A microbial method for improving salt swelling behavior of sulfate saline soil by an experimental study

Alexandria Engineering Journal (2019) xxx, xxx–xxx

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Alexandria University

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ORIGINAL ARTICLE

A microbial method for improving salt swelling behavior of sulfate saline soil by an experimental study Shuquan Peng a,1, Fan Wang a,2, Xibing Li a,3, Ling Fan a,*,4, Fengqiang Gong b,5 a b

School of Resources & Safety Engineering, Central South University, Changsha 410083, Hunan, China School of Civil Engineering, Southeast University, Nanjing 211196, Jiangsu, China

Received 8 October 2019; revised 6 November 2019; accepted 7 November 2019

KEYWORDS Sulfate saline soil; Sulfate-reducing bacteria; Salt swelling displacement; Reduction and reduceswelling effect; Microbial treatment

Abstract Services of soil environment system and safety production of pavements and railways are seriously affected and threatened by salt swelling behavior of sulfate saline soil. To improve the salt swelling, a microbial method using sulfate-reducing bacteria (SRB) was presented, based on the metabolism-reduction mechanism of SRB. First, the SRB was domesticated in an increasing sodium sulfate environment up to adapt the salinity of saturated sulfate saline soil. Then the salt swelling contrast tests of sulfate saline soil with and without SRB during the cooling process were conducted. Effects of temperature and salinity on salt swelling displacement, salt swelling reduction value, and salt swelling reduction rate of sulfate saline soil with and without SRB were investigated. The test results of X-Ray Diffraction (XRD) and Deoxyribonucleic Acid-Polymerase Chain Reaction (DNA-PCR) were used to verify and explain the reduction and reduce-swelling mechanism (RRSM) of SRB on sulfate saline soil. Test results as follows: (1) SRB strain-A resistant to a high concentration sulfate was obtained through multiple domestications, and had a significant reduction and reduce-swelling effect (RRSE) on sulfate saline soil. (2) Salt swelling displacement of sulfate saline soil with and without SRB increases with increasing salinity and decreasing temperature, but the maximum salt swelling displacement of sulfate saline soil containing SRB was less than that without SRB. (3) SRB has a significant inhibitory effect on salt swelling of sulfate saline soil. The maximum reduction rate of salt swelling was 32.96% (2% salinity) and the minimum one was

* Corresponding author at: Department of urban underground space engineering, School of resource & safety engineering, Central South University, Changsha 410083, China. E-mail addresses: [email protected] (S. Peng), [email protected] (F. Wang), [email protected] (X. Li), [email protected] (L. Fan), [email protected] (F. Gong). 1 Associate Professor, Department of Urban Underground Space Engineering, School of Resource and Safety Engineering, Central South University, Changsha, Hunan 410083, PR China. 2 Master Degree Candidate, School of Resource and Safety Engineering, Central South University, Changsha, Hunan 410083, PR China. 3 Professor, Department of Mining Engineering, School of Resource and Safety Engineering, Central South University, Changsha, Hunan 410083, PR China. 4 Associate Professor, Department of Urban Underground Space Engineering, School of Resource and Safety Engineering, Central South University, Changsha, Hunan 410083, PR China. 5 Associate Professor, School of Civil Engineering, Southeast University, Nanjing, Jiangsu 211196, PR China. Peer review under responsibility of Faculty of Engineering, Alexandria University. https://doi.org/10.1016/j.aej.2019.11.006 1110-0168 Ó 2019 Faculty of Engineering, Alexandria University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: S. Peng et al., A microbial method for improving salt swelling behavior of sulfate saline soil by an experimental study, Alexandria Eng. J. (2019), https://doi.org/10.1016/j.aej.2019.11.006

