Bio-Revegetation Impact on the Physicochemical Characteristics of a Sandy Quarry Soil in Terga Beach Region in Algeria

In order to define the impact of a bio-revegetation effect on soil physicochemical properties, we used Acacia Saligna in variants with bio-fertilizers such as rhizobia and mycorrhizae that play a key role in the productivity and sustainability of soil as well as the environmental protection. The area of study is a degraded sandy quarry in Terga, a coastal semi-arid area located in the northwestern part of Algerian. Our sampling and analysis of soil were made after each trimester of experiments in the fields, using four blocks, each one containing ten plots. Sampling is a composite of soil that was made in each plot diagonally on a depth of 10 cm and a diameter of 30 cm from the plant, at different times: first trimester (3 months), second (6 months), and third (9 months). Preliminary results showed a real and favorable modification of substrates by obtaining materials with less alkaline pH, there is a significant phosphorus increase in the second and third trimester compared to the first trimester, however the soil calcareous nature prevents the expression of some parameters resulting in a small improvement in total nitrogen and a deficiency in both exchangeable magnesium and organic matter.


Introduction
The soil is the living epidermis of earth, at the interface between the atmosphere, rocks and the living world.It is the meeting point of the plant world, animal and mineral that provides primary production on which human population, flora and fauna depend directly.Soil participates in the great cycles of energy, water and elements (Robert, 1996).It is essential to human activities and the functioning of terrestrial ecosystems.The soil is no longer considered an inert medium.It evolves in space and time.This development gives it variability in its morphological, physical, chemical and biological properties (Collin, 2006).However; it stays a nonrenewable resource because of the long time required for its formation process.Therefore, its preservation and restoration by biological and non-aggressive means to the environment is a major challenge of sustainable development.
In order to re-vegetate a degraded sandy quarry located in Terga (Province of Ain Témouchent in northwest Algeria), Acacia saligna plants with rhizobia (nitrogen fixing symbiont) and mycorrhizal inocula were used.

Inoculum Preparation
An isolated colony was inoculated in an Erlenmeyer flask of 500 ml containing 100 ml of (YMA) broth which was already sterilized at 120° C for 20 min (Vincent, 1970).After 5-7 days of incubation, 100 ml of each inoculum containing about 30X10 8 bacteria/ml was transferred into a 2 L erlenmeyer flask filled up to 1 L to allow aeration of medium during agitation (Mansouri, 2011).

Test Nursery Inoculation
Acacia Saligna seeds were already scarified with H 2 SO 4 for 90 min, after they were rinsed with sterile distilled water and then germinated in water agar 0.8% (Tillard & Drevon, 1988).After 6 days, seeds were transplanted into plastic bags and then transferred to nursery.The soil used was composed of 25% of the career's sand and 75% of peat (Mansouri, 2011).Each 5 ml of a pure culture of rhizobia containing approximately 30X10 8 bacteria/ml was used in the soil at the collar when transplanting.One week after the seedlings, plantlets received a second inoculation (Diouf et al., 2003;Mansouri, 2011).

Mycorrhizal Inoculums Preparation
Acacia Salina fresh roots were collected from 7 trees of Acacia Salina in Terga town and 7 other trees in the city of Oran.At each site (Terga and Oran) samples of fine roots were removed from Acacia Saligna root system, and then they were well washed, then submerged in a 20% KOH solution for 20 min at 90°C.They were then thoroughly rinsed with water and soaked in a 1% HCl solution for 5 minutes.Roots were then stained in a solution of 0.1% trypan blue in lactophenol for 20 minutes (Philipps & Hayman, 1970).About 50 random fragments of roots thus treated were cut to pieces of about 1cm length and compacted between slides and layers.Fragments were then observed under a light microscope 10x40 to estimate the endomycorhizal frequency (Mansouri, 2011).

Nursery Inoculation Tests
Roots mycorrhization whose frequency rate was 100% were used, an application of about 1g of fresh endomycorhizal roots against each of the root system at the time of transplanting plantlets (Mansouri, 2011).After 8 months of warehousing in the nursery, the plantlets were transferred to the field (Mansouri, 2011).

