delta Fe The regression equation is
delta As = 0,746 + 0,239 Delta Fe
Predictor Coef SE Coef T P
25 Constant 0,7464 0,5773 1,29 0,237 Delta Fe 0,23922 0,03378 7,08 0,000
S = 0,563739 R-Sq = 87,7% R-Sq(adj) = 86,0%
Analysis of Variance
Source DF SS MS F P Regression 1 15,935 15,935 50,14 0,000 Residual Error 7 2,225 0,318
Total 8 18,160
2.4.3 Bottles containing soil, water, sulphate, Na-lactate and Borregard sludge The release of arsenic between week 4 and week 8 is clearly correlated with sulfur and sulfate in dissolution. The regression explains 87% of arsenic behavior between week 4 and week 8. (Appendices regression 2)
The low contents of copper in week 4 and week 8, compared with control bottles, can lead to thinking that it has precipitated, in forms like Copper sulfide (CuS) (Kps=8×10-
37) or Copper (II) Arsenate (Cu3(AsO4)2) (Kps=7.95x10-36) which have a very low solubility product constant.
2.4.4 Bottles containing soil, water, sulphate, Na-lactate and Domsjo sludge
The release of arsenic between week 4 and week 8 is clearly correlated with other dissolved metals as well as with total sulphur and sulphate. (Appendices regression 3).
The low content of copper, leads to think that probably the copper has been precipitated.
2.4.5 Evidence of microbial activity in the bottles
It is supposed that Fe-reduction consumed organic matter during the first two weeks and then the activity stopped. Between week 6 and week 8 COD was decreased. This activity is still not correlated with sulphate decrease. COD decrease as predictor of sulphate concentration variation explains only a 7,4% of the behavior and the coefficient of regression (p-value=0.144) is not significant at a CI=95%.
Regression Analysis: Delta Sulphate versus Delta COD
The regression equation is
Delta Sulphate = - 292 + 0,133 Delta COD
Predictor Coef SE Coef T P Constant -291,93 70,86 -4,12 0,001 Delta COD 0,13252 0,08625 1,54 0,144
26
S = 181,001 R-Sq = 12,9% R-Sq(adj) = 7,4%
Analysis of Variance
Source DF SS MS F P Regression 1 77343 77343 2,36 0,144 Residual Error 16 524181 32761
Total 17 601524
27 DISCUSSION
3.1 Method discussion Limitations of the method:
-pH adjustment: All the bottles were pH-adjusted. The pH was measured when mixing the bottles, due to instability in the pH (it requires some hours or even days to achieve an equilibrium), so this pH adjustment was no as precise as expected.
-ICP analysis: The acidification of samples with HNO3 previous to ICP analysis oxidized the samples and some precipitates and suspended solids disappeared, which can suppose an increment in some elements concentrations. Even filtering the samples when taking them from the bottle some precipitates appeared in them.
-L/S ratio: Some extra ml were added to compensate the fact that some samples are taken. This may lead to a change in L/S ratio and in consequence in some elements concentrations.
-Iron Fe+2/Fe+3: The analysis of iron with the spectrometer gave very different results and a big variability. This is probably due to oxidation of samples or the appearance of precipitates in bottles. These results were discarded when considering ICP results as more reliable.
Sludge and soil acted as pH buffer, therefore the pH comparison between samples was discarded because the pH was not different enough to compare.
3.2 Arsenic immobilization
The addition of sludge, sulphate and Na-lactate significantly increase the concentration of dissolved As, while the addition of sulphate did not. The mechanism that released As into the solution can be considered to be similar under all conditions tested to the ones that release metals into solution (redissolution of Fe-hydroxides principally) , specially Fe and Al. The reducing conditions in the bottles and probably the activity of FeRB(Francis H.Chapelle, 2005) significantly affected arsenic solubility. The concentration of iron in week 4 was considerably higher in those bottles amended with sludge and Na-lactate, showing that probably the activity of FeRB was occurring. In week 8 the concentration of dissolved iron in water was still higher in the bottles amended with sludge, sulphate and Na-Lactate and those that were not.