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S. Peng et al. 11.54% (5% salinity). (4) Low temperature and high salt concentration environment inhibited SRB activity and hindered the SSRE of SRB. The reduction rate of salt swelling by SRB decreased continuously with the increase of salinity and presented an exponential function change trend. (5) Results of the DNA-PCR and XRD have effectively verified the occurrence of the RRSE of SRB. This research proves that the microbial method using SRB to improve the salt swelling is feasible and is of great significance for sulfate saline soil treatment. Ó 2019 Faculty of Engineering, Alexandria University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Saline soil is defined as the soil soluble salt content exceeds 0.3% [1], and diffusely existed throughout the world with an area of more than 1 million hectares [2]. High salinity in saline soil has adversely affected the safe use of land and threatened the growth of plant [3]. Especially, sulfate saline soil has many poor engineering characteristics such as melting, salt swelling and corrosion [1–3]. In China, the salt swelling increases the damage risk of subgrade and foundation construction, posing serious challenges to One Belt One Road (OBOR) initiative’s construction and Develop-the-West strategy. The research on the characteristic of sulfate saline soil has attracted the attention of many scholars. Previous studies indicated that the salt swelling behavior in sulfate saline soil was mainly caused by the cooling [4,5] and the drying process [6], which changed the sulfate saline soil environment [7–9]. For the cooling and drying process decreases the solubility of salinity in the sulfate saline soil, induces mirabilite precipitated from anhydrous sodium sulfate with volumes increasing by about 3.4 times, and then occurs salt swelling of sulfate saline soil [10,11]. And the salt swelling is affected by some factors such as salinity and water content of saline soil. [12,13]. It is necessary to improve the salt swelling of sulfate saline soil [8,14]. There are two kinds of methods to handle the salt swelling: one is to change the soil environment to block the behavior of salt swelling in sulfate saline soil [15,16], the other is to reduce of the initial salinity formed in sulfate saline soil, such as exchanging-fill method [17]. However, the enormous expenses, soil disturbance, pollution and destruction of the soil environment are the unavoidable disadvantages of the above two improvement ways [18–20]. And a microbial method has economic and ecological advantages. Sulfate-reducing bacteria (SRB) as an anaerobic microorganism widely existing in an anoxic environment and can produce sulfide by using sulfate as the final electron acceptor degrade [21]. The SRB can degrade organic compounds [22–25], precipitate heavy metals such as zinc ions, copper ions in wastewater [26–28]. And Sarma [29] immobilizes SRB with sodium alginate to remove more than 80% sulfate ions from wastewater depending on its outstanding metabolism-reduction mechanism. Thence, the SRB also can reduce the sulfate ions in sulfate saline soil. On account of the high salinity environment of sulfate saline soil is not conducive to the growth of microorganisms, there are few researches on SRB to improve salt swelling of sulfate saline soil. In this paper, SRB was inoculated from sludge and multiple domesticated in an increasing concentration sodium sulfate solution environment up to the salinity of saturated sulfate saline soil, and then the SRB was determined by gene sequencing

technology. The comparative tests of salt swelling of sulfate saline soil with and without SRB were conducted, and the tested samples were analyzed by X-Ray Diffraction (XRD) and Deoxyribonucleic Acid-Polymerase Chain Reaction (DNA-PCR). Therefore, a microbial method using SRB is proposed to improve salt swelling behavior of sulfate saline soil and the results of this research are of great significance to the sulfate saline soil treatment. 2. Materials and methods 2.1. Breeding and domestication of SRB SRB used in the experiment was extracted from activated sludge purchased by the biological laboratory, and an improved culture medium was prepared to promote SRB’s growth [30]. It is also worth noting that the pH was adjusted to 7.2 using acid-base reagents. All details of the medium are shown in Table 1. After primary culture, the extracted SRB is centrifuged and inoculated at 5% of the inoculation amount for further domestication. And after the first generation of strains was successfully domesticated, the initial generation SRB strain was isolated and purified by a laboratory centrifuge (rotating speed 4000 r/min). A high concentration salt solution environment was realized by controlling the concentration of sodium sulfate in the culture medium. According to the salt concentration of sulfate saline soil, the concentration of sodium sulfate in the culture medium was adjusted to 5–200 g/L, and the concentration interval was set to 1 g/L, a total of 6 batches. When the SRB was successfully acclimated at 5 g/L, it was inoculated to 10 g/L for culture until the inoculum reached 200 g/L and was successfully acclimated, as Table 2 for details. After inoculation, each batch of SRB was sealed and then put into FLY200B constant temperature shaking table (30 , 150 r/min) for culture. The culture results were tested and judged by lead acetate test paper and sulfur ion-selective electrode [30]. Finally, SRB was screened by OD665 value for further testing [30,31]. 2.2. Preparation of dry sulfate saline soil sample Sulfate saline soil sample was composed of standard sand and analytically pure anhydrous sodium sulfate. Standard sand was produced in ISO, Xiamen, China, with a particle size ranging from 0.08 to 2.00 mm, a non-uniformity coefficient of 6.3 and a curvature coefficient of 1.5. According to ASTM standard [32,33], the standard sand was a well-graded soil. See Fig. 1 for the particle grading curve. Anhydrous sodium

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A microbial method for improving salt swelling behavior

