Chemical and Isotopic Investigation of Abiotic Oxidation of Lactate Substrate in the Presence of Varied Electron Acceptors and Under Circumneutral Anaerobic Conditions

Abstract

Abiotic processes have ramifications in wastewater treatment, in situ degradation of organic matter, and cycling of nutrients in wetland ecosystems. Experiments were conducted to investigate abiotic oxidation of organic compounds (lactate) as a function of electron acceptors (ferric citrate and hydrous ferric oxide (HFO), media composition, and pH under anaerobic conditions, using sodium bicarbonate as the buffering agent. Dissolved inorganic carbon (DIC) was used as a proxy for the oxidation of substrates. HFO media generated more DIC compared to ferric citrate containing media. Light and pH had major roles in the oxidation of lactate in the presence of ferric iron. Under dark conditions in the presence or absence of Fe(III), the DIC produced was low in all pH conditions. Inhibition of DIC production was also observed upon photo exposure when Fe (III) was absent. Isotopically, the system showed initial mixing between the bicarbonate and the carbon dioxide produced from oxidation later being dominated by carbon isotope value of lactate used. These redox conditions align with previous studies suggesting cleavage of organic compounds by hydroxyl radicals. The slower redox processes observed here, compared to previous studies, could be due to the scavenging effect of chloride ion on the hydroxyl radical.

1. Introduction

The fate, mobility, and bioavailability of inorganic and organic compounds in the environment are heavily influenced by various geochemical processes, including sorption, precipitation, dissolution, and electron transfer [1,2,3]. Investigating the different geochemical processes and their reaction mechanisms would add to the understanding of natural systems and the fates of inorganic and organic compounds in pristine and contaminated environments. Electron transfer processes could be either enzymatically mediated microbial metabolism or abiotically controlled.

For studies related to biotic redox processes, strains (e.g., Shewanella putrefaciens) are cultured in the laboratory under favorable conditions of media, pH, buffering agents, etc., and suitable indicators of redox processes are analyzed. Since the redox process generates CO₂, an analysis of Dissolved Inorganic Carbon (DIC) is a convenient method to study this when the redox process is allowed to take place in vitro.

Abiotic electron transfer processes have drawn much attention in many fields, including wastewater treatment, enhancing in situ degradation of organic and inorganic compounds, cycling of nutrients in wetland ecosystems, and in atmospheric chemistry for investigating sources and sinks of chemicals in the troposphere [1,2,3,4,5,6,7,8,9,10,11,12,13].

Direct photolysis is an abiotic process in which organic compounds absorb light, and, as a result, they undergo photochemical transformation. With some organic compounds, exposure to a light source alone does not result in electron-transferring reactions [4,7,8]. Indirect photolysis takes place either due to energy transfer from another excited species (sensitized photolysis) or chemical reactions (e.g., Fenton like reactions) of nonexcited compounds. Non-excited compounds possess short-lived but very reactive species (e.g., OH- and peroxy- radicals, singlet oxygen) formed in the presence of light due to reactions of excited compounds (e.g., citrate, lactate). Sensitizing agents (e.g., Fe(III) and Zn) act as geocatalysts (acting as catalyzing agents) in natural ecosystems assisting photochemical reactions to transform pollutants [2,11]. One of these catalysts is ferric iron, which acts as a photosensitizer and participates in the formation of oxidants via photo-initiated transformations [2,11]. Under our experimental conditions discussed here, Fe⁺³ forms complexes with polycarboxylic acids (e.g., ferric citrate) and oxyhydroxides (e.g., HFO). Photochemical dissociation of ferric citrate complexes in aqueous solutions have been reported to involve the reduction of ferric iron to ferrous iron, coupled with the oxidation of citrate-producing acetone and carbon dioxide represented by the general reaction (1), shown below [5,6,13]. Along with microbial metabolism, photochemical processes and Fenton-like reactions enhance the reduction of metals and oxidation of organic compounds [14,15,16].

$$\begin{gathered} \mathrm{C}(\mathrm{OH})(\mathrm{COOH})({\mathrm{CH}}_2\mathrm{COOH}{)}_2+2{\mathrm{Fe}}^{+3} \stackrel{\mathrm{h}\mathrm{\upsilon }}{\to }{\mathrm{CH}}_3{\mathrm{COCH}}_3+2{Fe}^{+2}+3{\mathrm{CO}}_2+2{\mathrm{H}}^{+} \\ (\mathrm{Citrate})\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}(\mathrm{Acetone}) \end{gathered}$$

(1)

where hυ → photons or quanta, h = Planck constant, 6.63 × 10⁻³⁴ J.s, and υ = frequency.

