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Ammonium Cycling and Nitrification Stimulation during Oil Sludge Remediation by Gram-Positive Bacteria Lysinibacillus sphaericus Using Red Wiggler Earthworm Eisenia fetida

Ammonium Cycling and Nitrification Stimulation during Oil Sludge Remediation by Gram-Positive... AgriEngineering Article Ammonium Cycling and Nitrification Stimulation during Oil Sludge Remediation by Gram-Positive Bacteria Lysinibacillus sphaericus Using Red Wiggler Earthworm Eisenia fetida 1 1 , Juan Diego Acevedo and Jenny Dussán * Centro de Investigaciones Microbiológicas—CIMIC, Departamento de Ciencias Biológicas, Universidad de los Andes, Carrera 1 No. 18A-10 J-206, Bogotá PC 111711, Colombia; jd.acevedo245@uniandes.edu.co * Correspondence: jdussan@uniandes.edu.co; Tel.: +57-1-3394999 (ext. 3644) Received: 5 October 2020; Accepted: 3 November 2020; Published: 13 November 2020 Abstract: The performance of a mixture between L. sphaericus and E. fetida was evaluated for ammonium cycling and nitrifying bacteria stimulation during oil sludge remediation. The addition of E. fetida significantly increased ammonium concentration (p = 0.0218) and total Colony-forming units (CFU) count p = 0.02848). However, oil sludge with worms and L. sphaericus reached lower ammonium concentrations and CFU counts than sludge with worms alone. Sludge inoculated only with L. sphaericus presented higher ammonium concentration than sludge without inoculum, but the bacterial population reached a lower density during the final days. Final DNA and RNA extractions from all treatments amplified for L. sphaericus putative amoA and Gram-negative nitrifying bacteria amoA genes correlated with diminished ammonium concentrations during the final days of the experiment. Final RNA extractions for L. sphaericus amplified for Molybdenum transporter gene suggesting possible nitrogen fixation by L. sphaericus. The addition of Red Wiggler Earthworm to oil sludge remediation systems may provide better conditions for bacterial populations to carry out hydrocarbon degradation. The addition of E. fetida to a L. sphaericus crude oil biodegradation system may improve soil ammonium concentrations and nitrifying activity, and this could be crucial in oil sludge remediation because of bacterial inhibition due to high C:N ratios. The final product of this process may be used for soil enhancement due to its richness in nutrients and beneficial bacterial populations. Keywords: Lysinibacillus sphaericus; Eisenia fetida; bioremediation; crude oil sludge; ammonium; nitrification; amoA; molybdenum transporter 1. Introduction Crude oil extraction, transportation and refinement produces residual sludge composed by hydrocarbons, paran, produced water, sediments, and other possible contaminants such as heavy metals, sulfuric compounds and chemicals added during the operation [1]. The complexity of this mixture makes it very dicult and expensive to remediate, making crude oil sludge a persistent contaminant. Bioprospecting microbial communities found in this kind of sludge led to the discovery of di erent bacterial consortiums with the ability to break apart these compounds into less toxic molecules. Based on these findings, di erent crude oil bioremediation techniques have been developed resulting in a system that is cheaper, more ecient, and more sustainable compared to other remediation practices such as chemical degradation or incineration [2,3]. AgriEngineering 2020, 2, 544–555; doi:10.3390/agriengineering2040036 www.mdpi.com/journal/agriengineering AgriEngineering 2020, 2 545 Previous bioprospecting of native Colombian bacteria for crude oil degradation has led to the current use of di erent strains of Lysinibacillus sphaericus in remediation processes for oil companies around the country [4]. The strains used in these treatments are non-pathogenic and present high degradation rates for di erent oil hydrocarbons achieving significant results in the environmental recovery of contaminated soils, with removal eciencies up to 95% for C10 to C28 hydrocarbons in diesel oil assays and up to 84.1% and 60.1% for oil sludge in open air and closed arrays, respectively [5]. L. sphaericus is also involved in nutrient cycling. Genomic annotation has revealed the presence of several genes involved in the nitrogen cycle including Molybdenum transport system permease protein encoding gene (modB) needed to assemble Nif enzyme, nifU involved in the formation of Fe-S clusters for nitrogen fixation, Ammonium transporter and putative ammonia monooxygenase responsible for the first step in nitrification [6]. These abilities have also been proven by ammonium and nitrite quantification during in-vivo studies focused on nitrogen cycling [7,8]. Microbial remediation of crude oil sludge may be enhanced using earthworms [9,10]. Several studies have proven that adding Red Wiggler Worm Eisenia fetida to contaminated soils improves degradation rates of both, heavy [11] and light hydrocarbons [12]. Red Wiggler Worms also have the capacity to improve nutrient cycling significantly, raising ammonium and nitrite concentrations in soil [13] and bacterial populations related with nutrient cycling [14]. These abilities have been verified by gut bacterial genomic annotations that show abundance in genes related to aromatic hydrocarbon degradation, dehalogenation, nitrogen fixation, and ammonium oxidation [15]. E. fetida is commonly used in composting processes given its high degree of adaptability, resistance, and high reproductive rates, as well as its vast commercial availability worldwide [16]. Also, it has been widely used in remediation studies showing promising results for soil structure improvement, promotion of microbial numbers and hydrocarbon degraders and increased microbial respiration resulting in total petroleum hydrocarbons (TPH’s) degradation [17]. Several studies propose that nitrification is dominated by archaea or beta and gamma proteobacteria commonly classified as AOA (ammonia oxidizing archaea) and AOB (ammonia oxidizing bacteria). In the first stage of nitrification, ammonium is reduced to hydroxylamine by the ammonia monooxygenase enzyme (AMO), in AOB this enzyme is encoded by the genomic amoCAB operon [18,19]. The amoA gene of this operon is highly conserved, and thus, very useful for environmental studies in which nitrifying activity is evaluated [19]. Some Gram-positive bacteria have genes homologous to amoCAB, as is the case for L. sphaericus, where a putative amoA gene is present [6]. These findings suggest that bacteria other than beta and gamma proteobacteria may be involved in nitrification processes with significant e ects on ammonia oxidation rates and nitrite concentrations in soil [20]. Ammonia monooxygenase is also capable of degrading hydrocarbons via cometabolism, whereby electrons produced by ammonium oxidation are reused by AMO for hydrocarbon oxidation. Due to its wide range of substrates, this enzyme is capable of degrading alkenes, alkanes, and halogenated and aromatic hydrocarbons. Cometabolism of hydrocarbons by AMO is a complex phenomenon that generally causes AMO activity inhibition, and hydrocarbon byproducts may a ect cell physiology [21–23]. Cometabolism has been proved to be an important process in the degradation of xenobiotic hydrocarbons [24]. The objective of this study is to determinate ammonium cycling and nitrification stimulation during the biodegradation of petroleum hydrocarbons by L. sphaericus using E. fetida by monitoring bacterial populations, ammonium concentrations, and the expression of amoA and modB. 2. Materials and Methods 2.1. Summary This experiment was divided into two stages: Pretreatment and Treatment. During the Pretreatment the biological degradation was initiated at high crude oil concentrations and monitored by performing AgriEngineering 2020, 3 FOR PEER REVIEW 3 2. Materials and Methods 2.1. Summary AgriEngineering 2020, 2 546 This experiment was divided into two stages: Pretreatment and Treatment. During the Pretreatment the biological degradation was initiated at high crude oil concentrations and monitored by performing ammonium determinations, once ammonimun concentrations begun to raise the ammonium determinations, once ammonimun concentrations begun to raise the pretreatments were pretreatments were dilluted to permisibile TPH’s levels in order to inititate the Treatment, during dilluted to permisibile TPH’s levels in order to inititate the Treatment, during this stage the treatments were this stage the treatments were monitored for ammonimum concentration, vegetative cell count, and monitored for ammonimum concentration, vegetative cell count, and amoA and ModB gene presence amoA and ModB gene presence and expression. A brief summary of the methodology is presented and expression. A brief summary of the methodology is presented in Figure 1. in Figure 1. Figure 1. Methodology summary. Figure 1. Methodology summary. 2.2. Materials 2.2. Materials Heavy crude oil sludge and unpolluted soil samples were obtained from Biointech, a Colombian Heavy crude oil sludge and unpolluted soil samples were obtained from Biointech, a Colombian company dedicated to soil and water treatment for environmental recovery. The samples were collected company dedicated to soil and water treatment for environmental recovery. The samples were collected at Caño Limón, Arauca (N 07°05′25″–W 70°45′42″). 0  0 at Caño Limón, Arauca (N 07 05 25”–W 70 45 42”). Earthworm castings were bought from an E. fetida breeder and compost producer. The worms Earthworm castings were bought from an E. fetida breeder and compost producer. The worms were grown in a substrate made up of livestock manure, agricultural residue and vegetable residue were grown in a substrate made up of livestock manure, agricultural residue and vegetable residue from country markets. from country markets. The selection of the L. sphaericus strains used in this study was based on previous research carried The selection of the L. sphaericus strains used in this study was based on previous research out by the Center of Microbiological Research (CIMIC) at Universidad de los Andes (Bogotá, Colombia) [4]. Microbial bioaugmentation was performed using five strains of L. sphaericus: CBAM5, carried out by the Center of Microbiological Research (CIMIC) at Universidad de los Andes (Bogotá, OT4b.31, OT4b.49, 3(III)7 and 2362. Lab trials have shown great degradation results and survival Colombia) [4]. Microbial bioaugmentation was performed using five strains of L. sphaericus: CBAM5, rates at a concentration of 5% volume to volume ratio (v/v) of diesel [5]. This consortium is actually OT4b.31, OT4b.49, 3(III)7 and 2362. Lab trials have shown great degradation results and survival rates used by Biointech in soil remediation processes for the Colombian oil industry resulting in TPH´s at a concentration of 5% volume to volume ratio (v/v) of diesel [5]. This consortium is actually used by degradation to permissible levels in periods of time that span from 45 to 60 days under field Biointech in soil remediation processes for the Colombian oil industry resulting in TPH´s degradation conditions (Caño Limon, Arauca). to permissible levels in periods of time that span from 45 to 60 days under field conditions (Caño All the containers used for the experiment had holes in the bottom for drainage and were Limon, Arauca). covered with black plastic. The experiment was performed inside a 2 meters (m) wide, 1.2 m long All the containers used for the experiment had holes in the bottom for drainage and were covered and 0.8m tall light insulated incubator made with white carboard walls on the outside and black withplastic in black plastic. the inside. The experiment was performed inside a 2 meters (m) wide, 1.2 m long and 0.8m tall light insulated incubator made with white carboard walls on the outside and black plastic in the inside. 