Coral reefs and watersheds of the Gulf of Mexico in Veracruz: Hydrocarbon pollution data and bioremediation proposal

Regional Studies in Marine Science 35 (2020) 101155

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Coral reefs and watersheds of the Gulf of Mexico in Veracruz: Hydrocarbon pollution data and bioremediation proposal L. Narciso-Ortiz a , K.A. Vargas-García a , A.L. Vázquez-Larios a , T.A. Quiñones-Muñoz b , ∗ R. Hernández-Martínez c , M.A. Lizardi-Jiménez d , a

Instituto Tecnológico Superior de Tierra Blanca, Tierra Blanca, Ver., Méx. C.P. 95180, Mexico CONACYT- Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. (Centro de Investigación y Desarrollo en Agrobiotecnología Alimentaria: Consorcio entre el Centro de Investigación en Alimentación y Desarrollo A.C. y el Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C.), San Agustín Tlaxiaca, Hgo., Méx. C.P. 42162, Mexico c CONACYT- Colegio de Postgraduados Campus Córdoba, Mpio. de Amatlán de los Reyes, Ver., Méx. C.P. 94946, Mexico d CONACYT-Universidad Autónoma de San Luis Potosí, San Luis Potosí, Méx. C.P. 78210, Mexico b

article

info

Article history: Received 3 May 2019 Received in revised form 23 January 2020 Accepted 7 February 2020 Available online xxxx Keywords: Coral reefs Watersheds Hydrocarbon pollution Hydrocarbon bioremediation Hydrocarbonoclastic consortium

a b s t r a c t Coral reefs and watersheds are important ecosystems. Reefs provide many services to coastal communities, including services such as recreation and commercial fisheries. Watersheds support diverse life forms and provide clean domestic water. Hydrocarbon pollution threatens these ecosystems. Sources of hydrocarbon pollution in Veracruz, Mexico include the petroleum industry as well as tourism and maritime activities. The aim of this study was to evaluate hydrocarbon pollution in reefs and watersheds in the Gulf of Mexico along the coast of Veracruz and to propose effective bioreactor conditions for hydrocarbon bioremediation. We found more than 0.7 ppm of octane and more than 1.8 ppm of nonane in April 2018; and longer-chain and aromatic hydrocarbon in July 2018 including phenanthrene, dotriacontane, tetratriacontane, hexatriacontane, octatriacontane, tetracontane and tetratriacontane. Hydrocarbons concentration in water bodies exceeded the limit for discharge of produced waters of the oil industry of 15 ppm in fresh water and 40 ppm in sea water. A microbial consortium composed of Acinetobacter bouvetti, Defluvibacter lusatiensis, Xanthomonas and Shewanella was cultivated in a bioreactor with 20 gL−1 of diesel and gasoline as only carbon source. The results show that 91.39 ± 1.32% of diesel was degraded in a sea water medium and 97.55 ± 0.74% in a mineral medium after 14 days. Furthermore, 95.05 ± 4.75% of gasoline was degraded in a sea water medium and 98.79 ± 1.19% in a mineral medium over the same time period. Biomass production and hydrocarbon degradation were smaller when the culture medium was based on sea water, probably due to the salt concentration. Some microorganisms produce bioemulsifiers. These compounds increase the availability of hydrocarbons and allow the microorganisms to remediate them in the form of emulsified droplets. In all the experiments, consortium showed that emulsifier activity increased and the diameter of hydrocarbon droplets decreased with time. © 2020 Elsevier B.V. All rights reserved.

1. Introduction The Gulf of Mexico is a marine basin, bounded on the north by the United States (US) and on the west by the Mexican coast of Tamaulipas, Veracruz, Tabasco, Campeche, Yucatan and north of Quintana Roo. Important fluvial systems and watersheds drain into the area from 25◦ 57′ 15′′ N and 97◦ 08′ 00′′ W to 21◦ 36′ 17′′ N and 87◦ 06′ 15′′ W (INEGI, 2015). Mexico is organized into 37 hydrologic regions by the Mexican National Water Agency (CONAGUA) and 10 of them drain into the Gulf of Mexico (CONAGUA, 2018). The watersheds support diverse life forms and ∗ Corresponding author. E-mail address: [email protected] (M.A. Lizardi-Jiménez). https://doi.org/10.1016/j.rsma.2020.101155 2352-4855/© 2020 Elsevier B.V. All rights reserved.

provide clean domestic, farm and industrial water (Arthington et al., 2010). The Gulf of Mexico has 83 reefs (INEGI, 2015). The coral reef ecosystems are structures shallow-water dominated by scleractinian corals (Bellwood et al., 2004). Coral reefs are some of the most biodiverse ecosystems in the world and provide services to populations of people such as fisheries, tourism and coastal protection (Lamb et al., 2018), and to aquatic organisms as mechanisms for cycling energy and nutrients and organic matter in these systems (Haas et al., 2016). The Gulf of Mexico has coral reef systems located off the coasts of Veracruz, Mexico (Veracruz Reef System National Park PNSAV) and Yucatan, Mexico. PNSAV is located from 19◦ 02′ 24′′ N

