Use of dairy reject and fermented Aleo sp. Leaf Gel mixture in the biological Pre-treatment of landfill leachate

Landfill leachate contains large amounts of organic and inorganic compounds consisting the major source of groundwater contamination (Li et al. 2011). Leachate generated in municipal landfills contains high concentrations of organic substances, ammonia, heavy metals, inorganic salts and other contaminants (Modin et al. 2011). The treatment of landfill leachates (LFL) is very complicated, expensive and requires combined process applications due to their high organic matter content. Among the different methods used for LFL treatment, biological processes are gaining a great interest for their reliability, simplicity and high cost-effectiveness. Furthermore, such processes provide many advantages in terms of biodegradable matter and nitrogen compounds removal (Renou et al. 2008). Moreover, bacterial applications are gaining an increasing importance in the detoxification of wastewaters (Gomes et al. 2015). Few studies highlighted the efficiency of lactic acid bacteria as potential biosorbents of heavy metals (Kinoshita et al. 2013). In preceding works, the pretreatment and the reuse of wasted dairy products with combined physical-chemical and biological processes were investigated for promoting the treated effluents reuse (Kasmi et al. 2017a, 2017b).

Recent researches reported the use of natural resources for their clarification potential in the process of water treatment. Moringa seeds (Menkiti & Onukwuli 2010), peels of Lablab purpureus (Shilpa et al. 2012), Moroccan cactus extract (Abid et al. 2009), dates seeds (AL-Sameraiy 2012) and Opuntia dillenii (Nougbode et al. 2013) have been investigated for some rejects turbidity removal efficiency and interesting results were obtained. Additionally, Nougbode et al. (2016) confirmed that Aloe barbadensis Miller (Aloe vera) leaf gel can be used as natural flocculent for water treatment. It was also found that the use of this plant in low doses can rid the highly charged water of their suspended materials therefore their turbidity (Nougbode et al. 2016). Nevertheless, researchers reported that it could increase the organic matter content given the high levels of organic compounds in the Aloe plant (approximately 81.05%). According to literature, the chemical composition of Aloe plants is largely dependent on the species analyzed. Aloe leaves are well known by their mucilaginous jelly, which is referred to as Aloe gel. Aloe gel contains mainly monosaccharides such as glucose and mannose, vitamins, minerals, polysaccharides and phenolic compounds next to some organic acids (Mazzulla et al. 2012). Thus, fermentation could be an interesting alternative for the Aloe organic matter reduction and bioconversion. There have been very few studies conducted on Aloe fermentation (Cuvas-Limón et al. 2016). Kim et al. (2014) have developed a preliminary investigation of Aloe vera pulp fermentation and determined the presence of lactic acid bacteria (LAB). This supposed that this plant could be used as substrate for LAB fermentation as a starter culture (Cuvas-Limón et al. 2016). Besides, Cho et al. (2012) have shown that Aloe fermentation products may gain new proprieties compared to natural Aloe, where the former exhibited significant inhibition activity on Helicobacter pylori whereas the latter had no growth inhibition effects.

In the present study, a new approach for the use of bactofugate (dairy effluent) and Aloe sp. leaf gel mixture in the pre-treatment of landfill leachate of Jebel Chakir was investigated.

The site of Jebel Chakir is the first controlled landfill area in Tunisia (Tizaoui et al. 2007). It is mainly used for the disposal of domestic solid wastes from greater Tunis area and it is located at 10 km at the Western South of Tunis. The site occupies 47 ha over a reserve total area of 124 ha. Jebel Chakir landfill receives 1,800–2,000 tons/day of municipal solid waste of which 68% are organic materials (Ferchichi 2005). The volume of leachate generated from this site is estimated to be around 270 m³/day. Up-to date the leachate is piped and stocked without any treatment in 13 collecting pools of a total capacity of 300,000 m³. Leachate samples were collected in 20-l tanks and transported to the laboratory. The leachate was stored in obscurity at 4 °C in order to avoid its acidification, its chemical composition variation and notably to limit the microbial activity.

The dairy reject used in this study is the bactofugate collected from a regional dairy industry (Centrale Laitière du Cap Bon) which is part of Delice Group, leader of the food industry in Tunisia in the dairy sector. The bactofugate is a dairy release, generated following the bactofugation of raw milk. It consists in a special form of separation in which specific types of microorganisms are removed from milk and inactivated (Kolhe et al. 2009). The difference in density between spores and milk allows their sedimentation under the effect of a centrifugal force and the bactofugate is ejected outside (Hanno et al. 1991). According to the dairy industry statistical data, an average of 1,200 L of bactofugate was generated daily from bactofugation process as separated milk (Belwarda 2012).