2.3. Composted Soil, Worm Castings and Bacteria Growth 2.3. Composted Soil, Worm Castings and Bacteria Growth One month before starting the experiment, a 20 liter (L) plastic container was filled with 2 kilograms (kg) of worm casting mixed with 8 kg of unpolluted soil collected from the oilfield and 2 kg One month before starting the experiment, a 20 liter (L) plastic container was filled with of orange peels. The container was covered with black plastic and kept inside a greenhouse. All the 2 kilograms (kg) of worm casting mixed with 8 kg of unpolluted soil collected from the oilfield and 2 kg of orange peels. The container was covered with black plastic and kept inside a greenhouse. All the worms and amended soil used in this study were extracted from this container that will be referred to as (E0). L. sphaericus strains’ liquid cultures were prepared by growing 1 10 Colony-former units (CFU) of each strain in 20 milliliters (mL) of nutrient broth for 24 hours (h) at 30 C and 150 revolutions per minute (rpm), resulting in a final concentration of 1 10 CFU/mL. For the inoculation of soil samples, two trays were filled with 550 mL of SPC agar each. Using five sterile brushes (one for each strain), the liquid cultures at a concentration of 1  10 CFU/mL were AgriEngineering 2020, 2 547 evenly distributed along the surface of the agar occupying 300 square centimeters (cm ) per strain in each tray. Both trays were placed in an incubator for 24 h. On the day of inoculation, bacteria that had grown on the surface of the agar were collected using a spatula, resulting in 90 mL of solution with a concentration of 1 10 CFU/mL of the consortium. 2.4. Pretreatment Before starting the experiment, a Pretreatment was prepared in order to initiate the biological degradation system before diluting the samples to TPH´s concentrations tolerable for earthworms. A Pre-treatment master mix was prepared using a 1:1 weight to weight ratio (w/w) of crude oil sludge and unpolluted soil. Two days later, this mixture was divided into two equal bundles 2.4 kg each. One of the bundles was mixed with 480 grams (g) of SPC agar without inoculum and the other bundle was mixed with the same amount of agar and 90 mL of L. sphaericus consortium inoculum at a concentration of 1 10 CFU/mL. Each bundle was divided in half again resulting in four 1.77 kg bundles that were stored in 20-L plastic containers. Five days later, 30 g of E. fetida were added to one of the bundles without L. sphaericus inoculum and one with inoculum, this bundles corresponded to the final Treatments to be evaluated: E. feitda alone (E), L. sphaericus alone (L), E. fetida with L. sphaericus consortium (EL), and a Control (C) without worms or inoculum, this day was considered as day 0 of the pre-treatment. The pre-treatment was evaluated for a period of 41 days before starting the Treatment. 2.5. Treatment A Treatment master mix was prepared mixing 0.608 kg (4.4%) of crude oil sludge, 14.364 kg (53.2%) of unpolluted soil, and 11.394 kg (42.2%) of worm amended soil from the E0 container, and stored for stabilization for two days inside a 40 L plastic container covered with black plastic. On day 0 of the Treatment (day 41 for the Pretreatment), the Treatment master mix was divided into two equal bundles, one of the bundles was inoculated with 90 mL of consortium at a concentration of 1 10 CFU/mL and then both bundles were divided again into two equal parts resulting in four 20 L plastic containers filled with 6750 g of master mix each, two inoculated with L. sphaericus and two free of inoculum. After this, E and C pretreatments were diluted to 8.89% (w/w) each using the two Treatment master mix bundles that were not inoculated, and the same was done for L and EL using the two inoculated bundles. Following this, 250 g of worms were added to EL and E each resulting in the three final treatments and a control: E. feitda alone (E), L. sphaericus alone (L), E. fetida with L. sphaericus consortium (EL), and (C) without worms or inoculum. At this moment an additional control (CE) composed of 57.7% unpolluted soil and 42.3% amended soil with the same quantity of earthworms as Lo and LL was added to the experiment. The amended soil was added to all treatments and controls in order to elucidate the di erence between the e ects of adding amended soil only and adding the earthworms. The Treatment was evaluated for a period of 52 days. 2.6. Sampling and Analysis During the Treatment temperature and humidity were measured and recorded inside the incubator using an Arduino. TPH´s were determined on the Pre-treatments and oil sludge two days prior to the beginning of the Treatment in order to calculate the dilution needed to achieve, at least, a 50% earthworm survival rate. The 50% lethal dose was approximated at 20 g/kg TPH´s based on the toxicity trials made by Martinkosky et al. [11]. DNA was extracted from the earthworms’ tissue at the moment of purchase (E0). The day previous to the Treatment’s master mix preparation, DNA was extracted from crude oil sludge and unpolluted field soil. On day 52, DNA and RNA were extracted from all Treatments including worm tissue (E,EL,CE). Ammonium determinations and CFU counts were performed on days 0, 9, 22, 36 and 52. Ammonium determinations were also performed during the Pretreatment on days 0, 5, 20 and 41 [12]. Samples for ammonium and hydrocarbon determinations and CFU counts were taken AgriEngineering 2020, 2 548 in triplicate using compound sampling; soil samples for DNA and RNA extractions were taken in triplicate for each treatment and then combined in one compound sample for analysis. For worm samples, 1.5 g of worms were processed for each Treatment. 2.7. Ammonium and Hydrocarbon Determination and Microbial Count Ammonium concentrations were determined using Merck ammonium test kit catalog no. 114,752. One gram per soil sample was diluted by two orders of magnitude with distilled water prior to analysis. Hydrocarbon determinations were performed using an Oil and Water Retort, 10 g from each sample was processed following fabricator´s recommendations resulting in the separation of a liquid phased composed of oil and water and a solid phase [25]. The liquid phase, contained in the equipment´s test tube, was weighed using an analytical balance. Then, the oily fraction from the liquid phase was extracted from the test tube using a pipette and the tube was weighed again, this amount was subtracted from the initial weight of the liquid phase in order to calculate the concentration of the oily fraction. The solid phase was also weighed using an analytical balance, and the volatile fraction was calculated subtracting the solid and total liquid phase from the initial sample weight. The TPH’s concentration per kilogram was calculated as the sum of the oily and volatile fractions divided by the sample´s initial weight. CFU counts per gram of sample (CFU/g) were calculated by diluting 1 g of soil samples to 10 , 5 6 10 , 10 with distilled water and inoculating 10 microliter (uL) drops in SPC agar for each dilution in 5 6 7 triplicate, final orders of magnitude in each dish were to 10 , 10 , 10 Rstudio v 1.1.383 (RStudio Team 2016) software was used for statistical analysis. Di erences in ammonium concentrations and UFC counts between treatments were analyzed for each sampling day. The Shapiro-Wilcoxon test was used to evaluate the data for normality. None of the data was distributed normally so the Kruskal-Wallis test was performed to determine significant di erences between averages between treatments each day. Statistical analysis results are included in the Supplementary Materials. 2.8. DNA/RNA Extraction and PCR Total DNA was extracted from soil samples and worm tissue using the ZR Soil Microbe DNA MiniPrepTM kit (Catalog No D6001, ZYMO RESEARCH), and the same was conducted for total RNA with the ZR Quick-RNA tissue/insect Miniprep kit (Catalog No R2030, ZYMO RESEARCH). Worm samples were prepared by cleaning the worms’ surface by shaking the worms vigorously inside a falcon with distilled water. The water was changed until it maintained its transparency after shaking (around 3 cleaning cycles). Cleaned worms were ground using a mortar and the product was used for DNA/RNA isolation and plate count. DNA extraction from the five L. sphaericus strains was performed by microwaving a colony inside an eppendorf for one minute and then re-suspending the colony in 100 uL of molecular grade water. The eppendorf was then centrifuged for 30 seconds (s) and supernatant containing DNA was transferred to a new eppendorf avoiding the pellet. Two sets of primers were designed in order to search for the presence and activity of nitrifying bacteria [17]. The first set of primers was designed using L sphaericus genomic annotation where a putative amoA gene is reported [6]. The protein sequence was blasted in order to find conserved regions among L sphaericus populations. Two conserved regions flanking a variable region were selected 0 0 for primer design resulting in primers amoAputF (5 -GCAGGGTGGATTGCTCAAAAG-3 ) and 0 0 amoAputR (5 -GCACAATACTTAAAAAGCCTGTCCT-3 ) with an expected PCR product size of 313 base pairs (bp), This primers aligned 100% with several L. spahericus strains and a Lysinibacillus fusiformis strain´s complete genome. For the second set, 38 di erent amoA sequences from Gram-negative ammonia oxidizing proteobacteria were aligned in order to produce a consensus sequence. Most of the sequence’s conserved regions were selected for primer design using NBCI 0 0 primer blast resulting in primers amoAambF (5 -GGTGACTGGGATTTCTGGCT-3 ) and amoAambR (5´-GGCCAGTTACCCGGATAGAA-3 ) with an expected PCR product size of 350 (bp), this primers AgriEngineering 2020, 2 549 aligned 100% with more than fifty di erent ammonia oxidizing proteobacteria uncultured clones. Both primers were used with the same PCR protocol beginning with 3 minutes (min) of denaturation at 94 C followed by 35 cycles each with 30 s of denaturation at 94 C, annealing at 56 C for 30 s and 1 min of extension at 72 C, this protocol ended with 10 min of extension at 72 C. Main primer blasts for amoAput and amoAamb are included in the Supplementary Materials. The primers used to amplify modB were designed by Angelica Cajamarca from the Center of Microbiological Research, Universidad