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L. Narciso-Ortiz, K.A. Vargas-García, A.L. Vázquez-Larios et al. / Regional Studies in Marine Science 35 (2020) 101155

Fig. 1. Map of sampling points: 1. Sacrificios Reef (19◦ 10′ 38.233′′ , 96◦ 5′ 32.507′′ ); 2. De en medio Reef (19◦ 6′ 24.090′′ , 95◦ 56′ 17.574′′ ); 3. Gallega Reef (19◦ 13′ 19.235′′ , 96◦ 7′ 37.558′′ ); 4. Punta Gorda Reef (19◦ 14′ 41.970′′ , 96◦ 10′ 32.942′′ ); 5. Jamapa River (19◦ 05′ 39.0′′ , 96◦ 08′ 04.3′′ ); 6. La Antigua River (19◦ 19′ 08.4′′ , 96◦ 19′ 23.2′′ ); 7. Actopan River-Chachalacas Pole (19◦ 25′ 02.3′′ , 96◦ 19′ 17.7′′ ); 8. Papaloapan River (18◦ 36′ 47.2′′ , 95◦ 39′ 7.64′′ ).

to 19◦ 16′ 00′′ N and from 95◦ 45′ 19′′ W to 96◦ 12′ 01′′ W and is considered to be one of the most impacted reef areas in the Gulf of Mexico (Chazaro-Olvera et al., 2018). The PNSAV underwent a major modification in its boundary area in 2012 due to the expansion of the Port of Veracruz project (Ortiz-Lozano et al., 2018). Multiple pollutants, especially hydrocarbons, threaten the reefs and watersheds in Veracruz. There are several factors that contribute to hydrocarbon pollution in these reefs and watersheds, including the petroleum industry, fisheries (García-Cruz et al., 2018), maritime (local and tourism) and port activities that discharge crude oil and its derivatives from boat/ship traffic (Froehner et al., 2018). Additional hydrocarbon contamination can occur from accidental spills in open waters. One such example of this contamination is from the 2016, when Burgos vessel fire, discharged gasoline and diesel near the reef area (PROFEPA, 2016). The hydrocarbon pollutants in aquatic ecosystems affect surface water habitats (fish larvae and zooplankton), reduce sunlight penetration (impeding respiration and photosynthesis) and inhibit gas exchange rates between the water and the atmosphere by producing organic films (Silva et al., 2014; Liu et al., 2016). Thus, it is important to develop microbial remediation strategies for treating hydrocarbon pollution (Varjani, 2017). Bioremediation methods can be classified as in situ or ex situ processes. Ex situ methods allow removal of pollutants through the control of bioprocess parameters (Dzionek et al., 2016). Bioreactors are a type of ex situ bioremediation technique using containers in which pollutants are reduced or removed following a series of biological reactions (Chibueze-Azubuike et al., 2016). Hydrocarbonoclastic bacteria are reported to be the most active degradation organism, and a bacterial consortium has proven to have more potential than individual cultures (Varjani, 2017). Two general biological strategies have been suggested for microbial hydrocarbon degradation: adhesion and bioemulsification (Ron and Rosenberg, 2014). Some microorganisms produce bioemulsifiers. These compounds increase the availability of substrates by increasing the solubility of hydrocarbons (HernándezMartínez et al., 2018). Then, the microorganisms can transform

the hydrocarbon into emulsified droplets (Melgarejo-Torres et al., 2017). Information about hydrocarbon pollution and bioremediation alternatives in reefs and watersheds in Veracruz state is scarce. The aim of this work was to evaluate hydrocarbon pollution in reefs and watersheds along the Gulf Coast of Veracruz and to propose bioreactors for hydrocarbon bioremediation. 2. Materials and methods 2.1. Water sampling locations The sample points are shown in Fig. 1. The water samples were collected in April and July (rainy season) of 2018 at a depth of 25 cm directly in 30 mL amber glass bottles (the bottles were rinsed with site water prior to collection) and kept at 4 ◦ C. (PROY-NMX-AA-121/1-SCFI-2008, 2008). Water bodies were calm. Locations were selected due to proximity to human activities and the riverine discharge in the reef area. 2.2. Hydrocarbon identification The hydrocarbons were analyzed by gas chromatography (TRACE 1310 Thermo Scientific, USA) with a flame ionization detector and TR-5 column (TR-5 30 m × 0.32 mm × 1.0 µm). Temperatures of the injector and detector were 200 and 340 ◦ C, respectively, and the temperature program was 45 ◦ C for 1 min, increasing by 5 ◦ C min−1 until 110 ◦ C, then by 15 ◦ C min−1 until 330 ◦ C (Valdivia-Rivera et al., 2018). Twenty-eight hydrocarbon standard references (Restek, 2016) of aliphatic and aromatic hydrocarbons were used during the screening (Table 1). Quality assurance and quality control (QA/QC) procedures were followed to assure the data quality: the samples were analyzed in triplicate for the homogeneity of the aliquots taken and analytical precision, a solvent blank was analyzed between each triplicate sample using the same method for the samples, the same batches of solvent and hydrocarbons standard reference were used, and the average recoveries of hydrocarbons was calculated (96%).