Recent studies have reported that the photochemical reaction of ferric citrate and ferric oxyhydroxides form hydroxyl radicals via an intermediate process involving a modified Fenton-like reaction [6,16]. The redox potential and thermodynamic properties make ferric citrate and hydrous ferric oxide excellent electron acceptors to study biotic as well as abiotic oxidation of organic substrate [8]. Fenton chemistry (Fe⁺² + H₂O₂ → Fe⁺³ + .OH) plays a role in photochemical transformations of organic compounds in two ways. One is producing a hydroxyl radical that, in turn, attacks organic compounds, such as citrate and lactate, mineralizing them to lower-molecular-weight carbon compounds, such as carbon dioxide, both aerobically and anaerobically [2,6,10,11,12,13]. Second, Fenton chemistry oxidizes ferrous to ferric iron, an excited species, for further sensitized photo-oxidation of the organic compounds. Under anaerobic conditions, the presence of anions such as chloride inhibits abiotic reduction–oxidation couplings by forming halogenated compounds and by scavenging the hydroxyl radicals [17,18].

Iron is very reactive in near-surface environments, and its oxyhydroxides are common on the earth’s crust and can function as geocatalysts in speciation of organic and inorganic pollutants in the subsurface environment [2,19]. Recent studies have shown the importance of dissolved organic compounds (DOC) and iron ions involved in the photochemical formation of hydrogen peroxide and other photooxidants in degrading natural and anthropogenic compounds [1,9,11,13].

Carboxylic acids (e.g., oxalate, citrate) possess one of the most common functional groups (RCOO) of dissolved organic compounds present in natural waters and form strong complexes with ferric iron [6]. The photooxidation of these carboxylic acids could be sensitized by the presence of Fe(III) undergoing rapid photochemical reactions upon irradiation according to reaction 1 [20]. Deng et al. [6] used Fe(III)–citrate complexes as a photochemical sensitization material to promote the discoloration of dyes, forming more reactive species (e.g., H₂O₂ and .OH) compared to Fe(III)–hydroxyl and Fe(III)–oxalate complexes at acidic pH conditions. Deng et al. [6] reported that at a pH of 2, the photodegradation rates of organic compounds increased for varying citrate-to-Fe(III) ratios in the order of 4:1 > 2:1 > 1:4 > 1:1 > 1:2. They also pointed out that at neutral-to-alkaline conditions, Fe(III)–hydroxyl dominates the photoreactivity compared to Fe(III)–citrate complexes. Hydroxyl radicals produced in systems containing Fe(III)–ligands complexes could be responsible for the oxidation of organic compounds coexisting in aqueous solutions [6]. Deng et al. [6] concluded that under near-UV light, the efficiency of the Fe(III)–citrate complexes induced photoreduction reaction in aqueous solutions that depended on the pH and the initial citrate-to-Fe(III) ratio.

Several variables, including photo-formation of .OH radicals, .OH radicals attacking target groups, or bonds in the respective organic compounds, could result in different photodegradation rates for different organic compounds [9]. The latter might be a rate-controlling step in the photodegradation system. Wu and Deng [9] concluded that the photochemical nature of organic compounds coexisting in photo-reactive complexes containing solutions is strongly influenced by the pH, Fe(III) concentration, wavelength, and energy of the irradiation source of the solution. This observation concurred with the observation made by [4] that organic compounds dissolved in solutions rich in ions easily become oxidized upon illumination.

Understanding abiotically controlled electron transfer processes in a laboratory-based anaerobic system is the focus of this study. This can be carried out in vitro exactly the same manner as with biotic redox processes, with the difference being that no microorganisms are introduced into the system, and any DIC produced is expected to be from abiotic oxidation.