de los Andes based on the genomic annotation conducted by Gómez-Garzón et al. [7] with an expected PCR product size of 312 bp. The amplification protocol for this gene started with 3 min of denaturation at 94 C followed by 35 cycles each with 30 s of denaturation at 94 C, touchdown annealing from 54 C to 51 C for 30 s and 1 min of extension at 72 C, this protocol ended with 10 min of extension at 72 C. 3. Results and Discussion 3.1. Environmental Conditions and TPH’s Concentration Temperature and humidity readings showed a temperature oscillation between 28 C and 17 C and relative humidity between 50% and 85% during day, and night, respectively. Temperature and humidity changes during day and night promote earthworm mobilization from the bottom to the surface, thus, increasing substrate homogenization and oxygenation and microbial propagation. Optimal temperature for earthworm development is below the optimal temperature for hydrocarbon degradation by bacterial populations [4,26] but this problem may be solved by the propagation of the bacteria and the migration of earthworms to the bottom of the soil when temperature rise up during the day. No significant di erences were found between samples and the average TPH´s concentration was of 112.578 10.560 g/kg for the Pre-treatments and 237.197 22.528 g/kg for crude oil sludge, based on this, initial TPH´s concentration in all the Treatments was calculated at 19.593 1.350 g/kg. After day 25 of the Pretreatment earthworms begun to die ending in 100% mortality after one week, this suggest that earthworms are tolerable to high concentrations of heavy crude oil but once degradation starts and more volatile and toxic compounds begin to appear the worms start to die. No significant earthworm mortality was found during the Treatment, meaning that the application of E. fetida can be an ecient strategy for bioremediation and restauration of crude oil contaminated soils, especially during final stages of remediation [11]. 3.2. Bacterial Growth Aerobic bacteria cultivation based populations increased significantly in treatments with earthworms (Figure 2), and in the case of E and EL, bacterial populations were higher as from 7 7 day 0 (2.3 10 CFU/g and 1.03  10 CFU/g respectively p = 0.02848) thanks to the colonization that occurred during the pretreatment, while in CE, the presence of earthworms increased bacterial populations constantly from day 22 with an average of 2.65 10 CFU/g to day 52 with a final count of 1.45  10 CFU/g. The addition of amended soil also increased bacterial populations in treatments 6 6 without earthworms, C and L began with 1.96  10 CFU/g and 2.136 10 CFU/g on day 0 and 7 7 reached a maximum of 2.9810 CFU/g and 3.3 10 CFU/g respectively on day 36. AgriEngineering 2020, 2 550 AgriEngineering 2020, 3 FOR PEER REVIEW 7 Figure 2. CFU count in soil (Treatment). Figure 2. CFU count in soil (Treatment). All Altr l trea eatments tments wi with th crude crude oil oil seemed to seemed to be be af a f ected ected by the by the appearance appearance of reca of recalcitrant lcitrant compounds compounds resulting in the reduction of bacterial populations in the last days of the study [23]. Comparing E with resulting in the reduction of bacterial populations in the last days of the study [23]. Comparing E with EL, EL, and C w and C ith L show with L shows s that the presen that the presence ce of L. s ofpL. haer sphaericus icus affects bacteria a ects bacterial l populations; this ha populations; this s been has evidenced in previous studies where quorum quenching from L. sphaericus has been reported in oil been evidenced in previous studies where quorum quenching from L. sphaericus has been reported in sludge remediation [27]. On day 22, this difference was more evident with 2.22 × 10 CFU/g and 7.2 oil sludge remediation [27]. On day 22, this di erence was more evident with 2.22 10 CFU/g and 6 7 6 × 10 UFC/g for E, and C, respectively compared to 1.1 × 10 CFU/g and 2.6 ×10 UFC for EL and 7.2 10 UFC/g for E, and C, respectively compared to 1.1 10 CFU/g and 2.6 10 UFC for EL and L respectively (p = 0.01308). L respectively (p = 0.01308). Soil and worm tissue cultures were analyzed under the microscope (Figure included in Soil and worm tissue cultures were analyzed under the microscope (Figure included in Supplementary Materials) revealing that the transit of vegetative cells in the soil through worms’ guts Supplementary Materials) revealing that the transit of vegetative cells in the soil through worms’ guts promotes their sporulation [28]. These phenomena may improve the recycling of bacterial promotes their sporulation [28]. These phenomena may improve the recycling of bacterial populations populations maintaining greater abundance in sludge treated with E. fetida, which could be especially maintaining greater abundance in sludge treated with E. fetida, which could be especially beneficial for beneficial for L. sphaericus populations, as they tend to decay in oil sludge during the last phases of L. sphaericus populations, as they tend to decay in oil sludge during the last phases of remediation. remediation. 3.3. Ammonium Concentrations 3.3. Ammonium Concentrations During the Pretreatment (Figure 3), the presence of earthworms was related to higher ammonium During the Pretreatment (Figure 3), the presence of earthworms was related to higher concentrations on day 41 (p = 0.02162). During the previous sampling days, ammonium concentrations ammonium concentrations on day 41 (p = 0.02162). During the previous sampling days, ammonium seemed to remain stable between treatments, thus, this sudden di erentiation may be related concentrations seemed to remain stable between treatments, thus, this sudden differentiation may be to metabolic inhibition due to high hydrocarbon concentrations. The increment in ammonium related to metabolic inhibition due to high hydrocarbon concentrations. The increment in ammonium concentration during the final days may be evidence of better adaptation capability in degradation concentration during the final days may be evidence of better adaptation capability in degradation systems that include earthworms. systems that include earthworms. AgriEngineering 2020, 2 551 AgriEngineering 2020, 3 FOR PEER REVIEW 8 AgriEngineering 2020, 3 FOR PEER REVIEW 8 Figure 3. Ammonium concentration in soil (Pretreatment). Figure 3. Ammonium concentration in soil (Pretreatment). Figure 3. Ammonium concentration in soil (Pretreatment). Ammonium reached higher concentrations in treatments with E. fetida [29] and L. sphaericus Ammonium reached higher concentrations in treatments with E. fetida [29] and L. sphaericus Ammonium reached higher concentrations in treatments with E. fetida [29] and L. sphaericus (Figure 4), and differences were greater on day 22 (p = 0.0218) with average ammonium (Figure 4), and di erences were greater on day 22 (p = 0.0218) with average ammonium concentrations (Figure 4), and differences were greater on day 22 (p = 0.0218) with average ammonium concentrations of C: 26.3 miligrams per kilogram (mg/kgl), L: 75.0 mg/kgl, E: 152.3 mg/kgl, EL: 75.0 of C: 26.3 miligrams per kilogram (mg/kgl), L: 75.0 mg/kgl, E: 152.3 mg/kgl, EL: 75.0 mg/kgl and CE: concentrations of C: 26.3 miligrams per kilogram (mg/kgl), L: 75.0 mg/kgl, E: 152.3 mg/kgl, EL: 75.0 mg/kgl and CE: 64.33 mg/kgl. Earthworms with L. sphaericus inoculum reached only half of the 64.33 mg/kgl. Earthworms with L. sphaericus inoculum reached only half of the concentration reached mg/kgl and CE: 64.33 mg/kgl. Earthworms with L. sphaericus inoculum reached only half of the concentration reached by earthworms without inoculum. This could be explained by a higher by earthworms concentration without reached by inoculum. earthworms This could without in be explained oculum. Th byisa c higher ould be demand explaineof d by ammonium a higher for demand of ammonium for hydrocarbon catabolism by L. sphaericus and/or by ammonium demand of ammonium for hydrocarbon catabolism by L. sphaericus and/or by ammonium hydrocarbon concentra catabolism tions reachiby ng L. a threshol sphaericus d p and roduci /orn by g a ammonium transition frconcentrations om fixation to ni reaching trification by a threshold L. concentrations reaching a threshold producing a transition from fixation to nitrification by L. producing sphaericus. a transition The latter theory m from fixationato y be nitrification supported b byyL. th sphaericus. e fact thatThe L re latter ached t theory he samay me ma bexsup imuported m sphaericus. The latter theory may be supported by the fact that L reached the same maximum ammonium concentration as EL (75.0 mg/kgl, day 22). Ammonium concentrations in bioremediation by the fact that L reached the same maximum ammonium concentration as EL (75.0 mg/kgl, day 22). ammonium concentration as EL (75.0 mg/kgl, day 22). Ammonium concentrations in bioremediation systems tend to behave cyclically thus, such studies can provide evidence to better understand the Ammonium concentrations in bioremediation systems tend to behave cyclically thus, such studies can systems tend to behave cyclically thus, such studies can provide evidence to better understand the dynamics between nitrogen fixation and nitrifying bacterial populations and how ammonium and provide evidence to better understand the dynamics between nitrogen fixation and nitrifying bacterial dynamics between nitrogen fixation and nitrifying bacterial populations and how ammonium and nitrate concentrations regulate this cycle [29]. populations and how ammonium and nitrate concentrations regulate this cycle [29]. nitrate concentrations regulate this cycle [29]. Figure 4. Ammonium concentration in soil (Treatment). Figure 4. Ammonium concentration in soil (Treatment). Figure 4. Ammonium concentration in soil (Treatment). AgriEngineering 2020, 2 552 3.4. Presence and Expression of AmoA and Molybdenum Transporter Genes AgriEngineering 2020, 3 FOR PEER REVIEW 9 Putative amoA primers were tested with the five strains of L. sphaericus used in this study. 