processes Detoxiﬁcation of Copper and Chromium via Dark Hydrogen Fermentation of Potato Waste by Clostridium butyricum Strain 92

: The accumulation of various types of waste containing both organic and inorganic metal-containing compounds is extremely hazardous for living organisms. The possibility of polymer degradation, biohydrogen synthesis, and metal detoxiﬁcation via the dark fermentation of model potato waste was investigated. For this purpose, the strict anaerobic strain was isolated and identiﬁed as Clostridium butyricum . The high efﬁciency of dark hydrogen fermentation of potatoes with yield of hydrogen in 85.8 ± 15.3 L kg − 1 VS potato was observed. The copperand chromium salts solutions were added to the culture ﬂuid to obtain the concentrations of 50, 100, and 200 mg L − 1 Cu(II) and Cr(VI) in the active phase of growth (19 h of cultivation). Metals at a concentration of 200 mg L − 1 inhibited the fermentation process the most. The hydrogen yield decreased in 7.2 and 3.6 times to 11.9 ± 2.1 and 23.8 ± 5.6 L kg − 1 VSpotato in the presence of 200 mg L − 1 Cu(II) and Cr(VI), respectively. The efﬁciencies of the chromium bioremoval in all variants of the experiment were 100%, and those of copper bioremoval were about 90%. A pure culture of strict anaerobes Clostridium butyricum strain 92 was used for the ﬁrst time for the detoxiﬁcation of metals. The presented results conﬁrmed the possibility of this promising strain application for industrial H 2 production and the bioremediation of contaminated sites.


Introduction
Currently, a significant environmental problem is the accumulation of waste of organic and inorganic origins in the environment [1,2]. Both of them pose a significant threat to living organisms, from bacteria [3] to humans [4]. The accumulation of carbohydratecontaining organic waste in landfills causes a high metabolic activity of microorganisms and, as a consequence, the emergence of toxic compounds (organic acids, alcohols, mercaptans, hydrogen sulfide, ammonia, etc.) and the contamination of the environment [5]. However, organic, easy biodegradable wastes are promising for the obtaining of energy sources, C 6 H 12 O 6 → CH 3 CH 2 CH 2 COOH + 2CO 2 + 2H 2 .
This process is accompanied by the accumulation of reducing equivalents and the maximum decrease of the redox potential of the culture fluid (2H + + 2e = H 2 , E o = −414 mV). Such an anaerobic process can be used for the detoxification of inorganic metal-containing wastes due to the highly reducing conditions created by hydrogen-synthesizing microorganisms. Inorganic wastes, such as oxidizing metal salts [17], are even more hazardous than organic ones. They have a toxic effect immediately, as they have oxidizing [18], bactericidal [19], carcinogenic [20], fungicidal [21], hepatotoxic [22], and others properties. The most common metals pollutants are chromium [23] and copper [24]. They are released into the environment from deposits and mining sites, as well as from industrial wastewater. A new and previously little-studied direction of metal detoxification is the reduction or immobilization of metals to insoluble and therefore non-toxic compounds with the participation of strict anaerobic microorganisms. At first glance, oxidizing metals that create high redox potential values in the media are incompatible with the existence of strict anaerobes that grow at low redox potential values (E h = −200 ÷ −400 mV). However, spore-forming anaerobic bacteria (e.g., Clostridium) are able to autoregulated the redox potential in the absence of oxygen, followed by their active growth and development [25]. Clostridium strict anaerobic bacteria can be used for the purification of model metal containing sewage from toxic metals. The thermodynamic prediction allows one to theoretically substantiate the most effective mechanism of the microbial reduction of soluble toxic chromium(VI) and copper(II) ions to insoluble and non-toxic compounds such as insolvable Cr(OH) 3 ·nH 2 O and Cu 2 O. The precipitation of soluble forms of Cu(II) by microbial exometabolites without changing the valence (redox state) is another way of copper bioremoval (CuCO 3 (s), Cu(OH) 2 (s), etc.). The standard redox potentials of chromium(VI) and copper(II) reduction are equal to +555 mV and +480 mV, respectively [26]: CrO 4 2− + (n − 1)H 2 O + 5H + + 3e = Cr(OH) 3 ·nH 2 O↓ E o = +555 mV, 2Cu 2+ + H 2 O + 2e = Cu 2 O↓ + 2H + E o(pH = 4,6) = +480 mV.
We are considering a binary redox reaction of metal reduction by microorganisms. In this reaction, metabolically active microorganisms are the donor system, and highpotential compounds of chromium and copper are the acceptor system. It is obvious that the efficiency of metal reduction is proportional to the potential difference between the acceptor and donor systems. It follows that microorganisms with the lowest values of Processes 2022, 10, 170 3 of 16 the redox potential reduce chromium(VI) and copper(II) with the maximum efficiency. It is well-known that the lowest redox potential (E o = −414 mV) is created by hydrogenforming anaerobic bacteria of the Clostridium genus: 2H + + 2e = H 2 , E o = −414 mV [27]. It is obvious that a large potential difference between the acceptor and donor systems ensures the fastest possible reduction of chromium and copper compounds. From this, it is obvious that precisely clostridia is the most effective to remove soluble chromium and copper compounds from solutions with the simultaneous degradation of waste and the obtaining of high energy environment friendly fuel biohydrogen.
Therefore, the aim of the study was to investigate the efficiency of the potato fermentation and detoxification of chromium and copper compounds via dark hydrogen fermentation by Clostridium butyricum strain 92.