The low concentrations of sulphate found at the end of the experiment are probably due to the activity of SRB or precipitation of sulphates like CaSO4 or other salts. The low concentrations of Cu can be due to the precipitation of CuS (which would demonstrate SRB activity) or Cu-salts.
28
The high concentrations of arsenic found and the fact that the adaptation of these bacteria was probably difficult, probably inhibit SRB growth. According to Francis H.
Chapelle in the zone of Fe(III) reduction, sulfate reducing bacteria are at a disadvantage because the concentrations of organic matter are below the required levels for activity.
Concentrations of arsenic higher than >20 mg/l is stated to be strongly inhibitory for SRB (Aili TAN, 2004), so the production of sulfide was not as expected from the preliminary studies, and in consequence the precipitation of FeS and amorphous arsenic phases, so arsenic was not immobilized.
Previous reports and literature review indicated that arsenic was immobile in sulfate reducing conditions (Saunders et al., 2008) but it is important to consider that some of these reports were in situ immobilization. In these studies sulfate reducing conditions were achieved by adding SO4-2 and organic matter into a As-contaminated site and SRB were actively metabolizing (Keimowitz, 2007).
This experiment suggests how difficult it is to stabilize arsenic in soil. Each soil should be studied and treated differently. The idea of using sludge to treat arsenic may be a good idea to remediate As-Contaminated soils, but the adaptation and the activity in the new medium of the SRB should be checked before. All previous studies indicate that if SRB-activity is occurring arsenic gets immobilized.
29 CONCLUSIONS
It is clear that the addition of sludge and organic matter to the soil released arsenic from the soil. Arsenic was released principally because of the activity of Fe-Reducing Bacteria that use organic matter to reduce Fe-phases and cause the dissolution of arsenic present in the soil (sorbed or co-precipitated).
The high concentrations of arsenic at the end of the experiment in the soil amended with sludge and organic matter is due to the fact that the SO4-2
reducing conditions were not achieved or these conditions were not enough to immobilize arsenic in Fe-As or As-S phases.
The considerable difference between the organic matter consumed by the system sludge + water (preliminary experiment 2) and soil + water + sludge suggests that SRB metabolism was clearly inhibited.
30 FUTURE WORKS
In future work it would be worth to:
Develop a method in which pH does not play an important role in the experimental design.
Study further SRB species and adaptation to a soil media to avoid inhibition. In this work the activity that the sludge showed in the experiments with soil and those without it were very different.
31 PRE-STUDY 1
6.1 Objective
The objective of this pre-study was to determine the presence of SRB in the soil.
6.2 Theoretical basis
SRB, if present, in the soil will grow and consume dissolved SO4-2 and create reducing conditions. It is expected that dissolved sulphate concentration will drop and presence of SRB in microscope analysis.
6.3 Material and Methods
Three samples were prepared by adding distilled water into a bottle containing soil in the specified quantities in table 1. Then 0,42 g and 4,6 ml of sulphate source and lactic acid 1M were added respectively. The conditions were L/S=2, 4,5 g/l SO4-2 and 3,5g/l of lactate. pH was adjusted to a pH>5 by using a few ml of NaOH 1M. Then the bottles were closed tightly, vacuumed, flushed with N2 and stored in a dark room at 30ºC for two weeks.
Table 1 Quantities added per bottle in every bottle.
Per bottle Number of
bottles
Total
quantity Unit
Soil 71.36 3 214.08 g
Water 103.76 3 311.28 ml
Na2SO4 0.42 3 1.26 g
Lactic acid 4.6 3 13.8 ml
6.4 Results and discussion
The results of SO4-2 are shown in table 2. There is no evidence enough to say that sulphate concentration had changed.
N Mean StDev Mean S26-F 3 1072,2 87,2 50 S04-M 3 1137,4 38,3 22
Difference = mu (s1) - mu (s2) Estimate for difference: -65,1400
95% CI for difference: (-301,6379; 171,3579)
T-Test of difference = 0 (vs not =): T-Value = -1,19 P-Value = 0,358 DF = 2
32
Table 2 Results of sulphate analysis at days 7 and 14.