Sampling
Sampling is a composite of soil (from reworked materials) that was made in each plot diagonally on a depth of 10 cm and a diameter of 30 cm from the plant at different times T1 (3 months), T2 (6 months), and T3 (9 months) In the same way 10 ml of solution of ammonium oxalate was titrated, N was the number of milliliters of KMnO 4 poured for control (Aubert, 1978).
2.6.4Measurement of Total Nitrogen Content by the Kjeldahl Method 1 g of the soil was weighed and was put into a digestion flask containing 12-15 ml of concentrated sulfuric acid (H 2 SO 4 ), then 7g of potassium sulfate and a catalyst as Copper were added, the digestion was brought to a "rolling boil" (370°C to 400 o C) and the mixture was heated until white fumes could be seen; then 250 ml of water was added.The pH mixture was increased, with 45% NaOH solution, so the ammonium (NH 4 + ) ions were converted to ammonia (NH 3 ), which was a gas that was distilled and then trapped in a special solution of about 15 ml HCl in 70 ml of water.Afterward, an indicator color was added to the trapping solution showing an important amount of trapped acid was still present.Afterward, a standard solution of NaOH was put into the buret and the trapping acid solution was titrated with the sodium hydroxide solution (Blamir, 2003).
2.6.5 Measurement of Absorbed Phosphorus by the Olsen Method 1 g of soil was put into a 50 ml erlenmeyer flask, then 20 ml of extracting solution was added to each flask which was shaken at 200 epm or more for 30 minutes at a room temperature, afterward extracts were filtered through Whatmann filter paper # 42, then phosphorus was analyzed by colorimetry or inductively coupled plasma emission spectroscopy using a blank and standards prepared in the Olsen P extracting solution (Hodges, 2000).
2.6.6 Identification of Exchangeable Cations (Ca 2+ , Mg 2+ Atomic Absorption Spectrophotometry 10 g of soil was poured into a tube percolation mixing soil with a defined amount of quartz sand, after the bulb tube surmounting the percolation was filled of 500 ml ammonium acetate (77.08 g/l).The percolation took place during about 8 hours.Then percolate was collected into a 500 ml flask.Finally, the percolating was up to 500 ml with a solution of ammonium acetate (Aubert, 1978).
The Measurement of calcium rates was done by establishing a calibration range of 0, 4, 8, 12, 16 and 20 ppm from Ca +2 (40 ppm) solutions.The extract solution was diluted to obtain a concentration of less than 20 ppm (Aubert, 1978).The measurement of magnesium rates was done by establishing a calibration range of 1, 2, 3, 4 and 5 ppm from magnesium solutions (20 ppm) each complete 100 ml flask, with distilled water (Aubert, 1978).

Statistics Analysis
The average concentrations of various parameters analyzed are affected by an analysis of variance using the Fisher's exact test at P=5% using the software SPSS 8.0 for windows.