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Table 1 The specific composition of improved SRB culture medium. The unit standard of medium content was g/L. Eight kinds of substances were used in the whole culture medium, and acid and alkali regulators were not included. SRB medium

Content

K2HPO4 NH4Cl Yeast extract powder CaCl2
2H2O MgSO4
7H2O 50 ~ 60% Sodium Lactate (NH4)2Fe(SO4)2
6H2O Deionized Water

0.5 g 1.0 g 1.0 g 0.1 g 2.0 g 10.5 g 0.5 g 1000 ml

sulfate was 99% analytical grade (CAS: 7757-82-6) produced by China National Pharmaceutical Corporation. Above all, the standard sand and anhydrous sodium sulfates were respectively placed in an electric heating constant drying oven at 105 and were baked for 48 h until dried, and cooled to room temperature. And they were immediately sealed and preserved in a sealed polymer bag to ensure a drying state. After cooling, arid standard sand and anhydrous sodium sulfate were weighed by an electronic scale to control their quality, then those were put into a container and stirred for 15 min at a rate of 150 r/s by using a portable mixer so that salt was evenly distributed. Ultimately, the dried sulfate soil with salinity of 2%, 3%, 4%, and 5% was prepared [34]. 2.3. Experiment of salt swelling and SRB reduction The comparative salt swelling test of sulfate saline soil during the cooling process was carried out through controlling the presence or absence of SRB to verify the reduction and reduce-swelling effect (RRSE) of SRB on sulfate saline soil. The test unit consists of two parts: a plexiglass test cylinder and a bacteria liquid loading system (Fig. 2A). The test cylin-

Fig. 1 Grain size distribution of standard test sand. The abscissa used Lg function to represent the particle size of the tested sand, and the curve showed that the particle size of the sand was between 0.08 and 2.00 mm.

der was used to bear soil sample and test salt swelling displacement and divided into a top chamber and a bottom chamber by adopting a plexiglass partition plate. And the partition plate contains evenly distributed pores to facilitate bacteria liquid and water to pass through (Fig. 2C). The bottom part of the test cylinder wall was provided with a grouting import as an import of bacterial liquid and water. Accordingly, the top part of the test cylinder wall was provided with a grouting export to prevent bacteria liquid from overflowing and protect the LVDT sensor, an exhaust collection to collect gas and a siphon hole to prevent back suction. And the top of the specimen was provided with a movable plexiglass partition plate with evenly distributed pores for displacement measurement in cooperation with LVDT displacement meter. Specific parameters were shown in Fig. 2A–C.

Table 2 Experimental projects of artificial domestication of SRB. The content of sodium sulfate was the main variable and also the main controlling factor. The experiment in this stage was divided into 6 stages with duration of 18 months. Sample No.1

No.3

No.5

1 2 3 4 5 11 12 13 14 15 16 24 25 26 27 28 29

Na+(mol)

Na2SO4(g/L)

Sample

0.0704 0.1056 0.1408 0.2113 0.2817 1.2676 1.338 1.4085 1.4789 1.5493 1.6197 2.1127 2.1831 2.2535 2.3239 2.3944 2.4648

5.0 7.5 10.0 15.0 20.0 90.0 95.0 100.0 105.0 110.0 115.0 150.0 155.0 160.0 165.0 170.0 175.0

No.2

No.4

No.6

6 7 8 9 10 18 19 20 21 22 23 30 31 32 33 34

Na+(mol)

Na2SO4(g/L)

0.5634 0.7042 0.8451 0.9859 1.1268 1.6901 1.7606 1.831 1.9014 1.9718 2.0423 2.5352 2.6056 2.6761 2.7465 2.8169

40.0 50.0 60.0 70.0 80.0 120.0 125.0 130.0 135.0 140.0 145.0 180.0 185.0 190.0 195.0 200.0

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4 The bacteria liquid loading system for controlling the liquid flow rate to prevent serious interference with soil samples and to make the liquid evenly distributed. The bacteria liquid and equal volume of water were placed in the container, the flow rate was controlled by a peristaltic pump, the flow meter tests the flow rate to complete the liquid loading of the soil sample, and stops working after the set loading amount was completed. Considering the optimum water content of standard sand, the injected water content was set at 12.1% [15]. The cooling process of the experiment (35 °C-30 °C-25 °C 20 °C-15 °C10 °C-5 °C) was simulated by the constant temperature and humidity test chamber, and the corresponding test period