SO4-
2(mg/l) 26-feb 04-mar
1 971.58 1094.3
2 1121.4 1167.5
3 1123.7 1150.3
The results from the visual inspection using a microscope were negative. No SRB were found in the soil.
6.5 Conclusions
The sulphate analysis showed that there was not enough evidence to affirm that there was any change in sulphate concentration in the triplicate samples. When analyzing the samples in the microscope the hypothesis of not finding any SRB in the soil was then proved.
33 PRE-STUDY 2
7.1 Objective
The objective of this pre-study was to determine the presence of SRB and other types of bacteria in different sludges, in order to select a sludge for the final experiment.
7.2 Theoretical basis See literature review part 4.
7.3 Material and Methods
Table 3 shows the materials used for the experiment Table 3: Materials used for the experiment
Per bottle Number of
bottles
Total
quantity Unit
Sludge Uddebo 70.07 4 280.27 ml
Sludge Kaldnes 40.62 4 162.50 ml
Sludge Borregard 79.62 4 318.47 ml
Sludge Domsjo 77.88 4 311.53 ml
water 2927.24 ml
KH2PO4 0.13 16 2.00 g
NH4Cl 0.25 16 4.00 g
CaCl2 . 6H20 0.02 16 0.24 g
Sodium citrate 0.08 16 1.20 g
yeast extract 0.25 16 4.00 g
MgSO4 . 7H20 0.02 16 0.24 g
FeSO4 . 7H20 0.00 16 0.02 g
Na-Lactate * 0.55 16 8.75 g
Na2SO4 * 0.70 16 11.25 g
*Average per bottle
Four bottles of each sludge (16 bottles in total) were filled with 250 ml of water and sludge achieving a final solids concentration of 5 g/l. Then all the Postgate Medium C (see table 5) components were added one by one (except Na-Lactate and Na2SO4).
Na-Lactate and Na2SO4 were added in the quantities stated below (see table 4) in each of the four bottles of each one of the four sludges, in other words, each sludge had four bottles filled with different quantities of lactate and sulfate source.
34
Table 4: Different concentrations (g/l) of Na-Lactate and sulphate per each bottle in each sludge:
Type Lactate Na2SO4
1 0.875 1.125
2 0.875 4.5
3 3.5 1.125
4 3.5 4.5
High concentration of Na-Lactate and Na2SO4 correspond to the concentrations of Postgate’s Medium C (table 3) and low is a 25% of its concentration.
Table 5: Postgate Medium C composition. Na-Lactate and Na2SO4 concentrations are variable in the experiment. Na-Lactate and Na2SO4 3.5 and 4.5 g/l respectively in Postgate’s Medium C.
Postgate Medium C composition (g/l)
KH2PO4 0,5 Low high
NH4Cl 1 Na-Lactate 0.875 3.5
CaCl2 . 6H20 0.06 Na2SO4 1.125 4.5
Sodium citrate 0.3
yeast extract 1
MgSO4 . 7H20 0.06
FeSO4 . 7H20 0.004
Then al bottles were vacuumed and then flushed with N2 and stored for two weeks in a room at 30ºC.
7.4 Results and discussion 7.4.1 Results
In order to make a more accurate analysis and have better indicators and predictors for the further experiment, Kaldnes sludge was excluded due to very irregular behaviour during all the experiment. Table 6 shows the studied variables.
35
Table 6 Total Gas is the total quantity of gas produced during the two weeks experiment. Delta conductivity is the variation of this parameter between the first and the last measurement. Delta pH is the variation of pH between the first and the last measurement. Redox is the potential measured the last day of the experiment. SO4-2 decrease is the variation of sulphate concentration between the first and the last measurement. Delta COD is the COD consumed between the two COD measured per bottle. Conductivity and redox potential values were unstable in some measurements.
Uddebo bottle with high lactate and low sulphate exploited.