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Table 1 Hydrocarbon standard references.

Fig. 2. Airlift bioreactor. L1 : Bioreactor height; L2 : Draft tube height; D1 : Bioreactor diameter; D2 : Draft tube diameter.

Number

Hydrocarbon standard references (Restek, 2016)

Chemical formula

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

n-pentane n-heptane n-octane n-nonane n-decane n-undecane naphthalene n-dodecane 2-methylnaphthalene n-tetradecane n-pentadecane n-hexadecane phenanthrene n-heptadecane n-octadecane pyrene n-eicosane n-heneicosane n-docosane n-tetracosane n-hexacosane n-octacosane n-triacontane n-dotriacontane n-tetratriacontane n-hexatriacontane n-octatriacontane n-tetracontane

C5 H12 C7 H16 C8 H18 C9 H20 C10 H22 C11 H24 C10 H8 C12 H26 C11 H10 C14 H30 C15 H32 C16 H34 C14 H10 C17 H36 C18 H38 C16 H10 C20 H42 C21 H44 C22 H46 C24 H50 C26 H54 C28 H58 C30 H62 C32 H66 C34 H70 C36 H74 C38 H78 C40 H82

2.3. Bioreactor Hydrocarbons identification and concentration data were used for select carbon source as bioreactor design condition (Asenjo and Merchuk, 1994). The degradation kinetics were analyzed in a glass airlift bioreactor (Fig. 2) (Lizardi-Jiménez et al., 2012) in our laboratory, with 0.9 L of work volume with the following geometrical relations: D2 /D1 = 0.56, L2 /L1 = 0.77 and L1 /D1 = 3; the draft tube was located 0.011 m above the bottom. 1.13 cm s−1 air was supplied through a L-shaped air diffuser with a diameter of 0.004 m. The air diffuser had seven orifices,

each with 0.001 m diameter and 0.004 m of separation between them. The consortium selected was composed of four strains of microorganism: Acinetobacter bouvetti, Defluvibacter lusatiensis (before Aquamicrobium lusatiense), Xanthomonas and Shewanella (Tzintzun-Camacho et al., 2012), herein referred to as UAM-I consortium. Two culture media were used: the first was a mineral medium (MM) close to the salinity of the rivers, i.e., about 1–2 g L−1 (Landeros-Sánchez et al., 2012; Moran-Silva et al., 2005) using K2 HPO4 (2.15 g L−1 ), KCl (1.13 g L−1 ) and MgSO4 · 5H2 O

Table 2 Hydrocarbons concentration measured in April and July of 2018. Reef or watershed

April

July

Hydrocarbon

µg mL−1 (ppm)

Hydrocarbon

µg mL−1 (ppm)

Octane Nonane

1.882 ± 0.517 1.873 ± 0.244

Hexadecane Phenanthrene Dotriacontane Tetratriacontane Hexatriacontane Octatriacontane Tetracontane

0.035 ± 0.003 0.185 ± 0.019 51.765 ± 13.811 18.511 ± 1.779 78.017 ± 9.787 123.775 ± 31.709 155.032 ± 25.685

Chachalacas Pole (sea)

Octane Nonane

1.338 ± 0.233 2.308 ± 0.414

Hexatriacontane Tetracontane

90.825 ± 17.653 183.752 ± 44.010

La Antigua River

Nonane

2.033 ± 0.622

ND

–

Nonane

3.567 ± 0.436

Phenanthrene Eicosane Dotriacontane Hexatriacontane

0.078 ± 0.025 0.041 ± 0.007 67.914 ± 6.053 66.436 ± 21.562

Nonane

2.967 ± 0.302

Octane Triacontane Tetratriacontane

0.028 ± 0.005 0.038 ± 0.007 134.597 ± 9.578

Sacrificios Reef

Octane Nonane

0.766 ± 0.127 2.866 ± 0.523

Nonane

0.137 ± 0.002

Jamapa River Papaloapan River

Nonane Nonane

2.328 ± 0.112 2.545 ± 0.248

Nonane Nonane

0.037 ± 0.002 0.141 ± 0.033

De enmedio Reef

Octane Nonane

0.931 ± 0.186 2.37 ± 0.262

Nonane

0.115 ± 0.006

Actopan River-Chachalacas Pole

Gallega Reef

Punta Gorda Reef

ND — not detected.