S. Peng et al. (1d-1d-1d-1d-1d-1d-1d) was determined according to the temperature situation in the western region of China and the growth situation of SRB. The minimum temperature was set not only to consider the growth activity of SRB but also to prevent frost heaving from interfering with the test. Before the experiment, the tightness of the test cylinder was tested by water injection test, and the flow through the inlet and outlet of the test system was detected by flow meter. The test results prove that the tightness of the test device meets the test requirements. The salt swelling displacement was measured by Miran LVDT displacement meter (measuring range and accuracy are 0 ~ 50 mm and 0.001 mm respectively). Besides, according to the testing specification [30], the sodium

Fig. 2 Design of test equipment and physical drawing. (A) Test unit. 1-blender; 2-Bacterial fluid on water loader; 3-Flow meter; 4Peristaltic pump; 5-Grouting import; 6-LVDT displacement sensor; 7-Grouting export; 8- Cyclic loading tube; 9-Removable plexiglass partition; 10-Exhuast collection and siphon; 11-At the bottom of the device. (B) Test the whole device. (C) Test cylinder. Parts A and B formed the main testing device for the test, and C mainly reflects the specific physical parameters of the container carrying the test sample and the displacement test part.

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A microbial method for improving salt swelling behavior sulfate content of the tested specimen after the experiment was determined, and the effect of SRB on sulfate saline soil was further determined. 2.4. DNA detection and sequencing of SRB and XRD test of salt swelling The DNA-PCR [35] was used for high-throughput sequencing and bioinformatics analysis of the successfully tamed SRBs [36,37]. The 200 g specimen was provided for miseq library preparation and test experiments. Three PCR reactions were performed for each sample and test results were combined after PCR amplification. The DNA extraction was carried out by the Proteinase K Cleavage method, and 3 lL of the PCR product was electrophoresed using a 1.2% agarose gel. Purification was carried out using an AxyPrep DNA Gel Recovery Kit (TinyGene, Shanghai) and fluorescence quantification by FTC-3000TM real-time PCR instrument. According to the requirements of Illumina Miseq’s highthroughput sequencing, two-way sequencing was performed, and a library was constructed by a two-step PCR amplification method. The specific primer sequences adopted were (Amplification area: 16S V4-V5/dsrB): The hypervariable 16S V4 and V5 regions amplified by the primer pair 515F (50 -GTGCCAGCMGCCGCGG-3-30 ) and 926R (50 -CCGTCAATTCMTTTGG-30 ) through unique 12bit barcodes. This same technology was used to the hypervariable dsrB regions amplified by the primer pair dsrBF (50 -CA AYATTBGTBCCACCCA-30 ) and dsrBR (50 -GTGTGTAR CAGTTDCC-30 ). Then, operational taxonomic units (OUTs) were used to pick up a classification for PCA (Principal Component Analysis). Finally, bioinformatics analysis including principal component and species richness analysis was obtained [38]. XRD technique was used to determine the occurrence of salt swelling in sulfate saline soil [39]. After the test, 5-gram sulfate saline soil after the test was taken and an X-ray diffractometer was used for XRD analysis. The X-ray diffractometer had a starting scanning angle of 5 degrees, a stopping angle of 80 degrees and a scanning speed of 0.020 degrees/s. The image was recorded by a goniometer rotating 1 degree per minute.

5 3.2. Salinity reduction and XRD test results of sulfate saline soil XRD test was carried out by the sample of sulfate saline soil at the end of the test. Diffraction data were extracted and compared. And some components in the specimen were analyzed quantitatively. The results explained that the XRD spectrum was in good agreement with the FeS standard spectrum (JCPDS 75-000-2377) compared with the JCPDS database (Fig. 4). As one of the reduction products, Iron sulfide effectively proved that SRB can reduce sulfate into sulfur ions in sulfate saline soil. According to the International Standard Sample Card (72-000-0495 Mirabilite; 74-000-2036 Thenardite; 78-000-1253 Quartz), the comparison results and peak width analysis concluded that quartz, mirabilite, and thenardite were presented in the tested specimen. The effect of SRB on sulfate saline soil can also be obviously reflected in the change of salinity in the specimen after the test (Fig. 5). The experimental findings denoted that the salinity of sulfate saline soil was significantly reduced, and the final reduction value of salinity was similar to the reduction rate of salt swelling. Those have effectively verified the metabolism-reduction and RRSE of SRB on sulfate saline soil. Furthermore, it could be obtained that the degree of salt reduction slowed down as the increasing initial salinity [22], reflecting the inhibitory effect of high salt concentration on SRB growth. 3.3. Salt swelling behavior of sulfate saline soil It was clearly shown that the salt swelling displacement of sulfate saline soil with SRB (Fig. 6) and without SRB (Fig. 7) increased with the decrease of temperature as the cooling continues. In addition, an interesting phenomenon occurred in the temperature point of 35 °C, that is, the displacement value at this point was always zero. This is because the solubility of