Two-year-old Aloe sp plant leaves were harvested from the garden of the Biotechnology Center of Borj Cedria (CBBC), Soliman, Tunisia. Whole leaves were washed and spikes, placed along their margins, were removed before slicing the leaf to separate the skin from the filet. The resulting filets were mixed and homogenized using electric hand mixer and autoclaved (121 °C, 15 min). The obtained gel was fermented using commercial Saccharomyces cerevisiae yeast strain. The microorganism was reactivated on Sabouraud Broth medium (HiMedia) and a seed culture was prepared according to Kasmi et al. (2016a) instructions to be used for the Aloe sp leaf gel inoculation. Fermentation was carried out in batch using Erlenmeyer flasks (250 mL) containing 50 mL of Aloe sp leaf gel. Culture conditions were as follows: initial pH (5.0), fermentation time (5 days) (Cho et al. 2012), temperature (30 °C) and inoculum 9% (v/v). The rotary speed of shaker incubator (ZHWY- 103D) was fixed at 150 rpm.

Two mixtures were used in this study for the pretreatment of LFL:

• (i)

  90 ml of bactofugate was mixed with 10 ml of natural Aloe sp. leaf gel after sterilization. This mixture will be annotated as B-NAg.

• (ii)

  90 ml of bactofugate was mixed with 10 ml of yeast free fermented Aloe sp. leaf gel. The fermented gel was withdrawn at the end of the exponential cell growth phase and filtrated (0.45 μm) to remove cells. This mixture will be annotated as B-FAg.

B-NAg and B-FAg mixtures were maintained at 37–40 °C in static conditions until pellet formation.

Beakers containing aliquot volumes of LFL, were inoculated separately using the treatment mixtures B-NAg and B-FAg at the rates of 2, 6, 10 and 12% (v/v). Preparations were kept under magnetic stirring at room temperature during 48 h. Experiments were performed in triplicate and withdrawals analysis were carried out periodically each 2 h during the treatment.

The pH, conductivity (mS/cm) and total dissolved solids (TDS) (g·L⁻¹) of each sample was determined using multi-parameters device Consort C860. Chemical oxygen demand (COD) values were measured by the potassium dichromate colorimetric method using an opened reflex system (Rodier et al. 2009). Ammoniacal nitrogen was determined according to the NF T90-15 method (AFNOR 1999). As for yeast biomass estimation, it was determined using Sabouraud agar pour plates count. Plates were prepared beforehand using appropriate dilutions from the seed culture.

All the experiments were performed in normal conditions: ambient temperature and atmospheric pressure.

Table 1 summarizes the recorded results of the collected leachate analyses. pH values ranged from 8.0 to 8.6. The notable characteristic of this leachate was its high COD value exceeding 28,000 mg O₂/L. Furthermore, its ammonia nitrogen content (5.0 g/L), total dissolved solid (TDS) content (25 g/L) and conductivity (33 mS/cm) were relatively high compared to the Tunisian standards. Im et al. (2001) reported a similar composition of LFL with high organics concentration (21,000–26,000 mg O₂/L) and ammonia nitrogen content. Aydi et al. (2012) considered that the relatively low concentration of analyzed heavy metals and the high COD and nitrogen concentration confirmed the high organics content of the waste deposited in the landfill of Jebel Chakir.

In this study, bactofugate is proposed to be reused in leachate pretreatment. Analysis revealed that the pH value of the dairy reject was around 5 and its recorded COD value was 60,000 mg O₂/L. Bactofugate presents a high mineral content (36 g/L) and its conductivity was assessed at 36 mS/cm.

Aloe gel, which was used as a natural medium, was beige colored before being used as a substrate. After sterilization, its color became close to pink. When the Aloe medium was inoculated using Saccharomyces cerevisiae strain (10⁶ CFU/ml) and fermented for 5 days, the color of the aloe changed from pink to beige after just one fermentation day, which is Aloe's natural color. During the remaining fermentation period, the Aloe medium was going to be more and more decolorized. The Aloe color change was reported by Cho et al. (2012) who confirmed that on the 4th day it turned beige and the color persist for the 5^th day of fermentation. The highest microbial growth was reached at the end of the 4^th day where the cell density was estimated to be 3.3 10⁷ CFU/ml. It has been noticed that the fermented medium was mainly exhausted from its Mg content (91%). Thereafter, during the 5^th day, cells mortality was taking place due to the alcohol inhibition in the fermented medium. For this reason, the 4-days-fermented Aloe gel will be considered for further uses.