Isolation and Identification of the Hydrogen-Synthesizing Strain
The strain of strict anaerobic hydrogen-synthesizing microorganisms was isolated from the culture fluid during the fermentation of multicomponent organic wastes containing potato peel as one of the main substrates. A granular microbial preparation (GMP1) was used as an inoculum for the isolation of hydrogen-synthesizing microorganisms [8]. The pure culture of strict anaerobes was isolated by the ten-fold dilution method [28], followed by inoculation by the Hangate roll tube method (rotation of the vials with agarizated media in an inert gas atmosphere) [29]. The volume of the vials was 120 mL. To create anaerobic conditions in the vials, they were purged with argon at a rate of 0.5 L min −1 for 3 min. Reduced Fe(II) citrate was added to the medium to the final concentration of 500 mg L −1 to ensure a low redox potential (−220 ÷ −250 mV). A sodium resazurinate (0.01%) indicator was used to detect anaerobic conditions in an agar medium. NA medium (HiMedia Laboratories Pvt. Ltd., Mumbai, Maharashtra, India) with potato starch (10 g L −1 ) was used for the isolation of pure culture of strict anaerobic microorganisms-polymer destructors (starch). The culture was transplanted three times on the reduced agarizated nutrient medium under strict anaerobic conditions to ensure the purity. The taxonomic position of the investigated microorganisms was determined by molecular biological methods. The microorganisms were cultured for 24 h in a Nutrient Broth liquid nutrient medium (HiMedia Laboratories Pvt. Ltd., Mumbai, Maharashtra, India) for the obtaining of the microbial biomass. The isolation of bacterial DNA was performed from daily culture using the DNeasy UltraClean Microbial Kit (Qiagen) according to the manufacturer's instructions. The purity of the obtained DNA sample was determined spectrophotometrically using a NanoDrop OneC UV/Vis Spectrophotometer by absorption at wavelengths of 260, 230, and 280 nm, as well as using horizontal agarose gel electrophoresis. The visualization of electrophoresis was performed using a UV transilluminator MultiDoc-It Digital Imaging System (UVP). The obtained DNA was stored in TE buffer at −20 • C. Amplification of 16S rRNA gene sequences was performed by the classical method [30]. The amplification of the sequences was performed using oligonucleotide primers 8F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ). The BioRad thermal cycler was used for amplification. The polymerase chain reaction (PCR) reaction mixture consisted of 5.0 µL of PCR buffer (10X DreamTaq Green Buffer, Thermo Scientific TM , Thermo Fisher Scientific Inc., Waltham, MA, USA), 5.0 µL of dNTP, 2 µL of primer mixture, 0.5 µL of Taq polymerase (DreamTaq DNA polymerase, Thermo Fisher Scientific Inc., Waltham, MA, USA), added immediately before the reaction, 1.0 µL of DNA sample, and 36.5 µL of deionized water. Thirty cycles of the amplification of the following temperature profile were performed: DNA denaturation (94 • C, 20 s), primer renaturation (56 • C, 15 s), and elongation (polymerization) (72 • C, 90 s). Nucleotide sequences of 16S rRNA genes were sequenced at Eurofins Genomics (Louisville, KY, USA). The obtained nucleotide sequences of 16S rRNA genes of microorganisms were used for the determination of closely related species and phylogenetic analysis. To do this, the obtained sequences of 16S rRNA genes of bacterial isolates were compared with those deposited in the GenBank database using the BLAST software package. Related microorganisms were determined by calculating the pairwise similarity in percentage as the ratio of the number of matching/analyzed nucleotides of 16S rRNA genes of each strain with the compared bacteria. Nucleotide sequence correction, as well as the removal and replacement of degenerate nucleotides, were performed in the Bioedit program. A multiple alignment of the studied and closely related sequences was performed in ClustalX based on MEGA (Molecular Evolutionary Genetic Analysis). The phylogenetic position was determined by constructing trees (dendrograms), which showed the position of the studied strain among closely related and typical species (MEGAX program). The tree was constructed using the MEGAX program by the neighbor-joining method using 1000 bootstrap tests. Sequences of external groups of microorganisms (outgroups), which were more remotely related to the studied strains, were used for rooting the tree. The phylogenetic tree was built and edited in the MEGAX program.