3. Results 3.1. DNA sequencing results of SRB after domestication and culture After six generations of domestication, the SRB bacteria liquid turned black and the edge of lead acetate test paper turned black, which produced strong rotten egg flavor. Thus, SRB grew smoothly according to those results and could be used in the next batch of domestication experiments. The results of ultimate acclimatization experiments denoted that SRB could grow in high concentration sodium sulfate solution (30 °C, 200 g/L). PCR amplification genome DNA sequencing results demonstrated that the mixed bacteria of SRB were mainly composed of Desulfarculus_baarsii. It also included Desulfonatronospira, Desulfovirga, Desulfacinum, Desulfomicrobium, Alcaligenes, Desulfobulbus, Syntrophobacter, Desulfovibrio, etc., as shown in Fig. 3.

Fig. 3 Results of DNA-PCR gene test. The SRB colonies that have been successfully domesticated and have the most advantages are extracted for gene sequencing, and the distribution of genera and species is indicated by different colors. Among them, the yellow-dominated species of desulfurization occupy an important position.

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Fig. 4 Result of XRD diffraction. Diffraction analysis was carried out on the samples after the SRB comparative test with or without SRB. The tested samples were all from the samples with 5% salinity. The abscissa represents the diffraction angle and the ordinate represents the diffraction intensity. The existence of mirabilite (M) proved that the occurrence of salt swelling, precipitation of Iron sulfide (I) and relative reduction of mirabilite (M) reflected the effect of SRB.

anhydrous sodium sulfate varies with temperature. The temperature of precipitated crystals needed to be lower than 32.5 °C [7], while 35 °C was evidently higher than 32.5 °C, which had been fully reflected in previous studies [4,12]. The salt swelling displacement increased with the increase of the total salinity of sulfate saline soil with SRB (Fig. 6) and without SRB (Fig. 7). This was clearly because increases the salinity was equivalent to increasing the sodium sulfate content, causes more intense crystallization behavior, and then

increases in salt swelling displacement. In sulfate saline soil without SRB, the maximum displacement increased to 17.9 mm (2% salinity) and 20.8 mm (5% salinity), which was consistent with the results of previous researches about 15–30 mm [4,14]. The small increase is mainly due to the saturated state of sodium sulfate in the test sample and the heightdiameter ratio of the test sample. Salinity increases means that the amount of solute in the salt solution is increased, and the gradually saturated solution makes the increase smaller.

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A microbial method for improving salt swelling behavior

Fig. 5 Effect of reduction and reduce-swelling of SRB on salinity changes in sulfate saline soil. The abscissa was used to represent the initial salinity of the sample, while the salinity after the cooling process test was showed in the ordinate. The difference between vertical and horizontal coordinates directly indicated the extent of SRB’s influence on sulfate saline soil.

Fig. 6 The change curve of salt swelling displacement of sulfate saline soil without SRB during the cooling process. The abscissa indicated the test time, and the test temperatures of adjacent intervals differ by 5. Different colors were used to indicate the difference of salinity. The cooling process was reflected by the change of temperature.

Previous studies have also proved this point [6,8]. In addition, the height-diameter ratio of the test sample will also affect the growth of salt swelling displacement. The increase of salt swelling displacement is much more difficult for a sample with a diameter of 100 mm than for a sample with a diameter of 50 mm. Compared with the above results, the salt swelling dis-

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Fig. 7 The variation curve of salt swelling displacement of sulfate saline soil containing SRB bacteria liquid after cooling process. Related introduction is consistent with Fig. 6.