3.4. Presence and Expression of AmoA and Molybdenum Transporter Genes All strains amplified for this set of primers (Figure 5), confirming the genomic annotation calculated by Putative amoA primers were tested with the five strains of L. sphaericus used in this study. All strains amplified for this set of primers (Figure 5), confirming the genomic annotation calculated by Gómez-Garzón, et al. [6]. Gómez-Garzón, et al. [6]. Figure 5. Putative amoA amplification for the L. sphaericus strains used in this study. Figure 5. Putative amoA amplification for the L. sphaericus strains used in this study. No PCR products for amoAamb or amoAput were amplified from the crude oil sludge and the unpolluted field soil indicating that populations of AOB and L. sphaericus were too small or No PCR products for amoAamb or amoAput were amplified from the crude oil sludge and the completely absent, and amoAput presence may also go undetected due to L. sphaericus sporulation unpolluted field soil indicating that populations of AOB and L. sphaericus were too small or completely which impedes cell lysis. Initial DNA extraction and amplification from earthworm tissue from CE resulted in the absent, and amoAput presence may also go undetected due to L. sphaericus sporulation which impedes presence of both genes indicating that the addition of E. fetida increases AOB populations [14] and cell lysis. that some L. sphaericus strains may be associated with vermicomposting. Initial DNA extraction PCR of soil and and worm amplification DNA and RN fr A ext omrearthworm actions on day tissue 52 for am froAam om CE b and resulted amoAput in the presence resulted in positive amplification for all samples (Figure 6). Based on amoAput amplifications from of both genes indicating that the addition of E. fetida increases AOB populations [14] and that some earthworms’ tissue in E and CE, the idea that some strains of L. sphaericus were present previously in L. sphaericus strains may be associated with vermicomposting. the earthworms’ tissue is reinforced. The presence of this gene in the soil for all treatments suggests that these bacteria are not only associated to the earthworms’ tissue but that E. fetida may increase the PCR of soil and worm DNA and RNA extractions on day 52 for amoAamb and amoAput resulted populations of these bacteria in soil resulting in the presence of amoAput in the composted soil added in positive amplification for all samples (Figure 6). Based on amoAput amplifications from earthworms’ to each treatment. Despite nitrate determinations being impossible due to possible rapid assimilation and analytic interference, amoA RNA expression explains reductions in ammonium concentrations tissue in E and CE, the idea that some strains of L. sphaericus were present previously in the earthworms’ in all treatments. tissue is reinforced. The presence of this gene in the soil for all treatments suggests that these bacteria The expression of amoA RNA in worm tissue was unexpected based on the fact that nitrification is an aerobic process; these results suggest that some regions inside worms’ tissue or on the cuticle are not only associated to the earthworms’ tissue but that E. fetida may increase the populations provide enough oxygen for this process to occur [30]. of these bacteria in soil resulting in the presence of amoAput in the composted soil added to each treatment. Despite nitrate determinations being impossible due to possible rapid assimilation and analytic interference, amoA RNA expression explains reductions in ammonium concentrations in AgriEngineering 2020, 3 FOR PEER REVIEW 10 all treatments. (a) (b) Figure 6. (a) Electrophoresis gel for Gram-negative and Putative amoA PCR products from final soil Figure 6. (a) Electrophoresis gel for Gram-negative and Putative amoA PCR products from final soil and worm tissue DNA extraction; (b) Electrophoresis gel for Gram (-) and Putative amoA PCR and worm tissue products from DNA extraction; final soil an (b d )worm tissue R Electrophor NA extract esis gel ion. for Gram (-) and Putative amoA PCR products from final soil and worm tissue RNA extraction. Soil and worm RNA extractions on day 52 amplified for molybdenum transporter in all samples except for soil RNA from E and EL (Figure 7). The expression of molybdenum transporter may indicate nitrogen fixation by L. sphaericus and this would explain increased ammonium concentrations in all treatments [7,8]. Figure 7. Electrophoresis gel for Molybdenum transporter PCR products from final soil and worm tissue RNA extraction. The absence of the molybdenum transporter expression in E and EL may be related to theoxygenation of the soil by worm activity. Nitrogen fixation requires low oxygen concentrations; thus, in this case, the process may be inhibited. Nevertheless, expression was found in Co where the effect should be similar for oxygen concentrations, meaning that the inhibition of molybdenum transporter may also be related to compounds derived from hydrocarbon degradation or the nature of fixation-nitrification dynamics. modBexpression was also found in worm tissue from these treatments (•E and •EL) suggesting that worms’ guts may provide better conditions for nitrogen fixation [31] when soil contamination causes inhibition. 4. Conclusions The biodegradation processes are highly dependent on bacterial population diversity and dynamics. This study demonstrates the ability of E. fetida to improve the conditions for some of these populations resulting in the promotion of microbial numbers. Ammonia dynamics during the study and amoA genes expression suggests that bacteria associated with E. fetida are involved in nutrient cycling through nitrogen fixation and ammonia nitrification, and this activity is better when the earthworms are present than with worm-amended soil without earthworms. The increment of AgriEngineering 2020, 3 FOR PEER REVIEW 10 AgriEngineering 2020, 2 553 (a) (b) Figure 6. (a) Electrophoresis gel for Gram-negative and Putative amoA PCR products from final soil The expression of amoA RNA in worm tissue was unexpected based on the fact that nitrification and worm tissue DNA extraction; (b) Electrophoresis gel for Gram (-) and Putative amoA PCR is an aerobic process; these results suggest that some regions inside worms’ tissue or on the cuticle products from final soil and worm tissue RNA extraction. provide enough oxygen for this process to occur [30]. Soil and worm RNA extractions on day 52 amplified for molybdenum transporter in all samples Soil and worm RNA extractions on day 52 amplified for molybdenum transporter in all samples except for soil RNA from E and EL (Figure 7). The expression of molybdenum transporter may indicate except for soil RNA from E and EL (Figure 7). The expression of molybdenum transporter may nitrogen fixation by L. sphaericus and this would explain increased ammonium concentrations in all indicate nitrogen fixation by L. sphaericus and this would explain increased ammonium treatments [7,8]. concentrations in all treatments [7,8]. Figure 7. Electrophoresis gel for Molybdenum transporter PCR products from final soil and worm Figure 7. Electrophoresis gel for Molybdenum transporter PCR products from final soil and worm tissue RNA extraction. tissue RNA extraction. The absence of the molybdenum transporter expression in E and EL may be related to The absence of the molybdenum transporter expression in E and EL may be related to theoxygenation of the soil by worm activity. Nitrogen fixation requires low oxygen concentrations; theoxygenation of the soil by worm activity. Nitrogen fixation requires low oxygen concentrations; thus, in this case, the process may be inhibited. Nevertheless, expression was found in Co where the thus, in this case, the process may be inhibited. Nevertheless, expression was found in Co where effect should be similar for oxygen concentrations, meaning that the inhibition of molybdenum the e ect should be similar for oxygen concentrations, meaning that the inhibition of molybdenum transporter may also be related to compounds derived from hydrocarbon degradation or the nature transporter may also be related to compounds derived from hydrocarbon degradation or the nature of of fixation-nitrification dynamics. modBexpression was also found in worm tissue from these fixation-nitrification dynamics. modBexpression was also found in worm tissue from these treatments treatments (•E and •EL) suggesting that worms’ guts may provide better conditions for nitrogen (E and EL) suggesting that worms’ guts may provide better conditions for nitrogen fixation [31] fixation [31] when soil contamination causes inhibition. when soil contamination causes inhibition. 4. Conclusions 4. Conclusions The biodegradation processes are highly dependent on bacterial population diversity and The biodegradation processes are highly dependent on bacterial population diversity and dynamics. This study demonstrates the ability of E. fetida to improve the conditions for some of these dynamics. This study demonstrates the ability of E. fetida to improve the conditions for some of populations resulting in the promotion of microbial numbers. Ammonia dynamics during the study these populations resulting in the promotion of microbial numbers. Ammonia dynamics during the and amoA genes expression suggests that bacteria associated with E. fetida are involved in nutrient study and amoA genes expression suggests that bacteria associated with E. fetida are involved in cycling through nitrogen fixation and ammonia nitrification, and this activity is better when the nutrient cycling through nitrogen fixation and ammonia nitrification, and this activity is better when earthworms are present than with worm-amended soil without earthworms. The increment of the earthworms are present than with worm-amended soil without earthworms. The increment of nitrogen in bioremediation systems may be highly beneficial given the reduction of the C/N ratio, which may improve bacterial growth and activity [32]. Increased ammonium concentration may be beneficial for the biodegradation of volatile hydrocarbon compounds given the increased cometabolism by ammonia monooxygenase [21,23,24]. The expression of genes related with nitrogen fixation (modB) and ammonium nitrification (amoAput) in oil sludge remediation processes was demonstrated for L. sphaericus, indicating that this species has the ability to degrade hydrocarbons and, at the same time, may improve nitrogen levels in oil sludge bioremediation. Supplementary Materials: The following are available online at http://www.mdpi.com/2624-7402/2/4/36/s1, Table S1: Pretreatment data, Table S2: Treatment data, Annex S3: Ammonium concentration Pretreatment Statistical Analysis, Annex S4: Ammonium concentration Treatment Statistical Analysis, Annex S5: CFU Treatment Statistical Analysis, Annex S6: Main blast results for amoAamb primers, Annex S7: Main blast results for amoAput primers, Figure S8: Lysinibacillus sphaericus culture from soil and worm tissue samples. AgriEngineering 2020, 2 554 Author Contributions: Conceptualization, J.D.A. and J.D.; methodology, J.D.A. and J.D.; software, J.D.A.; validation, J.D.A. and J.D.; formal analysis, J.D.A.; investigation, J.D.A; resources, J.D.A. and J.D.; data curation, J.D.A.; writing—original draft preparation, J.D.A.; writing—review and editing, J.D.A. and J.D.; visualization, J.D.A.; supervision, J.D.; project administration, J.D.; funding acquisition, J.D. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Research Fund INV-2019-84-1827 at the Science Faculty at Universidad de los Andes and the Microbiological Research Center (CIMIC). Acknowledgments: We are thankful to Biointech for crude oil sludge samples and RETORT analysis. Conflicts of Interest: The authors declare no conflict of interest. References 1. Finer, M.; Jenkins, C.N.; Pimm, S.L.; Keane, B.; Ross, C. 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Goldman, J.C.; Caron, D.A.; Dennett, M.R. Regulation of gross growth eciency and ammonium regeneration in bacteria by substrate C: N ratio. Limnol. Oceanogr. 1987, 32, 1239–1252. [CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AgriEngineering Multidisciplinary Digital Publishing Institute