Sludge Type TOTAL Gas (ml)
Delta conductivity (mS/cm)
Delta pH Redox (mV)
SO4 decrease (mg /l)
Delta COD
Domsjo 1 68 0.29 0.2 -120 245 640
Domsjo 2 62 0.84 0.16 -188 118 580
Domsjo 3 133.5 0.62 0.29 -215 275 600
Domsjo 4 199.5 1.15 0.4 -222 1482 1440
Borregard 1 7 unstable -0.15 unstable 339 260
Borregard 2 0 unstable -0.17 unstable -308 310
Borregard 3 83 unstable 0.31 unstable - -
Borregard 4 0 unstable 0.05 unstable 690 180
Kaldnes 1 11 unstable -0.25 unstable 150 160
Kaldnes 2 0 unstable -0.44 unstable -1653 -960
Kaldnes 3 0 unstable -0.62 unstable 735 -3440
Kaldnes 4 0 unstable -0.76 unstable -430 0
Uddebo 1 120.5 0.51 0.07 -177 -136 1140
Uddebo 2 108.5 1.07 0.02 -177 216 780
Uddebo * 3 0 - - - - -
Uddebo 4 254 2 0.38 -242 1120 1480
The results from the microscope inspection are shown in the table 7. Borregard sludge has abundant MPB, but this specie of bacteria can consume cytrate, not lactate.
Table 7: Results of visual inspection made the last day of experiment in the bottles.
Uddebo Kaldnes Borreg Domsjö SRB not found a few some extent some extent
MPB abundant low cytrate MPB Abundant
The gas production (ml) is clearly high in the sludges from Domsjo and Uddebo as it is possible to observe in figure 1.
36
TOTAL Gas
Sludge Type
Uddebo Domsjo
Borregard
4 3 2 1 4 3 2 1 4 3 2 1 250 200 150 100 50 0
Individual Value Plot of TOTAL Gas vs Sludge; Type
SO4 decrease
Sludge Type
Uddebo Domsjo
Borregard
4 3 2 1 4 3 2 1 4 3 2 1 1500
1000
500
0
-500
Individual Value Plot of SO4 decrease vs Sludge; Type
Figure 3 Diagrams that show the gas production (ml) and SO4-2 decrease vs sludge and the different concentrations of Na-Lactate and Na2SO4 (see table 3).
The consumption of sulphate, as shows Table 8, is clearly the high in type 4(high conc.
of lactate and sulphate see table 2).
Table 8 Mean gas production depending in the quantities of Na-Lactate and Sulphate added.
Type Mean Gas
production
1 65.333
2 56.833
3 72.167
4 151.167
7.4.2 Discussion
It is possible to affirm that gas production and decrease of COD are correlated. Gas production as a predictor of COD variation predicts 86,9 % of the cases. This shows the possibility of finding a dominant population of MPB against SRB (methane produce gas when making its metabolism). (see Regression 4 in Appendices)
Regarding redox potential (only measured in Domsjo and Uddebo), there is no clear correlation between these variables and other. (see Regression 5 in appendices)
The ratio between SO4-2 and COD consumed are shown in the table 9:
37
Table 9 Variation of SO4-2 vs variation of COD, per each sludge and each Na-Lactate and Na2SO4 concentrations:
dSO4/dCOD
Uddeb Kaldnes Borreg Domsjö 1 0.12 -0.94 -1.30 -0.38 2 -0.28 -1.72 0.99 -0.20
3 0.21 -0.46
4 -0.76 -3.83 -1.03
Borregard sludge shows the highest ratio, while the high concentrations of Na-Lactate and sulphate show the highest ratios against the other concentrations tested.
7.5 Conclusions
SRB is not predominant in any sludge, as microscope analysis showed. The correlation between gas produced and the decrement of the COD is probably due to the presence of MPB, which in his metabolism produce gas (methane).
The sludges amended with Postgate’s Medium C sulphate and lactate concentrations showed the biggest sulphate consumption and biggest ratio sulphate/lactate consumed.
Regarding the sludges in particular, it is possible to affirm that the second sludge (Kaldnes) showed a irregular behaviour, while Borregard showed the biggest ratio sulphate/lactate consumed. Domsjo and Uddebo showed a clearly dominant population of MPB as demonstrated by the gas production.