placement of sulfate saline soil containing SRB was 13.1 mm (2% salinity) and 18.9 mm (5% salinity). And the salt swelling displacement was reduced by 33% to the maximum by using SRB (Figs. 10, 12, and 13). These results were obviously reflected that addition of SRB has a significant effect on the salt swelling displacement of sulfate saline soil [22,28,40], and these results also proved that a microbial treatment method using SRB of salt swelling of sulfate saline soil is feasible. The maximum displacement growth rate in the sulfate saline soil without SRB was 6.7%, 5.8% and 3.0% (Fig. 6). It has the same trend as the sulfate saline soil with SRB, which was 19.1%, 11.5% and 8.6% (Fig. 7). The main reason for this decrease in growth rate might be that the higher the salinity, the greater the salt swelling displacement. When the salinity is close to the salt saturation state, the corresponding growth rate becomes smaller. It was noteworthy that the high concentrations of salt significantly affected the rate and morphology of salt crystals [10]. The difference in growth rate may be that the added SRB changes the sodium sulfate content, resulting in a decrease in the maximum salt swelling displacement, which indirectly increases the percentage value of growth rate. The growth rate of the maximum displacement also reflected the significant growth of salt swelling by high salinity and low temperature (Figs. 6 and 7). 3.4. Influence of salinity on salt swelling Salinity was the main factor affecting the salt swelling behavior of sulfate saline soil. The salt swelling displacement of sulfate saline soil with 3% salinity (Fig. 8b) was larger than 2% (Fig. 8a). The same situation was also expressed in the salinity of 4% and 5% (Fig. 8c, and 8d). Besides (Fig. 8), the salt swelling rate (The slope of salt swelling displacement) of sulfate saline soil with and without SRB increased with the increase of salinity, as the environment with higher salt concentration is favorable for salt swelling. An interesting phenomenon appeared in this part (Fig. 8). The salt swelling rate of sulfate saline soil containing SRB

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Fig. 8 Displacement curves of sulfate saline soil with different salinity under with and without SRB conditions. (a) 2%; (b) 3%; (c) 4%; (d) 5%. Curves of two different colors were used to indicate the presence or absence of SRB test samples. The ordinate used salt swelling displacement to directly and clearly reflect the function of SRB.

was the slowest in the range of 2–4 d. This is mainly due to changes in SRB activity, and previous studies have shown that the most active growth phase of SRB was 48–72 h [40], which was consistent with the growth observed in the test (Fig. 8). It indicated that salt swelling displacement of sulfate saline soil with and without SRB has a difference under the same salinity condition (Fig. 8a). And a similar situation also occurred in other salinity (Fig. 8b, c, and d) and this difference shows a decreasing trend, as a higher salt concentration environment was not conducive to the growth of SRB. 3.5. Influence of temperature on salt swelling Temperature is one of the main factors that affect the solubility change of sodium sulfate in sulfate saline soil. During the

same cooling period, the maximum salt swelling displacement of sulfate saline soil containing SRB was smaller than without SRB (Fig. 9), as reflected in previous results (Sect. 3.3). Moreover, salt swelling displacement of sulfate saline soil containing 2%, 3%, 4%, and 5% salinity was smaller than that of the equivalently test soil sample without SRB at 30 . The same situation occurred in different temperatures (Fig. 9b–e), but except in 5 (Fig. 9f). The salt swelling displacement was the same when the salinity was 4% and 5%. This might be explained by the fact that the test time was the seventh day when SRB activity was relatively low and 5 was also not conducive to SRB’s RRSE. Moreover, a high concentration of salinity may restrain the growth (Fig. 8), which was the most likely cause of this effect, and previous studies have obtained similar upshots [22,29].

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Fig. 9 Salt swelling displacement of sulfate saline soil with and without SRB under different temperature intervals during the cooling process. (a) 30 °C; (b) 25 °C; (c) 20 °C; (d) 15 °C; (e) 10 °C; (f) 5 . The total salt swelling displacement at different temperature stages was represented by Fig. 9a–f. The influence of salinity and temperature on salt swelling was fully shown on the slope of the curve, while the change of yellow area reflected the influence of SRB.

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4. Discussions

3) In the oxidation process, sulfite was further reduced to sulfur ions.

4.1. Mechanism analysis of SRB’s RRSE on sulfate saline soil The salt swelling phenomenon of sulfate saline soil was mainly due to supersaturated crystallization of sodium sulfate. Mirabilite was generated along with the expansion of the crystallization force and the expansion of the volume. It caused the salt swelling and then volume expansion of about 3.4 times (Fig. 10) [8,41], damaging soil and buildings such as subgrade, agricultural soil. However, SRB can achieve the purpose of decreasing sulfate by reducing sulfate ions to sulfur ions and producing sulfide precipitates (Fig. 10). The metabolic process of sulfate ion in SRB could be divided into three parts [22,41]: 1) In the catalytic decomposition process, sulfate ion and adenosine triphosphate (ATP) generated adenosinephosphosulfate (APS) and pyrophosphate under the catalysis of ATP-sulfonyl enzyme. Pyrophosphate was hydrolyzed by pyrophosphatase to produce phosphoric acid quickly, which could promote the reaction equilibrium to move towards APS. 2) In the electron transfer process, APS was reduced by APS reductase to produce adenosine monophosphate (AMP) and sulfite.