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Table 3 Some hydrocarbon pollution studies in water bodies from 2010 to 2019 in Mexico country. Sampling point

Hydrocarbon

Concentration (ppm)

La Antigua River, Veracruz

Nonane Hexadecane

2.42 ± 0.62 0.41 ± 0.12

Martí Beach, Veracruz

Nonane Hexadecane Eicosane Heneicosane

6.07 1.17 0.52 0.22

Blanco River, Orizaba

Nonane Hexadecane

6.26 ± 0.95 0.93 ± 0.05

Underground water of Merida, Yucatan

Phenanthrene Anthracene Naphthalene Fluorene Pyrene

5 × 10−5 2.01 × 10−4 ± 1.67 × 10−4 2.44 × 10−4 ± 2.18 × 10−4 1.17 × 10−4 ± 6.2 × 10−5 6.5 × 10−5 ± 6.8 × 10−5

Total PAHs (16 priority-pollutant listed by US EPA: naphthalene, acenaphthylene, acenaphthene, flourene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3c,d]pyrene, benzo[g,h,i]perylene dibenz[a,h]anthracene)

0.98

Cancún

Anthracene Phenantrene Phenantrene

8.00 ± 0.13 6.2 ± 0.02 0.009 ± 0.00

Puerto Morelos

Pyrene Naphthalene Benz [a]anthracene

2.33 ± 0.07 5.03 ± 0.11 2.00 ± 0.69

Riviera Maya

Benzene Benzo[a] Pyrene Anthracene

1.00 ± 0.01 1.33 ± 0.07 4.20 ± 0.33

Tampamachoco lagoon system, Veracruz Mandinga lagoon system, Veracruz Alvarado lagoon system, Veracruz Terminos lagoon system, Campeche Yucateco lagoon, Tabasco Mecoacan, Tabasco

± 0.36 ± 0.22 ± 0.12 ± 0.11

García-Cruz et al. (2019)

López-Macías et al. (2019)

Vazquez-Botello et al. (2019) 5.68

2.00

6.12

3.85

0.15

Cozumel

Naphthalene

10.30 ± 0.33

Chetumal

Phenantrene Pyrene

1.08 ± 0.11 2.10 ± 0.11

Cancún,

Anthracene Icosane

0.1414 ± 0.00 1.0214 ± 0.00

Anthracene

0.0264 ± 0.00

Playa del Carmen (Xca-ha)

Reference

Tulum (Chaac-mo)l

Anthracene

0.0164 ± 0.00

Bacalar (Bacalar Lagoon)

Anthracene

0.0220 ± 0.00

Dos Bocas Terminal, Tabasco (Produced water)

Naphthalenes Fluorenes Pyrenes Chrysenes

2.88 × 10−2 ± 1.13 × 10−2 2.11 × 10−3 ± 7.1 × 10−4 3.6 × 10−4 ± 4 × 10−5 9.3 × 10−4 ± 9 × 10−5

Cancún

Naphthalene Phenanthrene Benzo-α -anthracene Dibenzo (α , β ) anthracene Indene Benzo-β -fluoranthene Benzo (k) fluoranthene

5.94 0.90 0.03 0.82 0.11 0.07 0.32

Playa del Carmen

Phenanthrene

1.54 ± 0.03

± ± ± ± ± ± ±

3.62 0.0 0.0 0.01 0.00 0.01 0.02

León-Borges and Lizardi-Jiménez (2017)

Lizardi-Jiménez et al. (2016)

Schifter et al. (2015)

Lizardi-Jiménez et al. (2015)

(continued on next page)

(0.54 g L−1 ) (Lizardi-Jiménez et al., 2012) and the second was

the coral reefs salinity). NaNO3 was used as the nitrogen source

a sea water medium (SM), using natural sea water from Gulf of Mexico with a salt concentration of 32.36 ± 0.45 g L−1 (close to

in a carbon to nitrogen ratio (C/N) of 12:1 (Salleh et al., 2003).

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Table 3 (continued). Sampling point

Hydrocarbon

Concentration (ppm)

Talleres, Cancún

Naphthalene Phenantrene

5.94 ± 3.62 0.09 ± 0.02

Reference

Rancho Viejo, Cancún

Hexadecane

2.02 ± 2.09

Mojarras, Puerto Morelos

Pyrene Phenantrene

4.96 ± 0.10 0.53 ± 0.19

Siete bocas, Puerto Morelos

Hexadecane Phenantrene

3.18 ± 0.02 2.54 ± 0.02

Xca - ha (Playa del Carmen), Riviera Maya

Benzene Benzo (a) Pyrene Decane Hexadecane

1.00 9.67 1.33 5.87

Naphthalene

2.57 ± 0.11

Naphthalene Hexadecane

3.48 ± 0.09 2.15 ± 0.19

Bacalar, Chetumal Lagoon

Naphthalene

2.18 ± 0.54

Milagros Lagoon, Chetumal

Pyrene

1.14 ± 0.11

Urban stormwater runoff, Tijuana

Naphthalene Phenanthrene Fluoranthene Pyrene Chrysene Benzo[a]pyrene

0.316 0.769 0.447 0.767 0.606 0.320

García-Flores et al. (2013)

Estero de Urias Estuary, Sinaloa

Naphthalene Fluorine Phenanthrene Anthracene Fluoranthene Pyrene Chrysene Benzo[b]fluoranthene; Benzo[a]pyrene Benzo[ghi]perylene Indeno[1,2,3-c,d]pyrene.