A literature review of such studies showed that photo-assisted oxidation of polycarboxylics (e.g., citrate, oxalate) and other organic compounds in the presence of ferric iron, other metals (Mn, Cd, and Ni), and semiconductors (TiO₂) is a well-documented phenomenon [1,5,6,13]. Most of these previous studies were performed under acidic and aerobic conditions. Our study was completed under anaerobic conditions, in the presence or absence of light, and at varying pH (5 to 9) conditions. Here, the oxidation of lactate and citrate with and without ferric iron was used to investigate the effect of (1) electron acceptor (ferric citrate vs. HFO) in a carbonate-buffered system; (2) media components; (3) buffers under neutral pH condition (4); pH conditions; and (5) fluorescent light. Most of these factors have environmental implications in natural and engineered remediation strategies of contaminated sites. It will also be important for adding data to the new and exciting ongoing research in the abiotic degradation of organic pollutants. Moreover, to the best of our knowledge, there are no reported abiotic studies on the oxidation of lactate. Here, we report the possible mechanisms of the DIC produced during the abiotic oxidation of lactate and citrate under strict anaerobic conditions, circumneutral pH, and a temperature of 30 °C. We also report carbon isotopic signatures associated with the abiotic electron transfer processes. The general schematic ferric-iron-sensitized photocatalytic processes in this study are represented by the reactions (Equations (2) and (3)):

$$\begin{array}{c}2{\mathrm{FeC}}_6{\mathrm{H}}_5{\mathrm{O}}_7 + {\mathrm{NaC}}_3{\mathrm{H}}_5{\mathrm{O}}_3 \stackrel{\mathrm{h}\mathrm{\upsilon }}{\to }{\mathrm{C}}_3{\mathrm{H}}_6\mathrm{O}+{\mathrm{NaC}}_3{\mathrm{H}}_5{\mathrm{O}}_3+2{\mathrm{Fe}}^{+2}+3{\mathrm{CO}}_2+2{\mathrm{H}}^{+}\\ (\mathrm{Citrate})\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}(\mathrm{Acetone})\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}(\mathrm{Lactate})\end{array}$$

(2)

$$\begin{array}{c}2{\mathrm{NaC}}_3{\mathrm{H}}_5{\mathrm{O}}_3+2\mathrm{Fe}(\mathrm{OH}{)}_3+{\mathrm{H}}_2\mathrm{O} \stackrel{\mathrm{h}\mathrm{\upsilon }}{\to }6{\mathrm{HCO}}_{3^{-}}+2{\mathrm{Fe}}^{2+}+2{\mathrm{Na}}^{+}+10{\mathrm{H}}^{+}{\mathrm{H}}_{2}O\\ (\mathrm{Lactate})\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}(\mathrm{HFO})\end{array}$$

(3)

2. Materials and Methods

2.1. Media Composition

Details of media composition and experimental setups are discussed elsewhere in detail [21,22]. Briefly, a defined circumneutral pH medium was prepared with components containing NH₄Cl, NaH₂PO₄H₂O, KCl, vitamins, Wolfe’s minerals, sodium lactate, ferric citrate, and sodium bicarbonate. Ferric citrate stock solution was prepared using autoclaved double-deionized water (R~17.6 MΩ cm) amended with reagent-grade ferric citrate (Fischer Chemical ^®, Pittsburgh, PA, USA) and 10 N NaOH [22,23].

An HFO (70 mM)-based experiment was performed using solid HFO prepared using the method discussed by Schwertmann and Cornell [19]. The solid HFO was added into each vacutainer tube. Fe (NO₃)₃9H₂O (80 g/L) was dissolved in DDIW(Double Distilled Ionized Water) with 330 mL/L of 1 M KOH, and the pH was adjusted to ~7.7. The solution was stirred for 1 h and centrifuged repeatedly at 5 min intervals at 1500 rpm to remove any impurities. Then, the solution was filtered using 0.2 μm sized filters before freeze-drying. HFO loses its structure on heating above 80 °C, so to preserve its structure, HFO was added in the anaerobic chamber after autoclaving the rest of the media components.