Ammonium Cycling and Nitrification Stimulation during Oil Sludge Remediation by Gram-Positive Bacteria Lysinibacillus sphaericus Using Red Wiggler Earthworm Eisenia fetida

Acevedo, Juan Diego; Dussán, Jenny
AgriEngineering , Volume 2 (4) – Nov 13, 2020
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2624-7402
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AgriEngineering Article Ammonium Cycling and Nitrification Stimulation during Oil Sludge Remediation by Gram-Positive Bacteria Lysinibacillus sphaericus Using Red Wiggler Earthworm Eisenia fetida 1 1 , Juan Diego Acevedo and Jenny Dussán * Centro de Investigaciones Microbiológicas—CIMIC, Departamento de Ciencias Biológicas, Universidad de los Andes, Carrera 1 No. 18A-10 J-206, Bogotá PC 111711, Colombia; jd.acevedo245@uniandes.edu.co * Correspondence: jdussan@uniandes.edu.co; Tel.: +57-1-3394999 (ext. 3644) Received: 5 October 2020; Accepted: 3 November 2020; Published: 13 November 2020 Abstract: The performance of a mixture between L. sphaericus and E. fetida was evaluated for ammonium cycling and nitrifying bacteria stimulation during oil sludge remediation. The addition of E. fetida significantly increased ammonium concentration (p = 0.0218) and total Colony-forming units (CFU) count p = 0.02848). However, oil sludge with worms and L. sphaericus reached lower ammonium concentrations and CFU counts than sludge with worms alone. Sludge inoculated only with L. sphaericus presented higher ammonium concentration than sludge without inoculum, but the bacterial population reached a lower density during the final days. Final DNA and RNA extractions from all treatments amplified for L. sphaericus putative amoA and Gram-negative nitrifying bacteria amoA genes correlated with diminished ammonium concentrations during the final days of the experiment. Final RNA extractions for L. sphaericus amplified for Molybdenum transporter gene suggesting possible nitrogen fixation by L. sphaericus. The addition of Red Wiggler Earthworm to oil sludge remediation systems may provide better conditions for bacterial populations to carry out hydrocarbon degradation. The addition of E. fetida to a L. sphaericus crude oil biodegradation system may improve soil ammonium concentrations and nitrifying activity, and this could be crucial in oil sludge remediation because of bacterial inhibition due to high C:N ratios. The final product of this process may be used for soil enhancement due to its richness in nutrients and beneficial bacterial populations. Keywords: Lysinibacillus sphaericus; Eisenia fetida; bioremediation; crude oil sludge; ammonium; nitrification; amoA; molybdenum transporter 1. Introduction Crude oil extraction, transportation and refinement produces residual sludge composed by hydrocarbons, paran, produced water, sediments, and other possible contaminants such as heavy metals, sulfuric compounds and chemicals added during the operation [1]. The complexity of this mixture makes it very dicult and expensive to remediate, making crude oil sludge a persistent contaminant. Bioprospecting microbial communities found in this kind of sludge led to the discovery of di erent bacterial consortiums with the ability to break apart these compounds into less toxic molecules. Based on these findings, di erent crude oil bioremediation techniques have been developed resulting in a system that is cheaper, more ecient, and more sustainable compared to other remediation practices such as chemical degradation or incineration [2,3]. AgriEngineering 2020, 2, 544–555; doi:10.3390/agriengineering2040036 www.mdpi.com/journal/agriengineering AgriEngineering 2020, 2 545 Previous bioprospecting of native Colombian bacteria for crude oil degradation has led to the current use of di erent strains of Lysinibacillus sphaericus in remediation processes for oil companies around the country [4]. The strains used in these treatments are non-pathogenic and present high degradation rates for di erent oil hydrocarbons achieving significant results in the environmental recovery of contaminated soils, with removal eciencies up to 95% for C10 to C28 hydrocarbons in diesel oil assays and up to 84.1% and 60.1% for oil sludge in open air and closed arrays, respectively [5]. L. sphaericus is also involved in nutrient cycling. Genomic annotation has revealed the presence of several genes involved in the nitrogen cycle including Molybdenum transport system permease protein encoding gene (modB) needed to assemble Nif enzyme, nifU involved in the formation of Fe-S clusters for nitrogen fixation, Ammonium transporter and putative ammonia monooxygenase responsible for the first step in nitrification [6]. These abilities have also been proven by ammonium and nitrite quantification during in-vivo studies focused on nitrogen cycling [7,8]. Microbial remediation of crude oil sludge may be enhanced using earthworms [9,10]. Several studies have proven that adding Red Wiggler Worm Eisenia fetida to contaminated soils improves degradation rates of both, heavy [11] and light hydrocarbons [12]. Red Wiggler Worms also have the capacity to improve nutrient cycling significantly, raising ammonium and nitrite concentrations in soil [13] and bacterial populations related with nutrient cycling [14]. These abilities have been verified by gut bacterial genomic annotations that show abundance in genes related to aromatic hydrocarbon degradation, dehalogenation, nitrogen fixation, and ammonium oxidation [15]. E. fetida is commonly used in composting processes given its high degree of adaptability, resistance, and high reproductive rates, as well as its vast commercial availability worldwide [16]. Also, it has been widely used in remediation studies showing promising results for soil structure improvement, promotion of microbial numbers and hydrocarbon degraders and increased microbial respiration resulting in total petroleum hydrocarbons (TPH’s) degradation [17]. Several studies propose that nitrification is dominated by archaea or beta and gamma proteobacteria commonly classified as AOA (ammonia oxidizing archaea) and AOB (ammonia oxidizing bacteria). In the first stage of nitrification, ammonium is reduced to hydroxylamine by the ammonia monooxygenase enzyme (AMO), in AOB this enzyme is encoded by the genomic amoCAB operon [18,19]. The amoA gene of this operon is highly conserved, and thus, very useful for environmental studies in which nitrifying activity is evaluated [19]. Some Gram-positive bacteria have genes homologous to amoCAB, as is the case for L. sphaericus, where a putative amoA gene is present [6]. These findings suggest that bacteria other than beta and gamma proteobacteria may be involved in nitrification processes with significant e ects on ammonia oxidation rates and nitrite concentrations in soil [20]. Ammonia monooxygenase is also capable of degrading hydrocarbons via cometabolism, whereby electrons produced by ammonium oxidation are reused by AMO for hydrocarbon oxidation. Due to its wide range of substrates, this enzyme is capable of degrading alkenes, alkanes, and halogenated and aromatic hydrocarbons. Cometabolism of hydrocarbons by AMO is a complex phenomenon that generally causes AMO activity inhibition, and hydrocarbon byproducts may a ect cell physiology [21–23]. Cometabolism has been proved to be an important process in the degradation of xenobiotic hydrocarbons [24]. The objective of this study is to determinate ammonium cycling and nitrification stimulation during the biodegradation of petroleum hydrocarbons by L. sphaericus using E. fetida by monitoring bacterial populations, ammonium concentrations, and the expression of amoA and modB. 2. Materials and Methods 2.1. Summary This experiment was divided into two stages: Pretreatment and Treatment. During the Pretreatment the biological degradation was initiated at high crude oil concentrations and monitored by performing AgriEngineering 2020, 3 FOR PEER REVIEW 3 2. Materials and Methods 2.1. Summary AgriEngineering 2020, 2 546 This experiment was divided into two stages: Pretreatment and Treatment. During the Pretreatment the biological degradation was initiated at high crude oil concentrations and monitored by performing ammonium determinations, once ammonimun concentrations begun to raise the ammonium determinations, once ammonimun concentrations begun to raise the pretreatments were pretreatments were dilluted to permisibile TPH’s levels in order to inititate the Treatment, during dilluted to permisibile TPH’s levels in order to inititate the Treatment, during this stage the treatments were this stage the treatments were monitored for ammonimum concentration, vegetative cell count, and monitored for ammonimum concentration, vegetative cell count, and amoA and ModB gene presence amoA and ModB gene presence and expression. A brief summary of the methodology is presented and expression. A brief summary of the methodology is presented in Figure 1. in Figure 1. Figure 1. Methodology summary. Figure 1. Methodology summary. 2.2. Materials 2.2. Materials Heavy crude oil sludge and unpolluted soil samples were obtained from Biointech, a Colombian Heavy crude oil sludge and unpolluted soil samples were obtained from Biointech, a Colombian company dedicated to soil and water treatment for environmental recovery. The samples were collected company dedicated to soil and water treatment for environmental recovery. The samples were collected at Caño Limón, Arauca (N 07°05′25″–W 70°45′42″). 0  0 at Caño Limón, Arauca (N 07 05 25”–W 70 45 42”). Earthworm castings were bought from an E. fetida breeder and compost producer. The worms Earthworm castings were bought from an E. fetida breeder and compost producer. The worms were grown in a substrate made up of livestock manure, agricultural residue and vegetable residue were grown in a substrate made up of livestock manure, agricultural residue and vegetable residue from country markets. from country markets. The selection of the L. sphaericus strains used in this study was based on previous research carried The selection of the L. sphaericus strains used in this study was based on previous research out by the Center of Microbiological Research (CIMIC) at Universidad de los Andes (Bogotá, Colombia) [4]. Microbial bioaugmentation was performed using five strains of L. sphaericus: CBAM5, carried out by the Center of Microbiological Research (CIMIC) at Universidad de los Andes (Bogotá, OT4b.31, OT4b.49, 3(III)7 and 2362. Lab trials have shown great degradation results and survival Colombia) [4]. Microbial bioaugmentation was performed using five strains of L. sphaericus: CBAM5, rates at a concentration of 5% volume to volume ratio (v/v) of diesel [5]. This consortium is actually OT4b.31, OT4b.49, 3(III)7 and 2362. Lab trials have shown great degradation results and survival rates used by Biointech in soil remediation processes for the Colombian oil industry resulting in TPH´s at a concentration of 5% volume to volume ratio (v/v) of diesel [5]. This consortium is actually used by degradation to permissible levels in periods of time that span from 45 to 60 days under field Biointech in soil remediation processes for the Colombian oil industry resulting in TPH´s degradation conditions (Caño Limon, Arauca). to permissible levels in periods of time that span from 45 to 60 days under field conditions (Caño All the containers used for the experiment had holes in the bottom for drainage and were Limon, Arauca). covered with black plastic. The experiment was performed inside a 2 meters (m) wide, 1.2 m long All the containers used for the experiment had holes in the bottom for drainage and were covered and 0.8m tall light insulated incubator made with white carboard walls on the outside and black withplastic in black plastic. the inside. The experiment was performed inside a 2 meters (m) wide, 1.2 m long and 0.8m tall light insulated incubator made with white carboard walls on the outside and black plastic in the inside. 