Results and Discussion
We note that the non re-vegetated plots recorded an increased pH values over time: the reported T1 (summer) pH is lower than that of autumn (T2) which is lower than that of winter (T3) (Figure 4.a).There is a significant increase of pH (p<0.05) in T2 compared to T1 on control plots (8.37 compared to 8.04), S14 (8.46 compared to 7.99), D10 (8.52 compared to 8.06), D14 (8.43 compared to 8.04), D24 (8.65 compared to 7.97).There is also a significant increase of pH (p<0.05) in T2 compared to T3 on the plots: D10 (8.52 compared to 8.05), D14 (8.43 compared 8.09) and D24 (8.65 compared 8.13).Moreover, there is a significant pH decrease at p<0.05 in T3 on the plots of D10 and D14 relative to the non Acacia plots (8.05, 8.09 compared to 8.29).In fact, these results are closely related with the seasonal variations in pH, since pH rates decrease in hot dry weather and increase in rainy and cold weather (Gasser, 2011).However, the decrease in pH levels in T3 under a vegetative cover may be due to a release of protons (H + ) during the uptake of cations by roots (Bye, 1999).In addition, the root exudates may indirectly lead to a decrease in pH because it stimulates the proliferation of soil microorganisms that synthesize organic acids and thus acidifies the soil (Davet, 1996;Waligora, 2010).The activity of soil microorganisms also depends on the temperature, moisture and soil texture (Lundquist et al., 1999;Steenwerth et al., 2008).Nevertheless, it was observed in laboratory conditions that rhizobia decreases soil pH; while Bradyrhizobia trends to increase it (Dubey, 2011).It was found however that the pH is a significant factor that affects the nodulation in soils mines with a high rate of nodulation at a pH which is between 5.5 and 7.2 and a low rate to a pH below 5.5 (Zahran, 1999).Meanwhile, alkaline soils with a pH greater than or equal to 7.8 limit accessibility to Iron, Zinc, Manganese and mainly Boron and Phosphorus in soil, thereby reducing the Nitrogen fixation (Graham & Allan, 2002) We found, a deficiency of exchangeable Magnesium for every plot at various times (T1, T2, T3) (Figure 4.b), with rates well below the standards that vary between 10% and 20% (Reuter et al., 1997).In fact, an excess of Calcium causes a Magnesium deficiency.However, an excess of Potassium inhibits the absorption of magnesium (Pousset, 2002).Erosion could also be one of the main causes of magnesium deficiency (Boyer, 1978).Thus, the soil is moderately calcareous (Figure 4.c), however, there is a significant decrease at p<0.05 in the total content of limestone at T2 in S14 plots (15.32%) compared to bare soil (18.55%) and a significant increase (P<0.05) at T3 in D24 plots (19.22%) compared to non Acacia plots (18.45%).Similarly, we recorded a significant increase (p<0.05) at T2 and T3 compared to T1 in Myc plots (18, 12, 12% and 19% compared to 12.83%) and mix (18.9% and 18.77% compared to 13.15%) and a significant increase (p<0.05) at T3 compared to T1 in S14 plots (19.1% compared to 14.53%).
The level of active limestone changes but not significantly.However its content is close to or exceeds 5% (Figure 4.e).Therefore, limestone solubilization and the gradual release of Calcium can be achieved by acid rainwater or by the biochemical activities of either microorganisms or plant roots (Wierzchos et al., 2003;Salomon, 2006;Coque, 2008).Moreover, the abundance of Calcium ions has an antagonistic effect on other nutrients availability such as Potassium, Iron, Boron, Copper, Manganese, Zinc and signs of deficiency or fading can appear on plants (chlorosis) (Pousset, 2002;Vasant et al., 2009;Ofme, 2011;Morel, 1996).In addition, it inhibits the mineralization of organic matter under the effect of coating (Morel, 1996).Although, land may be rich in total limestone, but relatively poor in active limestone (Pousset, 2002).
The Phosphorus level is low between 1 and 9 ppm.(According to AgSource Laboratories, 2013) (Figure 4.f).However there is an evolution in content over time, where there is a significant increase (p<0.05) of phosphorus in T2 compared to T1 in S10 (6.78 ppm compared to 5.2 ppm), S14 (7.59 ppm compared to 5.82ppm), S24 (7.16 ppm compared to 3.95 ppm), Myc (7.55 ppm compared to 4.67 ppm) and D24 (7.92 ppm compared to 5.32 ppm) plots.On The other hand, there is a significant increase (p<0.05) in T3 compared to T1 in D10 (7.45 ppm compared to 5.07 ppm) and Mix (7.77 ppm compared to 5.8 ppm) plots.In fact, Phosphorus is generally low in calcareous alkaline soils.It tends to be insolubilized by the Calcium (Calcium Phosphate and Magnesium) and it is possible that the phosphoric anions precipitate at the contact of the active limestone (Pousset, 2002;Ryan et al., 2001;Baize, 2000).However, Phosphorus is found in organic and inorganic forms.Low Phosphorus availability is due to the larger action of phosphoric anions soluble with Ca 2+ , Mg 2+ , Fe 3+ and Al 3+ , depending on the geochemical soil properties (Gyaneshwar et al., 2002).Some microorganisms are able to solubilize phosphate minerals while reducing the pH by the secretion of organic acids which are good chelator of divalent cations such as Ca 2+ and they can also form a complex with the metal ions associated with Phosphorus, thus releasing Phosphorus (Jones, 1998;Gyaneshwar et al., 2002;Pradipta, 2008).The mechanism of solubilization of inorganic Phosphorus is provided by organic acids; although the acid phosphatases play a major role in the mineralization of organic phosphorus (Goldstein , 1995;Kim et al., 1997;Rodriguez & Fraga, 1999).The genera Pseudomonas, Bacillus and Rhizobium are among the most powerful bacteria in the process of dissolution of Phosphorus (Rodriguez & Fraga, 1999).However, mycorrhizal hyphae make the Phosphorus and certain mineral traces such as Calcium and Zinc available to the host plant (Olsson et al., 1999).In agroforestry, mycorrhizal association contributes to the growth of Acacia species in infertile soils (Dart et al., 1991).Moreover, the dual inoculation by arbuscular mycorrhizal and bacteria that solubilize phosphorus increases the absorption of native soil Phosphorus as well as Phosphorus coming from Phosphate rocks (Goenadi et al., 2000;Cabello et al., 2005).
Total Nitrogen contents have not changed much except for the plots S14 where there has been a significant increase (p<0.05) in T3 compared to T1 (0.052% compared to 0.023%) (Figure 4.g).It is true that the bioavailability of Nitrogen depends primarily on the activity of nitrifying bacteria, but their activity is pH-dependent: they are, in fact, more active when the pH is between 6.5 and 7.5 and the optimum temperature between 30° to 35° Celsius (Biswas & Mukherjee, 2006).Although, microbiological antagonism may reduce nodulation (Kumara et al., 1974).
Organic matter levels determined are very low (<2%) (Figure 4.h).This result is in accord with Godwin (1992), who reported that soils in semi-arid regions contain little organic matter, of no more than one percent.Additionally, sandy soils are low in organic matter which generally would make them weak and unproductive (Pousset, 2002).In fact, minerals, organic matter and microorganisms are the key elements in pedogenesis and soil conservation (Bollag & Leyval, 1998).

Conclusion
After nine months of a bio-re-vegetation, we could achieve at a less alkaline pH soil and an improvement in phosphorus content.However, the soil calcareous nature and the rate of either active limestone or the exchangeable Calcium results in a small increase in total Nitrogen and organic matter, and makes exchangeable magnesium contents deficient.In perspective, the factor time may be important in this study, so the experimentation can be conducted over a period of two years or more.