7.6 Selection of the final sludges
According to the pre-study results, the sludges selected for the further experiment were Domsjo and Borregard.
Uddebo sludge contained no detectable amounts of SRB, while the results obtained with Kaldnes sludge were highly variable.
The conditions for the final experiment chosen are high concentrations of Na-lactate (3.5 g/l) and Na-sulphate (4.5 g/l).
38 LITERATURE REVIEW
8.1 Introduction
There are several methods for the removal of arsenic in water which include oxidation, coagulation or precipitation and adsorption. It is possible to achieve concentrations as low as 5 μg/l against a influent concentration as high 500 μg/l. [Jiang, 2001].
One of the most common mechanism to remove As is the oxidation to remove Fe +2 and Mn +2 that leads to the formation of hydroxides that remove soluble arsenic by coprecipitation and adsorption mechanisms. Without oxidation of Fe +2 or Mn +2 does not occur arsenic removal due to the coprecipitation into Fe or Mn hydroxides [Edwards, 1994].
There are three mechanisms which may occur during arsenic removal:1
• Precipitation (formation of insoluble compounds).
• Co-precipitation (incorporation of soluble arsenic species into precipitates).
• Adsorption (formation of surface complexes between soluble arsenic and external surfaces of insoluble metal hydroxides).
A problem occurs when, for example, shallow aquifer in reducing conditions makes more mobile arsenic, nowadays most of arsenic removal treatments cover oxidizing conditions.
Iron hydroxides in reducing conditions, for example in underground water, do not precipitate, so the immobilization of As due to these hydroxides is low compared to the adsorption in oxidizing conditions, which means that in typical values of pH in waters and reducing conditions As can become mobilized which supposes a environmental problem (Pedersen et al., 2006).
8.2 Toxicity of arsenic
8.2.1 Introduction
Arsenic-contaminated soils, sediments, and sludges are the major sources of arsenic contamination of the food chain, surface water, ground water, and drinking water (Frankenberger). Arsenic is known as carcinogen and mutagen which supposes a
1http://www.lenntech.com/periodic/water/arsenic/arsenic-and- water.htm#ixzz0nFr73m5A
39
serious health problem to human and animals, specially when long exposure to drinking water.
.According to W.T.Frankenberger, arsenic compounds can be found in the envinroment in several different ways, Arsenopyrite (FeAsS) is the most abundant, but Arsenolite (As2S3), olivenite (Cu2OHAsO4), cobaltite (CoAsS) and proustite (Ag3AsS3) are also present in big quantities. Arsenic average abundance in soil is 1.8 mg/kg but it is possible to fins concentrations of a few g/kg.
8.2.2 Legal limits and Lethal Dose of arsenic
The legal limits and lethal dose can show how hazard is arsenic for environment.
The next table shows the quantities which are considered to kill 50 % of animals exposed to the conditions shown. LD 50 defines the lethal dose to kill an animal by oral exposition. LC 50 defines the lethal concentration in air that kills 50 % of animals after 2 hours exposure. The limit for arsenic in water applied by the World Health Organization (WHO) is shown in table 1:
Table 1 LD 50, LC 50 and World Health organization limit for As2 LD 50 20 mg / kg
LC 50 400 mg/m3/2H WHO
Limit 10 mg / m3
8.2.3 Humans and animals
The principal routes of human and animal exposure to arsenic are ingestion and inhalation. Arsenic is accumulated in the body by chronic exposure concentration exceeded certain levels. It can cause conditions such as skin changes (relaxation of cutaneous capillaries and dilation of the same), skin lesions (neoplasms of skin), peripheral vascular disease ("black foot disease"), and respiratory diseases, neurological (peripheral neuropathy ), cardiovascular disease and various cancers (lung, corner, liver, bladder and skin). It has been reported very similar effects in animals.
Some organics compounds and Arsenate (As+5) can be removed by the kidneys, but some other compounds like Arsine or Arsenite are more toxic as shown in figure 13 and these will not be removed by the kidneys. Arsine is considered as the most toxic, and in general arsenite (As+3) compounds. Arsenic in his oxidate form (As+5) is less toxic than in his reduced forn.