8 × 10−6 7 × 10−6 2.52 × 10−5 2.1 × 10−6 5.5 × 10−6 9.7 × 10−6 3.4 × 10−6 2.4 × 10−6 0.7 × 10−6 1.6 × 10−6 1.9 × 10−6

Jaward et al. (2012)

Tecocomulco Lake, Hidalgo

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Chrysene Benzo[a]pyrene

0.153 2.400 0.325 0.224 1.500 1.770 0.086 0.200

Romo-Gómez et al. (2010)

Chaac - mol (Tulum), Riviera Maya Chanka – nab, Cozumel

Fig. 3. Experimental scheme of degradation kinetics.

The bioreactor was inoculated with 0.8 g L−1 of suspended solids (SS) of the bacteria consortium and the pH was adjusted to 6.5 with HCl 0.1 N (Lizardi-Jiménez et al., 2012). Analytic determinations were made in triplicate.

± ± ± ±

Medina-Moreno et al. (2014)

0.01 0.02 0.07 0.23

2.3.1. Degradation kinetics The degradation kinetics was carried out by inoculation of the microbial consortium in the bioreactor. The samples (10 mL) were taken from bioreactor in triplicate on days 0, 1, 2, 3, 7, 8, 10 and 14 and centrifuged in a Centrificient VI CRM Globe, USA at 3355 g for 15 min. Three phases were observed: hydrocarbons, aqueous and solid. The kinetics were adjusted by the mathematical Gompertz model. The kinetic parameters: – lag phase (Lag), maximum specific growth rate (µm ), maximum specific degradation rate (Qm ), maximum specific emulsifier activity units increase rate (EAUm ), maximum specific droplet diameter decrease rate (DDm ) and inflection time (Ti) – were determined (Zwietering et al., 1990), using data concentrations in g L−1 and time in days. 2.3.1.1. Suspended solids (SS). The SS were determined after centrifugation and included the bacterial consortium. The solid phase was washed in a 0.9% aqueous solution of NaCl (J.T. Baker, USA, ≥99%) (Valdivia-Rivera et al., 2019) and dried in an oven (Duo Vac, Lab-line Inc. Instruments, USA) at 90 ◦ C for 24 h until a constant weight was achieved (Denis et al., 2017).

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L. Narciso-Ortiz, K.A. Vargas-García, A.L. Vázquez-Larios et al. / Regional Studies in Marine Science 35 (2020) 101155

Fig. 4. Suspended solids (SS) production and diesel degradation in sea water (SM) and mineral medium (MM): A. Experimental data with standard error bars; B. Gompertz model.

Fig. 5. Emulsifier activity units (EAU) and diesel droplet diameter (DD) in sea water (SM) and mineral medium (MM): A. Experimental data with standard error bars; B. Gompertz model. Table 4 Coefficient of determination (R2 ) for the fit of the Gompertz model and the experimental data with diesel as the carbon source. Data

Sea water medium Gompertz (R2 )

Mineral medium Gompertz (R2 )

Suspended solids Diesel degradation Emulsifier activity units Diesel droplet diameter

0.962 0.886 0.995 0.851

0.979 0.868 0.983 0.946

unit of emulsifier activity (EAU) is defined as ‘‘the absorbance per 100 per dilution factor’’ (Patil and Chopade, 2001). The hydrocarbon droplet diameter (DD) was analyzed by the dynamic light scattering technique using a Nanobrook 90Plus PALS (Brookhaven Instruments Corporation, USA). Twenty-four hours before, free cell samples were mixed with the carbon source in a 6:1 ratio and stirred with a vortex (Thermo Scientific M37615Q USA) for two min. 3. Results and discussion

2.3.1.2. Residual hydrocarbons. The residual hydrocarbon from degradation kinetics was extracted from liquid phases with hexane (J.T. Baker, EUA, ≥ 99.5%) (1:1) and followed by gas chromatography with same detector and column used in hydrocarbon identification. The injector and detector temperature were 290 and 300 ◦ C, respectively, and the temperature program was 120 ◦ C for 1 min, increased by 10 ◦ C min−1 until 150 ◦ C (2 min), then by 15 ◦ C min−1 until 170 ◦ C (1.5 min) (Lizardi-Jiménez et al., 2012). Quality assurance and quality control (QA/QC) procedures were followed to assure the data quality: the samples were analyzed in triplicate for the homogeneity of the aliquots taken and analytical precision, a solvent blank was analyzed between each triplicate sample, the same batches of solvent and diesel and gasoline were used, and the average recoveries of hydrocarbons was calculated (96%). 2.3.1.3. Emulsifier activity and droplet size determination. Free cell samples were mixed with the carbon source (hydrocarbon) in a ratio of 5:1 and stirred with a vortex (Thermo Scientific M37615Q, USA) for two min. After 24 h, the optical density was measured in a spectrophotometer (Genesys UV–VIS 10 S, Thermo Scientific, USA) at 540 nm (Amaral et al., 2006), taking into account that one