Previous studies have demonstrated the antibacterial and antifungal activity of the natural Aloe vera gel. Such feature is highly recommended for pharmaceutical, medical and food industries (Bernardes et al. 2012). Nevertheless, Cho et al. (2012) have shown that the Aloe fermented gel is able to inhibit pathogen bacteria such as H. pylori. In this study, the fermented Aloe sp. gel using Saccharomyces cerevisiae strain was investigated for its antibacterial and antifungal activities on different microorganisms. The tested bacterial strains are Pseudomonas aeruginosa, Serratia marcescens, Salmonella anatum and Enterococcus faecalis. The tested fungal strains are Candida parapsilosis, Candida glabrata and Candida krusei. Despite that several works have demonstrated the Aloe vera antifungal activity against phytopathogens (Castillo et al. 2010), the fermented Aloe sp. gel in this study exhibit no inhibition effect on the selected strains. Those findings could be attributed not only to the studied plant species in this work, but also to the gel extraction methods; since researchers have usually used the gel organic solvent extract (i.e. ethanol, hexane, etc.) to investigate Aloe antimicrobial activities (Eugene et al. 2011).

As reported by Ghodhbane et al. (2016), several bacteria have been isolated and identified from bactofugate including: Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei, Enterococcus facecalis. In addition, it has been shown that L. plantarum strain ensured COD abatement rate exceeding 80% on dairy effluents (Kasmi et al. 2016b). Kasmi et al. (2017b) have also isolated Candida strains (C. tropicalis, C. paracasei and C. krusei) from bactofugate and investigated their performance for the biological treatment of dairy wastes and interesting results were achieved using C. krusei. Therefore, the mixture of Aloe gel with bactofugate is proposed for leachate treatment in this work. Such mixture is supposed to stimulate bacterial growth for the promotion of LFL biodegradation, since Aloe gel is rich in carbohydrates, vitamins and minerals.

The obtained results on leachate after the treatment mixture addition are illustrated in Figure 1. COD values exhibited some variation once the B-NAg mixtures were added. The recorded COD values changed from 25,000 to reach 26,400 mg/L within 2 h with 2 and 6% B-NAg mixtures. However, COD values reached 32,000 and 33,000 mg/L within 4 h with 10 and 12% B-NAg mixtures, respectively. This organic load ascension could be attributed not only to the mixtures high organic load but also to the microbial growth on LFL. Thereafter, COD values are going to decrease progressively with all B-NAg mixtures. The recorded COD removal rates were of about 62 and 67% with 2 and 10% of B-NAg mixture, respectively, after 28 h. No important organic load reduction was noticed thereafter. Whereas, the 6 and 12% B-NAg mixtures achieved COD abatement rates of 62 and 64.8%, respectively, after 44 h. By reference to the initial organic load of LFL and considering the treatment period of 48 h, the mixture 2% B-NAg exhibited the most interesting COD removal rates (63%). Nevertheless, no significant difference could be noticed using 6 and 10% B-NAg mixtures where the global abatement rates were 61 and 60%, respectively. With 12% B-NAg mixture, only 56% of organic load reduction was achieved.

Conductivity as well as total dissolved solids showed globally a decreasing profile during the treatment as illustrated in the Figure 2. Both conductivity and TDS followed non linear development shape. Some increases were detected during the first 4 h of the treatment with 2% B-NAg mixture and after 24 h with all of 2, 6 and 12% B-NAg mixtures. The highest dissolved solids content reduction during the treatment was obtained with 12% B-NAg mixture; then, 10, 6 and 2% B-NAg mixtures in the decreasing order. This order was also respected with the recorded values of LFL conductivity during the treatment. In contrast of the COD removal rates, the recorded reduction values in terms of TDS and conductivity indicating mainly minerals consumption rates in the LFL are less important. In this context, it has been reported that many important microbial processes can be influenced by minerals, including energy generation and nutrient acquisition (Brown et al. 2008). The vast majority of minerals contain metals that are directly and/or indirectly involved in all aspects of microbial growth, metabolism and differentiation (Gadd 2010). It is commonly accepted that toxic metals can have significant effects on microbial populations and, under toxic conditions, almost every index of microbial activity can be affected (Giller et al. 2009).