2.3. Composted Soil, Worm Castings and Bacteria Growth 2.3. Composted Soil, Worm Castings and Bacteria Growth One month before starting the experiment, a 20 liter (L) plastic container was filled with 2 kilograms (kg) of worm casting mixed with 8 kg of unpolluted soil collected from the oilfield and 2 kg One month before starting the experiment, a 20 liter (L) plastic container was filled with of orange peels. The container was covered with black plastic and kept inside a greenhouse. All the 2 kilograms (kg) of worm casting mixed with 8 kg of unpolluted soil collected from the oilfield and 2 kg of orange peels. The container was covered with black plastic and kept inside a greenhouse. All the worms and amended soil used in this study were extracted from this container that will be referred to as (E0). L. sphaericus strains’ liquid cultures were prepared by growing 1 10 Colony-former units (CFU) of each strain in 20 milliliters (mL) of nutrient broth for 24 hours (h) at 30 C and 150 revolutions per minute (rpm), resulting in a final concentration of 1 10 CFU/mL. For the inoculation of soil samples, two trays were filled with 550 mL of SPC agar each. Using five sterile brushes (one for each strain), the liquid cultures at a concentration of 1  10 CFU/mL were AgriEngineering 2020, 2 547 evenly distributed along the surface of the agar occupying 300 square centimeters (cm ) per strain in each tray. Both trays were placed in an incubator for 24 h. On the day of inoculation, bacteria that had grown on the surface of the agar were collected using a spatula, resulting in 90 mL of solution with a concentration of 1 10 CFU/mL of the consortium. 2.4. Pretreatment Before starting the experiment, a Pretreatment was prepared in order to initiate the biological degradation system before diluting the samples to TPH´s concentrations tolerable for earthworms. A Pre-treatment master mix was prepared using a 1:1 weight to weight ratio (w/w) of crude oil sludge and unpolluted soil. Two days later, this mixture was divided into two equal bundles 2.4 kg each. One of the bundles was mixed with 480 grams (g) of SPC agar without inoculum and the other bundle was mixed with the same amount of agar and 90 mL of L. sphaericus consortium inoculum at a concentration of 1 10 CFU/mL. Each bundle was divided in half again resulting in four 1.77 kg bundles that were stored in 20-L plastic containers. Five days later, 30 g of E. fetida were added to one of the bundles without L. sphaericus inoculum and one with inoculum, this bundles corresponded to the final Treatments to be evaluated: E. feitda alone (E), L. sphaericus alone (L), E. fetida with L. sphaericus consortium (EL), and a Control (C) without worms or inoculum, this day was considered as day 0 of the pre-treatment. The pre-treatment was evaluated for a period of 41 days before starting the Treatment. 2.5. Treatment A Treatment master mix was prepared mixing 0.608 kg (4.4%) of crude oil sludge, 14.364 kg (53.2%) of unpolluted soil, and 11.394 kg (42.2%) of worm amended soil from the E0 container, and stored for stabilization for two days inside a 40 L plastic container covered with black plastic. On day 0 of the Treatment (day 41 for the Pretreatment), the Treatment master mix was divided into two equal bundles, one of the bundles was inoculated with 90 mL of consortium at a concentration of 1 10 CFU/mL and then both bundles were divided again into two equal parts resulting in four 20 L plastic containers filled with 6750 g of master mix each, two inoculated with L. sphaericus and two free of inoculum. After this, E and C pretreatments were diluted to 8.89% (w/w) each using the two Treatment master mix bundles that were not inoculated, and the same was done for L and EL using the two inoculated bundles. Following this, 250 g of worms were added to EL and E each resulting in the three final treatments and a control: E. feitda alone (E), L. sphaericus alone (L), E. fetida with L. sphaericus consortium (EL), and (C) without worms or inoculum. At this moment an additional control (CE) composed of 57.7% unpolluted soil and 42.3% amended soil with the same quantity of earthworms as Lo and LL was added to the experiment. The amended soil was added to all treatments and controls in order to elucidate the di erence between the e ects of adding amended soil only and adding the earthworms. The Treatment was evaluated for a period of 52 days. 2.6. Sampling and Analysis During the Treatment temperature and humidity were measured and recorded inside the incubator using an Arduino. TPH´s were determined on the Pre-treatments and oil sludge two days prior to the beginning of the Treatment in order to calculate the dilution needed to achieve, at least, a 50% earthworm survival rate. The 50% lethal dose was approximated at 20 g/kg TPH´s based on the toxicity trials made by Martinkosky et al. [11]. DNA was extracted from the earthworms’ tissue at the moment of purchase (E0). The day previous to the Treatment’s master mix preparation, DNA was extracted from crude oil sludge and unpolluted field soil. On day 52, DNA and RNA were extracted from all Treatments including worm tissue (E,EL,CE). Ammonium determinations and CFU counts were performed on days 0, 9, 22, 36 and 52. Ammonium determinations were also performed during the Pretreatment on days 0, 5, 20 and 41 [12]. Samples for ammonium and hydrocarbon determinations and CFU counts were taken AgriEngineering 2020, 2 548 in triplicate using compound sampling; soil samples for DNA and RNA extractions were taken in triplicate for each treatment and then combined in one compound sample for analysis. For worm samples, 1.5 g of worms were processed for each Treatment. 2.7. Ammonium and Hydrocarbon Determination and Microbial Count Ammonium concentrations were determined using Merck ammonium test kit catalog no. 114,752. One gram per soil sample was diluted by two orders of magnitude with distilled water prior to analysis. Hydrocarbon determinations were performed using an Oil and Water Retort, 10 g from each sample was processed following fabricator´s recommendations resulting in the separation of a liquid phased composed of oil and water and a solid phase [25]. The liquid phase, contained in the equipment´s test tube, was weighed using an analytical balance. Then, the oily fraction from the liquid phase was extracted from the test tube using a pipette and the tube was weighed again, this amount was subtracted from the initial weight of the liquid phase in order to calculate the concentration of the oily fraction. The solid phase was also weighed using an analytical balance, and the volatile fraction was calculated subtracting the solid and total liquid phase from the initial sample weight. The TPH’s concentration per kilogram was calculated as the sum of the oily and volatile fractions divided by the sample´s initial weight. CFU counts per gram of sample (CFU/g) were calculated by diluting 1 g of soil samples to 10 , 5 6 10 , 10 with distilled water and inoculating 10 microliter (uL) drops in SPC agar for each dilution in 5 6 7 triplicate, final orders of magnitude in each dish were to 10 , 10 , 10 Rstudio v 1.1.383 (RStudio Team 2016) software was used for statistical analysis. Di erences in ammonium concentrations and UFC counts between treatments were analyzed for each sampling day. The Shapiro-Wilcoxon test was used to evaluate the data for normality. None of the data was distributed normally so the Kruskal-Wallis test was performed to determine significant di erences between averages between treatments each day. Statistical analysis results are included in the Supplementary Materials. 2.8. DNA/RNA Extraction and PCR Total DNA was extracted from soil samples and worm tissue using the ZR Soil Microbe DNA MiniPrepTM kit (Catalog No D6001, ZYMO RESEARCH), and the same was conducted for total RNA with the ZR Quick-RNA tissue/insect Miniprep kit (Catalog No R2030, ZYMO RESEARCH). Worm samples were prepared by cleaning the worms’ surface by shaking the worms vigorously inside a falcon with distilled water. The water was changed until it maintained its transparency after shaking (around 3 cleaning cycles). Cleaned worms were ground using a mortar and the product was used for DNA/RNA isolation and plate count. DNA extraction from the five L. sphaericus strains was performed by microwaving a colony inside an eppendorf for one minute and then re-suspending the colony in 100 uL of molecular grade water. The eppendorf was then centrifuged for 30 seconds (s) and supernatant containing DNA was transferred to a new eppendorf avoiding the pellet. Two sets of primers were designed in order to search for the presence and activity of nitrifying bacteria [17]. The first set of primers was designed using L sphaericus genomic annotation where a putative amoA gene is reported [6]. The protein sequence was blasted in order to find conserved regions among L sphaericus populations. Two conserved regions flanking a variable region were selected 0 0 for primer design resulting in primers amoAputF (5 -GCAGGGTGGATTGCTCAAAAG-3 ) and 0 0 amoAputR (5 -GCACAATACTTAAAAAGCCTGTCCT-3 ) with an expected PCR product size of 313 base pairs (bp), This primers aligned 100% with several L. spahericus strains and a Lysinibacillus fusiformis strain´s complete genome. For the second set, 38 di erent amoA sequences from Gram-negative ammonia oxidizing proteobacteria were aligned in order to produce a consensus sequence. Most of the sequence’s conserved regions were selected for primer design using NBCI 0 0 primer blast resulting in primers amoAambF (5 -GGTGACTGGGATTTCTGGCT-3 ) and amoAambR (5´-GGCCAGTTACCCGGATAGAA-3 ) with an expected PCR product size of 350 (bp), this primers AgriEngineering 2020, 2 549 aligned 100% with more than fifty di erent ammonia oxidizing proteobacteria uncultured clones. Both primers were used with the same PCR protocol beginning with 3 minutes (min) of denaturation at 94 C followed by 35 cycles each with 30 s of denaturation at 94 C, annealing at 56 C for 30 s and 1 min of extension at 72 C, this protocol ended with 10 min of extension at 72 C. Main primer blasts for amoAput and amoAamb are included in the Supplementary Materials. The primers used to