3.1. Hydrocarbon identification in reefs and watersheds The hydrocarbon concentrations found at the sample locations are shown in Table 2. Table 3 shows data from other studies in the region. Hydrocarbon pollution in Mexico has historically been a problem in rivers, beaches, lakes, lagoons, underground water and underwater sinkholes, with aromatic and aliphatic hydrocarbons in different concentrations. First research of hydrocarbon pollution in the beaches and watersheds of Veracruz state was in 2019, García-Cruz et al. (2019) found nonane and hexadecane in La Antigua River (Table 3), and nonane, hexadecane, eicosane and heneicosane in Martí beach. Some of these hydrocarbons were also found in this work (Table 2). The results show that nonane was found in all the samples in April, while octane was found in four of the samples. In this context, gasoline has the chemical formula C8 H18 (Liso et al., 2016) and consists of C4 – C10 hydrocarbons (Logeshwaran et al., 2018). The hydrocarbons C5 –C10 are considered light fractions and could indicate the presence of gasoline (NOM-138-SEMARNAT/SSAI-2012), 2012). For this reason, we analyzed a 2000 ppm gasoline sample (PEMEX, 2015) by gas chromatography under the same conditions. It was

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Table 5 Kinetic parameters of diesel degradation from the Gompertz model. Kinetics parameters

Lag (d) µm (d−1 ) Qm (d−1 ) EAUm (d−1 ) DDm (d−1 ) Ti (d)

Sea water medium

Mineral medium

SS

Diesel degradation

EAU

DD

SS

Diesel degradation

EAU

DD

0.176 0.361 – – – 1.196

0.100 – 17.732 – – 0.350

0.452 – – 2.281 – 1.097

0.092 – – – 21.948 0.355

0.000 0.384 – – – 1.111

0.125 – 17.805 – – 0.375

0.800 – – 1.214 – 1.800

0.050 – – – 22.809 0.300

Lag (d), lag phase; µm (d−1 ) maximum specific growth rate; Qm (d−1 ), maximum specific degradation rate; EAUm (d−1 ), maximum specific emulsifier activity units increase rate; DDm (d−1 ), maximum specific droplet diameter decrease rate; Ti (d), inflection time.

Fig. 6. Suspended solids (SS) production and gasoline degradation in sea water (SM) and mineral medium (MM): A. Experimental data with standard error bars; B. Gompertz model.

Fig. 7. Emulsifier activity units (EAU) and gasoline droplet diameter (DD) in sea water (SM) and mineral medium (MM): A. Experimental data with standard error bars; B. Gompertz model.

found to contain octane and nonane as well. Thus, the hydrocarbon pollution in the April samples is possibly derived from gasoline. This petroleum derivative is used principally for vehicles (Owagboriaye et al., 2017). The number of vehicles in Veracruz has increased about 3.5% every year since 2013 to 2018 (INEGI, 2018). Samples collected in July – the rainy season (INEGI, 2018) – also found long-chain (C16 –C40 ) hydrocarbons as well as phenanthrene (Table 3). About 50% of the hydrocarbons present exceeded the limit for discharge of produced waters of the oil industry (15 ppm in fresh water and 40 ppm in sea water) (NOM143-SEMARNAT-2003, 2003). Mexico does not currently have a standard for the permissible limit of hydrocarbons in water bodies. Both hexatriacontane and tetracontane were found in the Chachalacas Pole sea zone and the Actopan River-Chachalacas Pole suggesting that the runoff may transport the pollutants to the rivers and then discharge them at sea. In Gallega and Punta Gorda Reef, more hydrocarbons were detected in July than in April. These reefs are close to the new Veracruz Port construction

and maritime transport operations (Ortiz-Lozano et al., 2018). We analyzed a 2000 ppm diesel (PEMEX, 2018) and asphalt sample (473 g from 19◦ 11′ 35′′ N 96◦ 8′ 41′′ W) using the same gas chromatography conditions as the samples. The diesel consists of medium-fraction hydrocarbons (C10 –C28 ) and polycyclic aromatic hydrocarbons. The hydrocarbons present in the diesel sample were pentadecane, hexadecane, heptadecane, octadecane and pyrene. Asphalt sample contained phenanthrene, octadecane, dotriacontane, tetratriacontane and hexatriacontane. It is likely that the C15 –C18 hydrocarbons came from diesel and the C32 –C40 from asphalt. Also, bilge water that accumulates in the bottom of ships may contain residual fuel or compounds like diesel as well as lubricant oils, metals, degreasers and other chemicals derived from ship activities (Tiselius and Magnusson, 2017). This is not surprising considering that Veracruz has heavy marine traffic (SCT, 2017). In Mexico, the hydrocarbon concentration limit for the discharge of bilge water is 15 ppm, this concentration was regulated by NOM-015-SCT4-1994 (1994), but this regulation was canceled in 2010 (DOF, 2010), now bilge water discharge is