It's worthy to mention that the COD increase at the first 4 h of treatment is accompanied with sharp reduction in both TDS content and conductivity values within the first 2 h using 10 and 12% B-NAg mixtrures. This phenomenon could be attributed to the microbial nutrients acquisition in the environment (LFL). In the literature, informations about the microbial assimilation behavior and metabolism of minerals are barely available. Thus, Kasmi et al. (2017b) have confirmed the iron assimilation aptitude of some tested LAB strains that exceeded 65%. Nevertheless, the minerals content increased from 25 to 27–28 h during the treatment may also be explained by the biosynthesis of minerals in a way that all groups of microbes can mediate mineral formation by direct and indirect mechanisms (Gadd 2010).

The obtained results on leachate after bactofugate and fermented Aloe gel (B-FAg) treatment mixture addition are illustrated in Figure 3. COD values exhibited an increase for the first hours of treatment. The recorded COD values change from 25,000 to reach 28,800 mg/L within 4 h with 2% B-FAg mixture. However, COD values reached 33,600 and 32,400 mg/L within 6 and 8 h with 10 and 6% B-FAg mixtures, respectively. The organic load ascension during the treatment seems to be more important with B-FAg compared to B-NAg. Those findings may be explained by the effect of Aloe gel fermentation where nutrients were dissociated and depolymerized through baker's yeast fermentation. Indeed, Aloe gel fermentation ensured more availability of fermentable sugars and nutrients for better microbial growth on LFL. In this context, Cho and co-workers (2012) have detected the reduction of beta-glucan (D-glucose polymer) content after Aloe gel fermentation for 4 days or less. Thereafter, COD values are going to decrease progressively with all B-FAg mixtures. The highest COD removal rate (72%) was recorded with 2% of B-FAg mixture after 44 h. No important organic load reduction was noticed thereafter. Whereas, the 6 and 12% B-FAg mixtures achieved COD abatement rates of 68 and 64.8%, respectively, after 48 h. Globally, COD removal rates exhibited a slight improve when the treatment mixture of the fermented Aloe gel is used. Considering the recorded values during the whole treatment period (48 h), 2% B-FAg mixture achieve the better results in terms of organic matter reduction (68%). However, the global COD removal rates with 6, 10 and 12% B-FAg mixtures were limited at 50, 49 and 39%, since considerable organic matter increase was recorded at the beginning of the treatment.

Conductivity as well as total dissolved solids showed globally a decreasing profile during the treatment using B-FAg mixture as illustrated in the Figure 4. Both conductivity and TDS followed non linear development shape. Unlike the recorded profiles using the B-NAg mixtures, no increases were detected at the beginning of the treatment. Such behavior may confirm the microbial growth on the LFL which induces minerals and carbohydrates consumption. The most rapid dissolved solids content reduction during the treatment was obtained with 10% B-FAg mixture. Whereas, the TDS values of all the used treatment mixtures are going to be closer at the end of the treatment (after 48 h). Thus, no significant difference could be mentioned between 6, 10 and 12% B-FAg mixture in terms of TDS content reduction. In contrast, the conductivity profiles showed an important decrease with 10% B-FAg followed by 6, 12 and 2% B-FAg mixtures in the order at the end of treatment.

Most parameters of Jebel Chakir landfill leachate exceeded the permissible requirements for the treated wastewater discharge determined by the Tunisian local standards. Bactofugate and Aloe gel mixtures were used for the biological pretreatment of LFL. Results showed that such mixtures could achieve important COD removal rates in landfill leachate. The use of natural Aloe gel in the mixtures achieved organic matter abatement ranging from 56–63% in the LFL. An improvement in terms of the organic matter removal was recorded when the B-FAg mixture was used at an inoculum rate of 2% to reach 72% as global COD removal rate during 48 h of treatment. Those findings confirm that the biological pretreatment of LFL using bactofugate and Aloe gel mixture could be an interesting way. However, the microbial material growth and behavior investigations in terms of the valuable compounds bioconversion is highly required to control and promote the biodegradation of the residual organic matter of LFL.