amplify modB were designed by Angelica Cajamarca from the Center of Microbiological Research, Universidad de los Andes based on the genomic annotation conducted by Gómez-Garzón et al. [7] with an expected PCR product size of 312 bp. The amplification protocol for this gene started with 3 min of denaturation at 94 C followed by 35 cycles each with 30 s of denaturation at 94 C, touchdown annealing from 54 C to 51 C for 30 s and 1 min of extension at 72 C, this protocol ended with 10 min of extension at 72 C. 3. Results and Discussion 3.1. Environmental Conditions and TPH’s Concentration Temperature and humidity readings showed a temperature oscillation between 28 C and 17 C and relative humidity between 50% and 85% during day, and night, respectively. Temperature and humidity changes during day and night promote earthworm mobilization from the bottom to the surface, thus, increasing substrate homogenization and oxygenation and microbial propagation. Optimal temperature for earthworm development is below the optimal temperature for hydrocarbon degradation by bacterial populations [4,26] but this problem may be solved by the propagation of the bacteria and the migration of earthworms to the bottom of the soil when temperature rise up during the day. No significant di erences were found between samples and the average TPH´s concentration was of 112.578 10.560 g/kg for the Pre-treatments and 237.197 22.528 g/kg for crude oil sludge, based on this, initial TPH´s concentration in all the Treatments was calculated at 19.593 1.350 g/kg. After day 25 of the Pretreatment earthworms begun to die ending in 100% mortality after one week, this suggest that earthworms are tolerable to high concentrations of heavy crude oil but once degradation starts and more volatile and toxic compounds begin to appear the worms start to die. No significant earthworm mortality was found during the Treatment, meaning that the application of E. fetida can be an ecient strategy for bioremediation and restauration of crude oil contaminated soils, especially during final stages of remediation [11]. 3.2. Bacterial Growth Aerobic bacteria cultivation based populations increased significantly in treatments with earthworms (Figure 2), and in the case of E and EL, bacterial populations were higher as from 7 7 day 0 (2.3 10 CFU/g and 1.03  10 CFU/g respectively p = 0.02848) thanks to the colonization that occurred during the pretreatment, while in CE, the presence of earthworms increased bacterial populations constantly from day 22 with an average of 2.65 10 CFU/g to day 52 with a final count of 1.45  10 CFU/g. The addition of amended soil also increased bacterial populations in treatments 6 6 without earthworms, C and L began with 1.96  10 CFU/g and 2.136 10 CFU/g on day 0 and 7 7 reached a maximum of 2.9810 CFU/g and 3.3 10 CFU/g respectively on day 36. AgriEngineering 2020, 2 550 AgriEngineering 2020, 3 FOR PEER REVIEW 7 Figure 2. CFU count in soil (Treatment). Figure 2. CFU count in soil (Treatment). All Altr l trea eatments tments wi with th crude crude oil oil seemed to seemed to be be af a f ected ected by the by the appearance appearance of reca of recalcitrant lcitrant compounds compounds resulting in the reduction of bacterial populations in the last days of the study [23]. Comparing E with resulting in the reduction of bacterial populations in the last days of the study [23]. Comparing E with EL, EL, and C w and C ith L show with L shows s that the presen that the presence ce of L. s ofpL. haer sphaericus icus affects bacteria a ects bacterial l populations; this ha populations; this s been has evidenced in previous studies where quorum quenching from L. sphaericus has been reported in oil been evidenced in previous studies where quorum quenching from L. sphaericus has been reported in sludge remediation [27]. On day 22, this difference was more evident with 2.22 × 10 CFU/g and 7.2 oil sludge remediation [27]. On day 22, this di erence was more evident with 2.22 10 CFU/g and 6 7 6 × 10 UFC/g for E, and C, respectively compared to 1.1 × 10 CFU/g and 2.6 ×10 UFC for EL and 7.2 10 UFC/g for E, and C, respectively compared to 1.1 10 CFU/g and 2.6 10 UFC for EL and L respectively (p = 0.01308). L respectively (p = 0.01308). Soil and worm tissue cultures were analyzed under the microscope (Figure included in Soil and worm tissue cultures were analyzed under the microscope (Figure included in Supplementary Materials) revealing that the transit of vegetative cells in the soil through worms’ guts Supplementary Materials) revealing that the transit of vegetative cells in the soil through worms’ guts promotes their sporulation [28]. These phenomena may improve the recycling of bacterial promotes their sporulation [28]. These phenomena may improve the recycling of bacterial populations populations maintaining greater abundance in sludge treated with E. fetida, which could be especially maintaining greater abundance in sludge treated with E. fetida, which could be especially beneficial for beneficial for L. sphaericus populations, as they tend to decay in oil sludge during the last phases of L. sphaericus populations, as they tend to decay in oil sludge during the last phases of remediation. remediation. 3.3. Ammonium Concentrations 3.3. Ammonium Concentrations During the Pretreatment (Figure 3), the presence of earthworms was related to higher ammonium During the Pretreatment (Figure 3), the presence of earthworms was related to higher concentrations on day 41 (p = 0.02162). During the previous sampling days, ammonium concentrations ammonium concentrations on day 41 (p = 0.02162). During the previous sampling days, ammonium seemed to remain stable between treatments, thus, this sudden di erentiation may be related concentrations seemed to remain stable between treatments, thus, this sudden differentiation may be to metabolic inhibition due to high hydrocarbon concentrations. The increment in ammonium related to metabolic inhibition due to high hydrocarbon concentrations. The increment in ammonium concentration during the final days may be evidence of better adaptation capability in degradation concentration during the final days may be evidence of better adaptation capability in degradation systems that include earthworms. systems that include earthworms. AgriEngineering 2020, 2 551 AgriEngineering 2020, 3 FOR PEER REVIEW 8 AgriEngineering 2020, 3 FOR PEER REVIEW 8 Figure 3. Ammonium concentration in soil (Pretreatment). Figure 3. Ammonium concentration in soil (Pretreatment). Figure 3. Ammonium concentration in soil (Pretreatment). Ammonium reached higher concentrations in treatments with E. fetida [29] and L. sphaericus Ammonium reached higher concentrations in treatments with E. fetida [29] and L. sphaericus Ammonium reached higher concentrations in treatments with E. fetida [29] and L. sphaericus (Figure 4), and differences were greater on day 22 (p = 0.0218) with average ammonium (Figure 4), and di erences were greater on day 22 (p = 0.0218) with average ammonium concentrations (Figure 4), and differences were greater on day 22 (p = 0.0218) with average ammonium concentrations of C: 26.3 miligrams per kilogram (mg/kgl), L: 75.0 mg/kgl, E: 152.3 mg/kgl, EL: 75.0 of C: 26.3 miligrams per kilogram (mg/kgl), L: 75.0 mg/kgl, E: 152.3 mg/kgl, EL: 75.0 mg/kgl and CE: concentrations of C: 26.3 miligrams per kilogram (mg/kgl), L: 75.0 mg/kgl, E: 152.3 mg/kgl, EL: 75.0 mg/kgl and CE: 64.33 mg/kgl. Earthworms with L. sphaericus inoculum reached only half of the 64.33 mg/kgl. Earthworms with L. sphaericus inoculum reached only half of the concentration reached mg/kgl and CE: 64.33 mg/kgl. Earthworms with L. sphaericus inoculum reached only half of the concentration reached by earthworms without inoculum. This could be explained by a higher by earthworms concentration without reached by inoculum. earthworms This could without in be explained oculum. Th byisa c higher ould be demand explaineof d by ammonium a higher for demand of ammonium for hydrocarbon catabolism by L. sphaericus and/or by ammonium demand of ammonium for hydrocarbon catabolism by L. sphaericus and/or by ammonium hydrocarbon concentra catabolism tions reachiby ng L. a threshol sphaericus d p and roduci /orn by g a ammonium transition frconcentrations om fixation to ni reaching trification by a threshold L. concentrations reaching a threshold producing a transition from fixation to nitrification by L. producing sphaericus. a transition The latter theory m from fixationato y be nitrification supported b byyL. th sphaericus. e fact thatThe L re latter ached t theory he samay me ma bexsup imuported m sphaericus. The latter theory may be supported by the fact that L reached the same maximum ammonium concentration as EL (75.0 mg/kgl, day 22). Ammonium concentrations in bioremediation by the fact that L reached the same maximum ammonium concentration as EL (75.0 mg/kgl, day 22). ammonium concentration as EL (75.0 mg/kgl, day 22). Ammonium concentrations in bioremediation systems tend to behave cyclically thus, such studies can provide evidence to better understand the Ammonium concentrations in bioremediation systems tend to behave cyclically thus, such studies can systems tend to behave cyclically thus, such studies can provide evidence to better understand the dynamics between nitrogen fixation and nitrifying bacterial populations and how ammonium and provide evidence to better understand the dynamics between nitrogen fixation and nitrifying bacterial dynamics between nitrogen fixation and nitrifying bacterial populations and how ammonium and nitrate concentrations regulate this cycle [29]. populations and how ammonium and nitrate concentrations regulate this cycle [29]. nitrate concentrations regulate this cycle [29]. Figure 4. Ammonium concentration in soil (Treatment). Figure 4. Ammonium concentration in soil (Treatment). Figure 4. Ammonium concentration in soil (Treatment). AgriEngineering 2020, 2 552 3.4. Presence and Expression of AmoA and Molybdenum Transporter Genes AgriEngineering 2020, 3 FOR PEER REVIEW 9 Putative amoA primers were tested with the five strains of L. sphaericus used in this study. 