2.2. Media Preparation

Eight 500 mL/L Pyrex bottles were autoclaved and filled with defined salt media by omitting one media component at a time. Each bottle was set to a pH of ~7.1 by adding HCl dropwise. All respective media components, with the exception of minerals and vitamins, were added before autoclaving. For each experimental setup, six vacutainer tubes of 15 mL/L volume were loaded with 8 mL/L of the media solution within the anaerobic chamber after applying nitrogen to each for an hour. All samples were taken in duplicates, and a pair of samples from each Pyrex bottle was taken before starting to shake to know the starting values. This set of experiments continued for about 2900 h (~121 days).

A set of experiments with ferric citrate or HFO as Fe(III)-containing compounds were used to examine the effect of ferric iron–complex compounds on the oxidation of the organic compounds under the experimental conditions discussed above.

The effect of two buffering compounds, NaHCO₃ on the generation of carbon dioxide and soluble ferrous iron, was compared under both dark (wrapped by aluminum foil) and fluorescent light conditions. Each sample was treated similarly, and the experiments were carried out for pH 5 and 7 to check the relative importance of two of the controlling factors, pH and fluorescent light.

The effect of pH on the photo-oxidation of ferric citrate and sodium lactate was investigated in the presence of light for pH varying from acidic (pH~5) to alkaline pH (pH~9) conditions for the bicarbonate buffered system.

Photooxidation of organic compounds were investigated in the presence and absence of light and Fe(III) in the acidic (pH~5) to neutral pH (pH~7) conditions. This portion of the experiment was conducted to compare and contrast the effect of pH, Fe(III), and light on redox processes.

2.3. Chemical Analyses

Details of chemical analyses are explained in detail in earlier publications [21,22], and repetition is deemed unnecessary and beyond the scope of this paper. The iron concentration was determined using UV/VIS spectrophotometer absorbance wavelength of 562 nm via the ferrozine method under anaerobic conditions and is reported in mg/L of Fe (II)/Fe(III) [24]. The pH of each sample was determined using a portable pH/mV meter.

2.4. Stable Carbon Isotope Analyses

The carbon isotopic composition of sodium lactate, sodium acetate, citric acids and ferric citrate was determined from CO₂, as discussed in Gebrehiwet & Krishnamurthy [21]. The stable carbon isotopic ratio of sodium bicarbonate used as a buffering agent was analyzed by reacting with 85% phosphoric acid [25], and DIC was analyzed via the gas evolution method [26]. Carbon isotope measurements were made using a VG IRMS (Isotope Ratio Mass Spectrometer), and the results are reported in the traditional δ notation relative to Vienna PeeDee Belemnite (V-PDB) standard: (Equation (4)).

$${\delta }^{13}\mathrm{C}‰={\displaystyle \frac{R_{Sample}}{R_{Standard}}}-{10}^3$$

(4)

where R_Sample and R_Standard represent the ¹³C/¹²C ratio of the sample and standard (VPDB), respectively. The overall precision of the analytical processes are better than 0.1‰ for δ¹³C, δ¹³C_org, and δ¹³C_Inorg and 1% for carbon dioxide measurements. All abiotic samples were treated with cupric oxide at ~900 °C to purify other gases produced during the photochemical reactions before subsequent isotope ratio measurements. This purification resulted in much cleaner isotope ratio traces.

3. Results and Discussion

3.1. Extent of Iron Reduction and Organic Compound Oxidation

Soluble iron and DIC measurements indicated that, in all experimental conditions, except when covered with aluminum foil (not exposed to light), both ferrous iron and CO₂ yield increased as a function of the time of reaction, as shown in Figure 1 and Figure 2. Figure 1 shows the ferrous iron concentration of the ferric citrate media exposed to fluorescent light at pH~7.

The DIC production, which is assumed to be a proxy for organic compound oxidation also increased with the reaction time in all experiments except in the combined absence of light or Fe(III)-containing compounds (Figure 2).

The behavior observed in Figure 1 and Figure 2 means there should be a correlation between DIC and ferrous iron concentration. That this indeed is the case is evident from Figure 3. Figure 3 is a cross-plot of ferrous iron as a function of the DIC produced in the bicarbonate buffed conditions in the ferric-citrate-containing media.

There is linear relationship between the concentrations of the reduced form of iron and the production of carbon dioxide, consistent with a photo-induced abiotic redox process. Somewhat low correlation between DIC and Fe(II) values (R² = 0.67) could be explained by the possible re-oxidation of ferrous to ferric iron via the Fenton-like reaction (Equation (5)) with increased generation of hydrogen peroxide (H₂O₂) and ferrous iron [6].