2World Health Organization
40 8.2.4 Plants
Arsenic is phytotoxic. Plants take up arsenic in proportion to the soil concentration, except at very high soil concentrations. Plants growing on mine or smelter wastes have developed resistance to arsenic toxicity; such plants sometimes have high concentrations of arsenic (6000 mg/kg has been found) that may be toxic to animals who eat the plants. Arsenic taken up by plants is distributed to all tissues.3
Toxicity symptoms in plants include stunted, blackened roots and blackened leaf margins. Edible portions of plants seldom accumulate high concentrations of As due to that most backyard vegetable plants are sensitive to As in soil and will either be killed or severely stunted long before the As concentrations in their tissues reach concentrations that pose a health risk.
8.3 Factors affecting arsenic mobility
8.3.1 General conditions
The conditions that show a high mobility in some shallow waters of Arsenic are the following according to some experiments made in several contaminated places (BGS y DPHE , 2001; Anawar et al., 2003), This factors should be considered while making the final lab experiment.
- Reducing conditions - pH ≈ 7 or slightly higher - High alkalinity
The compounds that seem to have a huge correlation with Arsenic concentration are:
- SO4-2 and NO3- (negative correlation) - Total Organic Carbon
In the sediments there is a huge relation between arsenic and the following elements:
- Fe
- Al
- Mn
3 IPCS INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY Health and Safety Guide No. 70
41 8.3.2 Arsenic stabilization
Factors such as the concentration of certain elements, control the speciation of arsenic and in consequence As mobility. For example, in presence of high concentrations of sulfur, it is possible to find precipitates of amorphous As-S. When establishing reducing and acidic conditions precipitate arsenic sulfide (orpiment, As2S3, and Realgar, AsS) (Sherwood, 2005).
Arsenic is known for his affinity with sulphides. In slightly sulphidic zones can form Arsenian pyrite and highly sulphidic zones As2S3. In presence of other trace metals it can occur competitive sorption if the sulphide concentration is below orpiment saturation, so arsenic may not precipitate (Sadiq, 1997).
“Ferrihydrite has long been known as a principal sorbent of As, and transformations to secondary minerals in the presence of Fe2+ influence the fate of As.” (Kocar,2006) The association of As is apparent because FeAsS formation occurs on FeS and FeS2 mineral surfaces (Cooper, 1996). Under highly reducing conditions, reduced aqueous Fe2+ reacts with H2S to form arsenian pyrite (instead of pure pyrite), which can remove As from groundwater by co-precipitation. However, the reduction of these co- precipitates can lead to the liberation of As due to the redissolution of Fe-hydroxides and consecuent increase of As in water.
Figure 1(A) Eh–pH diagrams for As drawn at 25 ºC and fixed As and SO4-2 activities of 10-2. Dashed lines show stability limits of water at 1 bar pressure.
(B) Eh–pH diagrams for As drawn at 25 ºC with fixed As and SO4-2
42
activities of 10-2 and Fe2+ activity of 10-8. Arsenian pyrite (FeS1.99 As0.01–FeS1.90 As0.1) solid solution is included in the thermodynamic database. (Saunders et al.,
2008).
“These transformation pathways, which commonly include Fe (hydr)oxide dissolution when reducing conditions, have been involved to account for enhanced mobilization of As under reducing conditions.”(Kocar, 2009) Under sulfate reducing conditions (S-2) As may react with excess sulfide to first form soluble sulfarsenites salts, which may then precipitate As-S solid phases such as amorphous As2S3 or realgar (AsS) as dissolved sulfide concentrations increase (Bostick et al.,2005).
8.3.3 Reduction of As(V) to As(III)
According to Frankenberger the speciation and solubility of As(III) in soils in the presence of dissolved is controlled by sulphides. This speciation is clearly correlated with microbial activity present in the soil, pH and redox conditions. However As(III) has been repeatedly observed in highly oxidized environments (Macur et al.2001). H2S and HS- might contribute to As(V) reduction specially at pH below neutral.