3.4. Presence and Expression of AmoA and Molybdenum Transporter Genes All strains amplified for this set of primers (Figure 5), confirming the genomic annotation calculated by Putative amoA primers were tested with the five strains of L. sphaericus used in this study. All strains amplified for this set of primers (Figure 5), confirming the genomic annotation calculated by Gómez-Garzón, et al. [6]. Gómez-Garzón, et al. [6]. Figure 5. Putative amoA amplification for the L. sphaericus strains used in this study. Figure 5. Putative amoA amplification for the L. sphaericus strains used in this study. No PCR products for amoAamb or amoAput were amplified from the crude oil sludge and the unpolluted field soil indicating that populations of AOB and L. sphaericus were too small or No PCR products for amoAamb or amoAput were amplified from the crude oil sludge and the completely absent, and amoAput presence may also go undetected due to L. sphaericus sporulation unpolluted field soil indicating that populations of AOB and L. sphaericus were too small or completely which impedes cell lysis. Initial DNA extraction and amplification from earthworm tissue from CE resulted in the absent, and amoAput presence may also go undetected due to L. sphaericus sporulation which impedes presence of both genes indicating that the addition of E. fetida increases AOB populations [14] and cell lysis. that some L. sphaericus strains may be associated with vermicomposting. Initial DNA extraction PCR of soil and and worm amplification DNA and RN fr A ext omrearthworm actions on day tissue 52 for am froAam om CE b and resulted amoAput in the presence resulted in positive amplification for all samples (Figure 6). Based on amoAput amplifications from of both genes indicating that the addition of E. fetida increases AOB populations [14] and that some earthworms’ tissue in E and CE, the idea that some strains of L. sphaericus were present previously in L. sphaericus strains may be associated with vermicomposting. the earthworms’ tissue is reinforced. The presence of this gene in the soil for all treatments suggests that these bacteria are not only associated to the earthworms’ tissue but that E. fetida may increase the PCR of soil and worm DNA and RNA extractions on day 52 for amoAamb and amoAput resulted populations of these bacteria in soil resulting in the presence of amoAput in the composted soil added in positive amplification for all samples (Figure 6). Based on amoAput amplifications from earthworms’ to each treatment. Despite nitrate determinations being impossible due to possible rapid assimilation and analytic interference, amoA RNA expression explains reductions in ammonium concentrations tissue in E and CE, the idea that some strains of L. sphaericus were present previously in the earthworms’ in all treatments. tissue is reinforced. The presence of this gene in the soil for all treatments suggests that these bacteria The expression of amoA RNA in worm tissue was unexpected based on the fact that nitrification is an aerobic process; these results suggest that some regions inside worms’ tissue or on the cuticle are not only associated to the earthworms’ tissue but that E. fetida may increase the populations provide enough oxygen for this process to occur [30]. of these bacteria in soil resulting in the presence of amoAput in the composted soil added to each treatment. Despite nitrate determinations being impossible due to possible rapid assimilation and analytic interference, amoA RNA expression explains reductions in ammonium concentrations in AgriEngineering 2020, 3 FOR PEER REVIEW 10 all treatments. (a) (b) Figure 6. (a) Electrophoresis gel for Gram-negative and Putative amoA PCR products from final soil Figure 6. (a) Electrophoresis gel for Gram-negative and Putative amoA PCR products from final soil and worm tissue DNA extraction; (b) Electrophoresis gel for Gram (-) and Putative amoA PCR and worm tissue products from DNA extraction; final soil an (b d )worm tissue R Electrophor NA extract esis gel ion. for Gram (-) and Putative amoA PCR products from final soil and worm tissue RNA extraction. Soil and worm RNA extractions on day 52 amplified for molybdenum transporter in all samples except for soil RNA from E and EL (Figure 7). The expression of molybdenum transporter may indicate nitrogen fixation by L. sphaericus and this would explain increased ammonium concentrations in all treatments [7,8]. Figure 7. Electrophoresis gel for Molybdenum transporter PCR products from final soil and worm tissue RNA extraction. The absence of the molybdenum transporter expression in E and EL may be related to theoxygenation of the soil by worm activity. Nitrogen fixation requires low oxygen concentrations; thus, in this case, the process may be inhibited. Nevertheless, expression was found in Co where the effect should be similar for oxygen concentrations, meaning that the inhibition of molybdenum transporter may also be related to compounds derived from hydrocarbon degradation or the nature of fixation-nitrification dynamics. modBexpression was also found in worm tissue from these treatments (•E and •EL) suggesting that worms’ guts may provide better conditions for nitrogen fixation [31] when soil contamination causes inhibition. 4. Conclusions The biodegradation processes are highly dependent on bacterial population diversity and dynamics. This study demonstrates the ability of E. fetida to improve the conditions for some of these populations resulting in the promotion of microbial numbers. Ammonia dynamics during the study and amoA genes expression suggests that bacteria associated with E. fetida are involved in nutrient cycling through nitrogen fixation and ammonia nitrification, and this activity is better when the earthworms are present than with worm-amended soil without earthworms. The increment of AgriEngineering 2020, 3 FOR PEER REVIEW 10 AgriEngineering 2020, 2 553 (a) (b) Figure 6. (a) Electrophoresis gel for Gram-negative and Putative amoA PCR products from final soil The expression of amoA RNA in worm tissue was unexpected based on the fact that nitrification and worm tissue DNA extraction; (b) Electrophoresis gel for Gram (-) and Putative amoA PCR is an aerobic process; these results suggest that some regions inside worms’ tissue or on the cuticle products from final soil and worm tissue RNA extraction. provide enough oxygen for this process to occur [30]. Soil and worm RNA extractions on day 52 amplified for molybdenum transporter in all samples Soil and worm RNA extractions on day 52 amplified for molybdenum transporter in all samples except for soil RNA from E and EL (Figure 7). The expression of molybdenum transporter may indicate except for soil RNA from E and EL (Figure 7). The expression of molybdenum transporter may nitrogen fixation by L. sphaericus and this would explain increased ammonium concentrations in all indicate nitrogen fixation by L. sphaericus and this would explain increased ammonium treatments [7,8]. concentrations in all treatments [7,8]. Figure 7. Electrophoresis gel for Molybdenum transporter PCR products from final soil and worm Figure 7. Electrophoresis gel for Molybdenum transporter PCR products from final soil and worm tissue RNA extraction. tissue RNA extraction. The absence of the molybdenum transporter expression in E and EL may be related to The absence of the molybdenum transporter expression in E and EL may be related to theoxygenation of the soil by worm activity. Nitrogen fixation requires low oxygen concentrations; theoxygenation of the soil by worm activity. Nitrogen fixation requires low oxygen concentrations; thus, in this case, the process may be inhibited. Nevertheless, expression was found in Co where the thus, in this case, the process may be inhibited. Nevertheless, expression was found in Co where effect should be similar for oxygen concentrations, meaning that the inhibition of molybdenum the e ect should be similar for oxygen concentrations, meaning that the inhibition of molybdenum transporter may also be related to compounds derived from hydrocarbon degradation or the nature transporter may also be related to compounds derived from hydrocarbon degradation or the nature of of fixation-nitrification dynamics. modBexpression was also found in worm tissue from these fixation-nitrification dynamics. modBexpression was also found in worm tissue from these treatments treatments (•E and •EL) suggesting that worms’ guts may provide better conditions for nitrogen (E and EL) suggesting that worms’ guts may provide better conditions for nitrogen fixation [31] fixation [31] when soil contamination causes inhibition. when soil contamination causes inhibition. 4. Conclusions 4. Conclusions The biodegradation processes are highly dependent on bacterial population diversity and The biodegradation processes are highly dependent on bacterial population diversity and dynamics. This study demonstrates the ability of E. fetida to improve the conditions for some of these dynamics. This study demonstrates the ability of E. fetida to improve the conditions for some of populations resulting in the promotion of microbial numbers. Ammonia dynamics during the study these populations resulting in the promotion of microbial numbers. Ammonia dynamics during the and amoA genes expression suggests that bacteria associated with E. fetida are involved in nutrient study and amoA genes expression suggests that bacteria associated with E. fetida are involved in cycling through nitrogen fixation and ammonia nitrification, and this activity is better when the nutrient cycling through nitrogen fixation and ammonia nitrification, and this activity is better when earthworms are present than with worm-amended soil without earthworms. The increment of the earthworms are present than with worm-amended soil without earthworms. The increment of nitrogen in bioremediation systems may be highly beneficial given the reduction of the C/N ratio, which may improve bacterial growth and activity [32]. Increased ammonium concentration may be beneficial for the biodegradation of volatile hydrocarbon compounds given the increased cometabolism by ammonia monooxygenase [21,23,24]. The expression of genes related with nitrogen fixation (modB) and ammonium nitrification (amoAput) in oil sludge remediation processes was demonstrated for L. sphaericus, indicating that this species has the ability to degrade hydrocarbons and, at the same time, may improve nitrogen levels in oil sludge bioremediation. Supplementary Materials: The following are available online at http://www.mdpi.com/2624-7402/2/4/36/s1, Table S1: Pretreatment data, Table S2: Treatment data, Annex S3: Ammonium concentration Pretreatment Statistical Analysis, Annex S4: Ammonium concentration Treatment Statistical Analysis, Annex S5: CFU Treatment Statistical Analysis, Annex S6: Main blast results for amoAamb primers, Annex S7: Main blast results for amoAput primers, Figure S8: Lysinibacillus sphaericus culture from soil and worm tissue samples. AgriEngineering 2020, 2 554 Author Contributions: Conceptualization, J.D.A. and J.D.; methodology, J.D.A. and J.D.; software, J.D.A.; validation, J.D.A. and J.D.; formal analysis, J.D.A.; investigation, J.D.A; resources, J.D.A. and J.D.; data curation, J.D.A.; writing—original draft preparation, J.D.A.; writing—review and editing, J.D.A. and J.D.; visualization, J.D.A.; supervision, J.D.; project administration, J.D.; funding acquisition, J.D. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Research Fund INV-2019-84-1827 at the Science Faculty at Universidad de los Andes and the Microbiological Research Center (CIMIC). Acknowledgments: We are thankful to Biointech for crude oil sludge samples and RETORT analysis. Conflicts of Interest: The authors declare no conflict of interest. References 1. Finer, M.; Jenkins, C.N.; Pimm, S.L.; Keane, B.; Ross, C. 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AgriEngineering – Multidisciplinary Digital Publishing Institute

Published: Nov 13, 2020

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