At present, the theories to explain the reduction process include: Theory one was that sulfite-thiosulfate-sulfur ion. This process included three two-electron reduction processes. Theory two was that sulfite- sulfur ion. This process was a six-electron direct reduction process [22,28,42,43]. Considering the above two and making full use of the metabolism-reduction process of SRB, the sulfate ion in sulfate saline soil was reduced to sulfur ion, thus reducing the content of sodium sulfate [24,29]. With the continuous progress of sulfate radical reduction by SRB, the content of sodium sulfate in sulfate saline soil was bound to further decrease and the salt swelling effect will be weakened. The decrease of sulfate by SRB reduced the total amount of sodium sulfate in sulfate saline soil, resulting in a decrease in the amount of essential substances for salt swelling. Thereby the fundamental reduction in the amount of sodium sulfate crystals was achieved and the occurrence of salt swelling was further prevented. In this paper, by the cooling process test for seven days, the salt swelling displacement was reduced by 33% to the maximum by using SRB (Figs. 12–14), which provided a new idea for the treatment of salt swelling of sulfate saline soil.

Fig. 10 Reduction and reduce-swelling process of SRB on sulfate saline soil. The situation of soil was represented by circles of different sizes and colors. Arrows were used to indicate the next reaction and action. Finally, the occurrence of salt swelling and the reduction of mirabilite production were highlighted by larger circles.

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4.2. Effects of salinity, temperature, and SRB on the salt swelling reduction value of sulfate saline soil The value of salt reduction swelling is one of the most directly reflected test parameters for the RRSE of SRB on sulfate saline soil. An increase in salt concentration (sodium sulfate) causes an increase in sodium ions, changes the osmotic pressure of the bacteria, and inhibits the growth activity of SRB. In addition, increasing sulfates will continue to weaken the activity of SRB. Therefore, the maximum salt swelling reduction value showed a decrease with the increase of salinity (Figs. 11 and 12). At different temperature intervals, higher salt concentrations also produced the same effect and also denoted the same downward trend (Fig. 13). The decrease in temperature is not conducive to the growth activity of SRB, and previous studies have shown that the optimum temperature for SRB growth is around 25 [29]. It could be clearly seen that the salt swelling reduction value first increased with increasing temperature and then decreased (Fig. 13). Specifically, it rose at 30–25 (Fig. 13a and b) and decreased at 25–5 (Figs. 13c, 13d, 11e and 13f). The reasons for this result may be, on the one hand, a temperature range of 30–25 °C was favorable for the growth of SRB and stimulated the activity of SRB. On the other hand, the temperature range of 30–25 was the second to the third day of the experimental project. This period of SRB having the strongest growth activity favors the RRSE of SRB on sulfate soil. Maintaining a decrease in salt expansion at 0 again indicated that low temperatures are detrimental to the activity of SRB (Fig. 13f).

Fig. 12 The reduction of the maximum salt swelling displacement of sulfate saline soil with and without SRB under different salinity. The different values of overall salt swelling displacement at the completion of the whole cooling process was used to reflect the reduction effect of SRB on salt swelling of sulfate saline soil.

4.3. Effects of salinity, temperature, and SRB on the salt swelling reduction rate The salt swelling reduction rate is a relatively clear parameter reflecting the RRSE of SRB. It was indicated that the reduction rate of salt swelling caused by SRB action decreased with the increase of salinity (Fig. 14a). The reason for this result lied not only on the activity of SRB reduced by a high salinity concentration and gradual cooling (Fig. 13) but also on the inhibition of SRB activity by terminal reduction products. Among them (Fig. 14b), the reduction rate of maximum salt swelling was 32.96% (2%). And the reduction rate of minimum salt swelling was 11.54% (5%). The salt environment with higher concentration formed due to the increase of salinity and the bacterial growth and metabolism behavior passivated due to the decrease of temperature, the combined action of the two inhibits SRB activity, which is the main reason for the difference in salt swelling and reduction rates. And the overall reduction rate of salt swelling was shown an exponential function change trend (Fig. 14). The fitting results were as follows: (RSRB represents the reduction rate of swelling, S% represents the salinity.) RSRB % ¼ 63:302e34:22S% ; R2 ¼ 0:9927

ð1Þ

4.4. Analysis and prospect of the influence of SRB on sulfate saline soil Fig. 11 Changes in the maximum salt swelling displacement of sulfate saline soil with and without SRB under different salinity during the cooling process. The two curves mainly reflect the change process of total salt swelling displacement under different salinity. The change in the size of the yellow area was seen as the effect of salt content on salt swelling and the slope of the curve reflects the growth relationship between salinity and salt swelling.