As-O-S species like H2AsO3S- or H2AsO2S- might control the reduction rate of As(V), but it is not known how these species serve as redox specie for microbial metabolism. It is reported that at pH around 5, reduction rates due to dissolved sulphide is more significant than anaerobic respiration where As(V) is used as electron acceptor.
However, it is important to observe that As concentration in soils is normally very low which means that it is very difficult to find a As-reducing bacteria dominating population.
8.3.4 Oxidation of As(III) to As(V)
Recent works suggest that oxidation of As(III) takes place but only at pH as high as 9 and it is clearly dependant in microbial activity of the soil. The oxidation pathway, as commented, is clearly pH dependant and at pH<8 this pathway is unlikely to contribute significantly to oxidation rates of As(III).
Additional work is necessary to understand the diversity of bacterial species in soils and waters that are able to oxidize As(III), and the mechanism by As oxidation occur in the environment.
It is very difficult to make predictions regarding the fate of As in real soils and waters.
The identification of the processes (biotic or abiotic) will provide information in order to create models that may be necessary for predicting As(III)/As(V) ratios
43
The interrelationship among biotic and abiotic factors is what controls As(V)/As(III) cycling, and in consequence, As mobility and solubility.
8.3.5 Role of Sulphate-Reducing Bacteria (SRB) in As mobility SRB reduce SO4-2
into S-2, and as mentioned As has a big affinity for sulphide compounds to form As-S solid phases or adsorbed into Fe-compounds like pyrite.
The addition of organic compounds into shallow waters containing As-Fe-(hydro)oxides can cause significant release of As to groundwater due to the redissolution of hydroxides. This mobilization is caused by the metabolism of FeRB like Geobacter, which use organic C as an electron donor causing the reductive dissolution of HFO phases and the release of sorbed and coprecipitated As (Saunders,2008).
“When anaerobial metabolism is optimized by providing both electron donors and acceptors, As is mobile under Fe-reducing conditions, inmobile under SO4-2 reducing conditions, and arsenian pyrite is the likely mineral phase formed under these conditions, instead of pure As-S phases” (Saunders,2008).
Several problems have been reported in Bangladesh, where some waters are contaminated with As. These waters are typically low in dissolved SO4-2 which can seriously inhibit the metabolism of SRB and then become rich in dissolved As. As is normally lower in groundwaters that exist under SO4-2
reducing conditions (Saunders, 2008).
8.4 Sulphate-Reducing Bacteria
8.4.1 Introduction
SRB are anaerobes bacteria that require organics nutrients and a highly reducing conditions to function. It is possible to find these bacteria in several type of water, even in oxidized water. SRB are SRB are implicated in problems of corrosion in irons and steels.4
“SRB reduce sulphate to sulphide, utilizing sulphate as the terminal electron acceptor under anaerobic conditions and oxidizing hydrogen or organic sources.”
(Kleikemper,2002).
Equation 1 shows the theoretical stoichiometric ratio for acetate degradation by SRB:
4www.corrosionsource.com
44
CH3OO- + SO4-2 2HCO3- + HS- Eq.1
Lactate is known to be used by a whole range of SRB (Vanbroekhoven) and between other typical carbon sources (acetate, propionate, citrate and citrate) is the one which produces an average higher SO4-2 consumption (Kleikemper et al.,2002).
It is important to observe that lactate is not a common source in the environment, so the bacteria depend on other organism to provide lactate.
Most common strains of SRB grow best at temperatures from 25° to 35°C. A few thermophilic strains capable of functioning efficiently at more than 60°C have been reported.
The presence of SRB have been always demonstrated by growing them in laboratoy, normally in a very different media in which they were found. These laboratory media will only grow certain strains of SRB, and even then some samples require a long time before the organisms are adapted to the new conditions. (Roberge).
8.4.2 SRB competitors
The principal competitors in sludge waters of SRB are MPB (Methanogenic Bacteria), which grow in anaerobic environments, consume organic source and form methane as product of their metabolism. It is demonstrated that SRB can out-compete for organic source in some media, specially in those that have a high concentration of sulphate and low concentration of organic carbon. When high concentration of organic source MPB dominate against SRB(Yoda et al.,1987).