H₂O₂ + Fe⁺² → Fe⁺³ + 2·OH

(5)

3.2. Effects of Media Composition

Experiments were carried out to examine if media components had an effect. For this, one media component was removed from the rest, and the amount of DIC produced was measured. For instance, M-MIN was the media without the minerals, M-VITM was the media without the vitamins, and so on. Generally, the removal of a media component in all the samples was observed to have comparable carbon dioxide generation with time. The significant change occurred when ferric citrate was removed from the media (M-FC). The M-FC samples gave the least amount of DIC from the degradation of lactate, the only significant carbon-containing organic compound. The amount of DIC in the M-FC samples was less by a factor of about 5 compared to the other set of samples in the current experimental setup. Some plausible explanation is provided based on the following experiments.

3.3. Effect of Fe(III)-Containing Compounds

Two sets of experiments with two different iron-containing electron acceptors, ferric citrate and HFO, were carried out to compare the importance of the form of ferric iron in organic carbon oxidation. As shown in Figure 4, HFO in the presence of lactate produced twice as much DIC, compared to ferric citrate in the presence of lactate, and increased with time. The observed difference between the two sets of samples could be attributed to the efficiency of HFO in oxidizing organic compounds at near-neutral pH conditions compared to ferric citrate [4,6].

3.4. Effect of pH

The effect of pH on the abiotic oxidation of organic compounds has been investigated by various researchers [6,11,27,28]. Most of these studies have been carried out at low pH conditions (<5). Here, our study focused on the effects of pH, ranging from 5 to 9, on the redox process for the bicarbonate-buffered system. Fe(II), Fe(III), pH, and DIC were determined in each sample. Figure 5 shows the DIC as function of time for the acidic (pH = 5), neutral (pH = 7), and alkaline (pH = 8 and 9) conditions. DIC production was the highest at acidic conditions followed by the neutral and alkaline conditions, respectively. Similarly, the highest Fe(II) accumulation was observed in the acidic condition compared to the neutral and alkaline pH conditions, respectively (Figure 6).

3.5. Effect of Light Under Acidic and Neutral Conditions

A set of experiments were conducted to investigate the effect of light, pH, and ferric iron. Experiments were carried out under fluorescent light and dark conditions, at acidic and neutral pH, and in the presence and absence of ferric iron. In the absence of ferric iron, no DIC generation occurred under any photo-exposure or different pH conditions. Figure 7 and Figure 8 compare the effect of light and pH on the DIC and Fe(II) generations for the bicarbonate buffered condition, respectively, in the presence of ferric iron.

The main conclusion from these experiments is that, under our experimental conditions, fluorescent light had a greater effect on the redox process than pH in the presence of ferric iron. Regardless of the pH conditions, in the absence of light or ferric iron, both DIC and ferrous iron accumulate slowly or not at all, indicating the absence of organic compounds oxidation.

3.6. Stable Carbon Isotopes in Abiotic Systems

Results of isotopic analyses at neutral pH and anaerobic conditions of the extracted DIC are shown in Figure 9 and Figure 10. Figure 9 shows a plot of δ¹³C_DIC as a function of total DIC in the bicarbonate system without correcting for the initially added bicarbonate buffer that had a d¹³C value of 9.3‰. At lower pH conditions (pH~5), there were no systematic relationships between the DIC and its respective carbon isotopic values. Figure 10 shows graph of the δ¹³C_DIC values as a function of inverse of the total DIC in the bicarbonate system. Such plots are useful in estimating the source of additional carbon dioxide produced from oxidation of organic compounds [29]. The y-intercept of such a plot provides clues regarding the original source of organic carbon involved in the redox process. In this case, the lactate which was the organic carbon source had a δ¹³C value of −25‰.

Figure 7. Influence of light under acidic and neutral conditions on DIC production in the bicarbonate-buffered system; UC (open symbols) = uncovered, exposed to light; C (filled symbols) = covered, dark.

Figure 7. Influence of light under acidic and neutral conditions on DIC production in the bicarbonate-buffered system; UC (open symbols) = uncovered, exposed to light; C (filled symbols) = covered, dark.