The salt swelling of sulfate soil is mainly caused by the expansion of the mirabilite to destroy the soil structure, and the result of soil salinization is extremely unfavorable for agricultural production (Fig. 4). The added SRB uses adenosinephosphosulfate reductase to reduce sulfate to sulfur ions, thereby reducing sulfate and improving salt swelling (Fig. 10). The activity and growth activities of SRB are

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Fig. 13 The reduced value changes of salt swelling displacement caused by sulfate saline soil with and without SRB under different salinity and temperature intervals during the cooling process. (a) 30 ; (b) 25 ; (c) 20 ; (d) 15 ; (e) 10 ; (f) 5 . The change of total salt swelling displacement difference at the completion of different temperature stages in the cooling process was shown in Fig. 10a–f. The effect of salinity and temperature on salt swelling was fully reflected in the height of the column, while the change of dark color column reflects the effect of SRB.

Fig. 14 Effect of SRB on salt swelling reduction rate of sulfate saline soil after the cooling process. (a) Histogram; (b) Curve fitting. Both (a) and (b) were test results of test samples containing SRB. Histogram (a) directly reflected the change trend of salt swelling reduction rate with salinity (b) obtained the specific mathematical model of the change trend through exponential fitting.

affected by many factors [29,43,44] such as temperature, salinity and pH and the reduced product. And low temperature and high salinity are not conducive to the activity of SRB (Figs. 11 and 12). Among them, the temperature is an external factor of the soil and affects the environment in which the bacteria

grow. High salinity is an internal factor of the soil that affects the osmotic pressure of the bacteria. In addition, the reduced product also has a bad influence on the growth activity of SRB [45]. Overall, the RRSE of SRB on sulfate saline soil was reported in this research, but it was not comprehensive.

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A microbial method for improving salt swelling behavior In the test results, the maximum displacement was reduced by nearly 33% (Fig. 14), and the author considers that the better effect will be obtained by adding bacterial liquid repeatedly. Moreover, the high salinity environment of sulfate saline soil has more stringent requirements on SRB in reality. The domesticated and cultured SRB in this paper needs to be further improved to meet the needs of actual engineering. In addition, the temperature was considered to change the solubility of sodium sulfate in sulfate saline soil, thus affecting salt swelling, and then microbial experiments were carried out. This has a certain gap with the actual temperature environment of sulfate saline soil, which requires further research and analysis. Moreover, the selection of bacteria and the inhibition of SRB activity by terminal reduction products have a significant impact on reducing the salt swelling of sulfate saline soil, which still needs further research to improve. 5. Conclusions In this study, by using the RRSE of SRB on sulfate saline soil, a series of reduction and reduce-swelling laboratory experiments were carried out on sodium sulfate-based sulfate saline soil with and without SRB. The change curves of salt swelling with and without SRB under temperature and salinity changes were obtained. The effects of temperature and salinity on the reducing value of salt swelling and the reduction rate of salt swelling under SRB were further studied. The mechanism of SRB’s RRSE on sulfate saline soil and its further research direction were summarized. The specific conclusions are as follows: 1) A microbial treatment method using SRB for reducing the salt swelling of sulfate saline soil proved feasible. And the research results have important guiding significance for the treatment of sulfate saline soil foundation and the mechanism of reducing swelling. 2) SRB strain-A resistant to high-concentration sulfate was obtained through multiple domestication and culture, and DNA-PCR gene detection confirms that the strain belonged to Desulfarculus_baarsii. The XRD results had effectively verified the occurrence of salt swelling and the RRSE of SRB. 3) The salt swelling displacement of sulfate saline soil with and without SRB increased with the increase of salinity and increased with the decrease of temperature. SRB had a significant SSRE on sulfate saline soil. The maximum salt swelling displacement of sulfate saline soil containing SRB was less than that of sulfate saline soil without SRB. 4) SRB has a significant inhibitory effect on salt swelling of sulfate saline soil. Low temperature and high concentration salt environment can inhibit SRB activity and hinder the RRSE of SRB. The reduction rate of salt swelling decreased continuously with the increase of salinity and presented an exponential function change trend considering SRB. The maximum reduction rate of salt swelling was 32.96% (2%) and the minimum reduction rate of salt swelling was 11.54% (5%). The overall reduction rate of salt swelling presented an exponential function change trend.

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