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Table 6 Coefficient of determination (R2 ) for the fit of Gompertz model and the experimental data with gasoline as the carbon source. Data

Sea water medium Gompertz (R2 )

Mineral medium Gompertz (R2 )

Suspended solids Gasoline degradation Emulsifier activity units Diesel droplet diameter

0.979 0.900 0.931 0.970

0.967 0.902 0.964 0.961

regulated by the International Maritime Organization (IMO, 1992, 2003). Asphalt related to tourism has already been considered as a likely hydrocarbon pollutant by León-Borges and Lizardi-Jiménez (2017). Possible asphalt-related hydrocarbons were found in July samples during the height of the rain and tourism season (INEGI, 2017). It is probable that the asphalt is transported by runoff from rain into the watersheds and then to the ocean reef systems. Hydrocarbons can diffuse through membranes and accumulate in the tissues of aquatic organisms thus affecting the food chain (Qari and Hassan, 2017). 3.2. Bioreactor design conditions: carbon source selection. The hydrocarbon identification supports the second objective (bioreactors design) due to hydrocarbons identification and concentration data were used for select carbon source as bioreactor design condition. Gasoline and diesel (20 g L−1 ) were selected as carbon sources for the two culture media: a mineral medium (MM) as a liquid phase close to the salinity of freshwater and a sea water medium (SM) close to the salinity at the reefs. The experimental scheme of degradation kinetics is shown in Fig. 3. 3.3. Degradation kinetics 3.3.1. Diesel as carbon source Experimental data (Fig. 4A) and the corresponding fit to the Gompertz model (Fig. 4B) show a rapid increase in SS and a dramatic decrease in residual diesel (g L−1 ) in both culture media. Nearly complete diesel degraded (91.39 ± 1.32% of reduction) was observed in the SM (Fig. 4A). In addition, the degradation percentage obtained was lower than previously reported (ValdiviaRivera et al., 2019). However, a diesel concentration up to 7 g L−1 was used in this study as compared with the literature cited. The coefficients of determination for the fit are shown in Table 4. Experimental data (Fig. 5A) show that the diesel droplet diameter stabilized at about 520 nm on day 7. Similar results were reported by Melgarejo-Torres et al. (2017) who used diesel as a carbon source (13 g L−1 ) with other microorganisms. Diesel in the MM had a degradation of 97.55 ± 0.74% at day 14. Dutta et al. (2018) reported a diesel percentage degradation lower than this study (about 80%) with the same bacterial consortium, but the initial concentration was different (130 g L−1 ) relative to the concentrations used in this study (20 g L−1 ). From kinetic parameters (Table 5), it was observed that the degradation of diesel in the sea water medium began on day 0.100 (2.4 h), and biomass production started on day 0.176 (4.2 h). The maximum rate of biomass growth occurred at day 1.196 (28.7 h), but diesel degradation began before day 0.350 (8.4 h). It is likely that diesel degraded by the bacterial consortium was from emulsified droplets because the diesel droplet diameter had a lag phase of 0.092 (2.2 h) and the maximum velocity of decrease was at day 0.355 (8.5 h), similar to other diesel degradation parameters. The emulsifier activity started at day 0.452 (10.8 h),

after the biomass production, but the maximum velocity of emulsifier and biomass was about the same day (∼ day 1). Thus, the diesel degradation started before the biomass production when the droplet diameter decreased, and the biomass was related to emulsifier productivity. The SS in the MM does not have a lag phase (Table 5). The biomass production started immediately, but the maximum specific growth rate was similar to the SM experiment. Kinetic parameters for diesel degradation were similar too. In the MM, the diesel droplets started with a diameter of 1200 nm and the final size was about 600 nm. In the SM, the final drop size was a little higher. In the MM, the emulsifier activity had a longer lag phase and the maximum specific emulsifier activity rate was minor (Table 5). This factor could be the reason for the difference in the final droplet size. The diesel percentage degradation and the biomass production were higher in the MM, but it is important to remember that biomass production was not the aim. The salt concentration in the SM decreased the diesel degradation (Nápoles-Álvarez et al., 2017), but both media presented a diesel degradation greater than 90% in 14 days. 3.3.2. Gasoline as carbon source The results of this work confirmed that both diesel and gasoline degradation by the bacterial consortium was successful (Figs. 6 and 7). The coefficients of determination for the fit are shown in Table 6. Experimental data (Fig. 6A) and the Gompertz model (Fig. 6B) show an increase in SS and a decrease in residual gasoline (g L−1 ) in both culture media. In addition, this research is the first to show gasoline degradation by the consortium UAM-I, which indicated that 95.05 ± 4.75% of the gasoline degradation in the sea water medium occurred in 14 days and in the mineral medium a degradation of 98.79 ± 1.19% was observed simultaneously. In this case, the culture mediums did not have statistical difference in percentage of gasoline degradation. Otherwise, experimental data (Fig. 7A) and the Gompertz model (Fig. 7B) show an increase in the emulsifier activity unit (mL−1 ) and a decrease in gasoline droplet diameter (nm) in 14 days in both culture media. Table 7 shows that gasoline degradation in the SM has a lag phase of 0. The gasoline degradation started at day zero and accelerated at day 0.27. The biomass production had a 1.2-day lag phase and accelerated until day 3.2, that is, the consortium started the gasoline degradation first and then started to grow. In this case, the gasoline droplet diameter decreased, while the emulsifier activity increased (Melgarejo-Torres et al., 2017). In the MM, the biomass production had a minor lag phase (0.444 day) with a major maximum specific growth rate in the SM. A difference with diesel as the carbon source was the emulsifier activity units; the gasoline did not have a lag phase. The consortium UAM-I can grow with gasoline as the carbon source in both media. In all the experiments, the consortium had emulsifier activity and the hydrocarbon droplet diameter decreased. Acenitobacter spp, is known as a biosurfactant microorganism producer (Silva et al., 2014) and Acenitobacter bouvetti was reported as the only biosurfactant producer in the UAM-I consortium (TzintzunCamacho et al., 2012). Diesel and gasoline can be degraded by UAM-I consortium by more than 90% and probably the degradation is in emulsion form. Our research team has studied other airlift bioreactors volumes: 3 L (Dutta et al., 2018; Tec-Caamal et al., 2018); 7 L (Denis et al., 2017) and 10 L (Medina-Moreno et al., 2013; Lizardi-Jiménez, 2011; Lizardi-Jiménez et al., 2012) and we could observe, in those studies, that productivity and yield are not affected by scale up to 200 L of working-volume. 200 L could be considered as pilot scale, maybe semi industrial