3.7. Chemical Factors Controlling Redox Processes

Experiments were carried out in anaerobic conditions under different controlling factors. Our results agreed well with previous studies reporting the importance of pH, light, and ferric iron for the abiotic oxidation of organic compounds coupled to metal reduction. In addition, the concentration of the ferric iron, wavelength, and energy of the irradiation source could influence the rate of redox processes [7]. The type of photo-oxidizing catalysts, the strength of the reducing agents, and the presence of multidentate ligands also affect the rate of photo-assisted redox processes [4]. Moreover, different organic compounds exhibit different photodegradation rates involving several intermediate reactions in aqueous solutions [6,7]. Our results could be affected by a combination of the multiple controlling factors mentioned above and by other researchers.

In the experimental setups containing all media components, both Fe(II) and DIC increased with time. The rate of redox processes observed in our experimental conditions was slower compared to most of the previous studies both at acidic and neutral conditions [6,16]. Deng et al. [6] studied at a lower pH (2) compared to our pH (ranging from 5 to 9), so considering the effectiveness of ferric citrate at lower pH could partially explain the observed discrepancies in the two studies. Northup and Cassidy [16] used modified Fenton mechanism using hydrogen peroxide at a pH of 8. Such a modification could explain the faster rate observed in their study compared to our study. Moreover, in these two studies [6,16], the concentration of chloride ion is low compared to our experimental conditions, where we used KCl (1.4 mM) and NH₄Cl (4.7 mM) in the media preparation. Recent studies [17,18] have shown that the presence of chloride ion in anaerobic conditions inhibits indirect photolysis by scavenging hydroxyl radicals in the systems. Hence, the chloride ions could be partially responsible for the slow rate of oxidation observed.

Figure 8. Ferrous iron production in the bicarbonate buffering system in the presence or absence of light at pH 5 and 7 as a function of time. UC (open symbols) = uncovered, exposed to light; C (filled symbols) = covered, dark.

Figure 8. Ferrous iron production in the bicarbonate buffering system in the presence or absence of light at pH 5 and 7 as a function of time. UC (open symbols) = uncovered, exposed to light; C (filled symbols) = covered, dark.

Figure 9. Carbon isotopic values of DIC vs. DIC concentration under neutral pH conditions in the bicarbonate buffered system as a function of total DIC.

Figure 9. Carbon isotopic values of DIC vs. DIC concentration under neutral pH conditions in the bicarbonate buffered system as a function of total DIC.

Figure 10. Carbon isotopic values of DIC vs. inverse of DIC concentration relationship of the bicarbonate-buffered system under neutral pH conditions. The y-intercept of the regression of the line provides the estimate of the starting δ¹³C value of the organic compound being oxidized in the bicarbonates system.

Figure 10. Carbon isotopic values of DIC vs. inverse of DIC concentration relationship of the bicarbonate-buffered system under neutral pH conditions. The y-intercept of the regression of the line provides the estimate of the starting δ¹³C value of the organic compound being oxidized in the bicarbonates system.

The HFO experiments (70 mM total Fe) generated more DIC than the ferric citrate experiments (50 mM total Fe) showing the effect of the concentration of initial Fe(III). The ratio of ligand to Fe(III) is also reported to influence the rate of photodegradation of organic compounds, at least at pH values less than 2 [2]. In general, a larger ligand-to-sensitizing-agent ratio results in a greater rate of organic compound photodegradation. In case of the two ligands (OH and citrate) containing electron acceptors used in our experiments, the ligand-to-Fe(III) ratios are 3:1 in HFO compared to 1:1 in ferric citrate. This could also contribute to the difference in the rates of lactate/citrate oxidation observed for these two media compositions.