In sediments, lakes and some others places where it is able to find iron sources, Fe- Reducing bacteria can inhibit SRB in certain environments by consuming organic matter to reduce iron (III) to iron (II). However, when Fe(III) becomes the limitant factor of FeRB, SRB increase their activity with the consequent iron sulphides precipitation (Chapelle,2005 ). Furthermore, high aqueous Fe(II) concentrations may result in chemical conditions where sulphate reduction can be favoured over Fe reduction. FeRB activity and presence has been demonstrated in the common temperatures and soils (Kostka, 2002) and the better reduction occurs in aerobic conditions in pure cultures and enrichment cultures (Kostka, 2002).
Figure 2 shows a simplified model to explain the activity and interaction of these bacteria in soils:
45
Figure 2: Model for carbon and electron flow with Fe(III) reduction, sulfate reduction and methanogenesis as potential terminal-electron accepting processes. (Chapelle,
2005).
8.4.3 SRB competitors
SRB growth can be inhibited by some types of bacteria as it has been explained before, but this and other types of bacteria can be inhibited by the presence of some trace metals like Cu(II), Cd(II), Zn(II) or Pb(II) which can inhibit seriously the SRB activity and growth if these metals reach a concentration of 50 mg/l (Aili TAN,2005), as well as very high concentrations of sulphate and carbon source can make inhibitory too. Even, concentrations as low as 5–10 mg/l have been reported as inhibitory to bacteria. The impact of the metals on microbial activity could be due to:
(1) a decrease in viable cell numbers resulting from death of the less tolerant species due to toxicity; and (2) the metals could decrease the metabolic activity of the survivors in the population (Teclu, 2007).
D.Teclu (2007) reported that the effect of heavy metals on the growth of sulphate- reducing bacteria can be stimulatory at lower concentrations and toxic/inhibitory at higher concentrations. Additional complications might be the effects of metal hydroxides and sulphide precipitation, biosorption, and complexation with the constituents of the growth media.
The absence of the potentially toxic metals Cd, Pb and Ni and presence of only very small amounts of Al, As, Cu, Fe, Mg, Mn and Zn could be beneficial since, in addition to serving as carbon source it would also supply many of the essential trace elements required by the bacteria for balanced growth. (Teclu, 2007)
There are no reports concerning the maximum concentrations of arsenic species that can be tolerated by growing cultures of SRB. Arsenic at concentrations of 1 and 5 mg/l for
46
both species, (As(III) and As(V)), did not affect the reduction of sulfate. However, if the concentration is increased to 20 mg/l the level of sulfate reduction is greatly reduced (Teclu, 2007).
The growth of SRB is clearly influenced by the pH, pH below 5 inhibit seriously the SRB metabolism (Aili TAN, 2005) and also pH above 9 (Philp et al.1991).
8.5 Hypothesis
-As will become mobile under Fe-reducing conditions, and as soon as the Sulphate reduction begins it will be formed arsenian pyrite (Saunders, 2008).
-Fe will be reduced, so As will be then released. As soon as sulfate reductions begins As will become inmobile (Saunders,2008). Figure 3 shows the approximate expected behavior of the system.
Figure 3: Arsenic, iron and sulphate approximate expected evolution during the experiment.(Saunders, 2008)
-SRB will not be inhibit by the presence of some trace elements (Aili TAN, 2004).
47 REFERENCES
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Masuzawa, T., Handa, N., Kitagawa, H., Kusakabe, M. (1992). Sulfate reduction using methane in sediments beneath a bathyal “cold seep” giant clam community off Hatsushima Island, Sagami Bay, Japan. Earth and Planetary science letters, 110 (1992), 39-50.
Kleikemper, J., Oliver, P., Schorth, M.H., Zeyer, J. (2002). Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms. Fems Microbiology Ecology 42 (2002) 109-118.
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Kocar, B.D., Borch, T., Fendorf, S.(2009). Arsenic repartitioning during biogenic sulfidization and transformation of ferrihydrite. Geochimica et cosmochimica Acta 74 (2010), 980-994.
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