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Table 7 Kinetic parameters of gasoline degradation. Kinetics parameters

Lag (d) µm (d−1 ) Qm (d−1 ) EAUm (d−1 ) DDm (d−1 ) Ti (d)

Sea water medium

Mineral medium

SS

Gasoline degradation

EAU

DD

SS

Gasoline degradation

EAU

DD

1.200 0.166 – – – 3.200

0.000 – 16.470 – – 0.270

0.000 – – 0.706 – 1.667

0.025 – – – 23.103 0.275

0.444 0.430 – – – 1.556

0.000 – 12.515 – – 0.357

0.125 – – 0.868 – 1.375

0.000 – – – 26.265 0.200

Lag (d), lag phase; µm (d−1 ) maximum specific growth rate; Qm (d−1 ), maximum specific degradation rate; EAUm (d−1 ), maximum specific emulsifier activity units increase rate; DDm (d−1 ), maximum specific droplet diameter decrease rate; Ti (d), inflection time.

and could be useful in a real world application due to low cost manufacturing of this kind of reactors. Information contained in our manuscript is important because 200 L is not laboratory scale and could be used as ‘‘minor’’ scale in scale-up process finding ‘‘major scale’’. 4. Conclusions There is hydrocarbon pollution in reefs and watersheds in Veracruz, Mexico. More than 0.7 ppm of octane and more than 1.8 ppm of nonane were found in April 2018; and longer-chain and aromatic hydrocarbons such as phenanthrene, dotriacontane, tetratriacontane, hexatriacontane, octatriacontane, tetracontane, and tetratriacontane were found in July 2018 (Table 2). Hydrocarbons concentration in water bodies, even exceeded the limit for discharge of produced waters of the oil industry of 15 ppm in fresh water and 40 ppm in sea water. These hydrocarbon pollutants likely came from gasoline, diesel and/or asphalt through rain runoff, river discharge, bilge water, marine transport and tourism-related activities. Under the bioreactor conditions selected, diesel and gasoline degradation by the UAM-I consortium was successful (20 g L−1 in 14 days). Diesel degradation was 91.39 ± 1.32% in a sea water medium and 97.55 ± 0.74% in a mineral medium; gasoline degradation was 95.05 ± 4.75% in a sea water medium and 98.79 ± 1.19% in a mineral medium. Biomass production and hydrocarbon degradation were minor when the culture medium was sea water, probably due to the salt concentration (NápolesÁlvarez et al., 2017). In all the experiments, the consortium showed emulsifier activity and the hydrocarbon droplet diameter decreased. Results from this study shed further insight on the effect of bacterial consortiums on carbon source degradation like gasoline and diesel, emphasizing the applicability of bioremediation with microorganisms for hydrocarbon pollution. Several possibilities are due here; one approach could be use these systems in continuous mode (with the same volume but a continuous entry of nutrients an exit of products) or try to test bigger scales using information of this manuscript. Both possibilities could be explored in future works. Processing runoff water from impervious surface retaining ponds and some sort of in situ deployment on the reefs are excellent trends of action Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement L. Narciso-Ortiz: Formal analysis, Investigation, Methodology, Writing - original draft. K.A. Vargas-García: Methodology, Formal analysis, Writing - review & editing. A.L. Vázquez-Larios: Methodology, Formal analysis, Writing - review & editing. T.A.

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