No measurable differences were observed when using either KH₂PO₄ or NaHCO₃ as buffering agents in the DIC or soluble ferrous iron production. These observations showed that as long as the pH conditions are maintained, the type of buffering agent has little effect on the redox process. This was contrary to microbially mediated systems [21,22], where bicarbonate clearly enhanced the oxidation and reduction process at a near-neutral pH. The complex enzymatic reactions in biotic systems could be responsible for the observed difference due to enhanced metabolism in the bicarbonate system. Whereas in abiotic systems, both bicarbonate and phosphate buffers affect the formation of ^.OH radical at a neutral pH equally enhancing the effect of Fenton mechanisms.

pH determines the dominance of different metal–ligand complexes influencing their photo reactivity and participation in redox reactions. Under acidic conditions, Fe(III)–citrate complexes photochemically produce more reactive species (e.g., H₂O₂ and ^.OH) compared to Fe(III)–hydroxyl and Fe(III)–oxalate complexes [4,6]. This observation could explain the greater DIC yield and ferrous iron production measured at a pH of 5 compared to the neutral and alkaline pH conditions. At near-neutral-to-alkaline conditions, Fe(III)–hydroxyl becomes more photoreactive than the Fe(III)–citrate complex [6]. Thus, the greater DIC production observed in HFO media compared to the ferric citrate media at near neutral pH conforms well to the observation of Deng et al. [6].

The effects of light and sensitizing agents (Fe(III) and minor concentrations of transition metals in the Wolfe’s minerals) were clearly demonstrated in our experimental conditions. In the absence of both Fe (III) and fluorescent light, minimal oxidation of lactate and citrate were observed, indicating that, in abiotic conditions, these two organic compounds could only obtain oxidization via indirect (sensitized) photocatalytic processes. Omission of Wolfe’s minerals from the media also affected the DIC yield, indicating that the transition metals (e.g., Mn, Co, Zn, and Cu) might make some contribution to the oxidation of the organic compounds used.

Understanding the importance of microbially mediated and abiotic mechanisms, such as the synergistic combination of microbial–Fenton systems [14,16,27] and sonochemically induced Fenton reactions [10], would help remediation of contaminants. These abiotic mechanisms could be important in the degradation of less microbially degradable compounds (e.g., PCE and TCE) by increasing the production of H₂O₂, which, in turn, produces more hydroxyl radicals.

3.8. Isotopic Factors Controlling Redox Processes

The trend of isotopic values in Figure 9 indicated the presence of two isotopically distinct carbon sources (e.g., bicarbonate vs. organic compounds) and their sequential time-dependent contribution of carbon, indicating mixing of these isotopically distinct sources. As time progresses, the isotopic contribution of the bicarbonate became exponentially less influential, whereas the DIC coming from the oxidation of organic compounds increased.

The graph of the δ¹³C_DIC of the samples as a function of the inverse of the total DIC (DIC⁻¹) shown in Figure 10 suggests clearly that the source of the starting component in the bicarbonate system is lactate (δ¹³C_DIC = −25.4‰). At lower pH conditions (pH~5), there were no systematic relationships between the DIC and its respective carbon isotopic values. The reason for such a behavior under lower pH conditions is unclear and warrants more quantitative work, including the usage of isotopically tagged organic carbon compounds.

4. Conclusions

Most previous studies were conducted under aerobic and acidic conditions (pH = 2–5), with a few under anaerobic conditions. Such acidic pH conditions are less likely in the natural environment due to the buffering capacity of soils in natural ecosystems. Here, this research reported results from experimental work performed under circumneutral and anaerobic conditions. The effects of light, Fe(III) presence and concentrations, ligand-to-Fe(III) ratio, and pH on the redox processes involving lactate and citrate as model organic compounds were observed in our study, confirming previous observations made mostly under acidic and aerated conditions. The practical implications of abiotic processes are in relation to totally anaerobic conditions, which are not ubiquitous in nature but do exist. Atmospheric processes can also be included here.

Both lactate and citrate undergo oxidation by indirect photolysis involving sensitizing agents and the modified Fenton reaction. Hydroxyl radicals play a vital role in recycling the oxidized form of the sensitizing agents (e.g., Fe(III)) and could accelerate organic matter mineralization, producing bioavailable intermediate products. The rate of oxidation of the organic compounds in our experimental condition was slowed down due to the presence of inhibiting ions, such as chloride, by the scavenging of the hydroxyl radical produced via the Fenton-like mechanism.

Stable carbon isotopic signatures in bicarbonate-buffered systems in neutral pH conditions were observed to approach δ¹³C values of the lactate used (≈−25.0‰) with the increasing DIC yield. The isotopic signatures in acidic conditions did not show a similar distinctive pattern to those observed in neutral pH conditions and need more quantitative work.