Preparation of Chromium and Copper Solutions and Their Analytical Determination
To prepare 250 mL of a copper citrate solution, 29.4 g of CuSO 4 ·5H 2 O (Sigma Aldrich, pure for analysis) was dissolved in 100 mL of distilled water. Then, 30 g of dry trisubstituted sodium citrate was added and stirred until completely dissolved. The solution had a pH value of 5.5, and therefore, it was neutralized with Na 2 CO 3 to pH = 7.0. The resulting solution was added to a 250 mL Mora flask and made up to the mark with distilled water. The citrate chelation procedure was performed to stabilize the Cu 2+ cation in a soluble form in the neutral or alkaline pH range. The final concentration of Cu(II) was 30.0 mg L −1 . The concentration of Cu(II) in the solution was determined by a qualitative reaction with a PAR (4-(2-Pyridylazo) resorcinol; Sigma-Aldrich), followed by titration with ethylenediaminetetraacetic acid (EDTA) [31].
To prepare 250 mL of a chromate solution, 28 g of K 2 CrO 4 salt (Sigma Aldrich, pure for analysis) were dissolved in 200 mL of water. The solution was poured into a Mora flask after complete dissolution and made up to 250 mL. The final concentration of Cr(VI) was 30.0 mg L −1 . The concentration of Cr(VI) in the solution was determined by a colorimetric method using a qualitative reaction with 1,5-diphenylcarbazide (DFC) [32]. Metal solutions were sterilized by heating in a water bath in a hermetically sealed vial for 15 min at a temperature of 100 • C.

The Process of Fermentation and Metals Insertion
Clostridium butyricum strain 92 was cultivated in a mineral nutrient medium with the addition of sterile potato (0.5 × 0.5 cm) as the model substrate for a common potato waste and source of carbon and energy in a ratio of 3:1 (150 mL of the nutrient medium and 50 g of potatoes). The potato was sterilized, because it is (especially potato peels) a source of mixed facultative and strict anaerobic microorganisms. Mineral salts NH 4 Cl (1 g L −1 ), K 2 HPO 4 (2 g L −1 ), and K 2 SO 4 (0.5 g L −1 ) were used as sources of basic nutrients. The inoculum was also grown on a potato medium for 24 days. The inoculum (3 mL, 2% from the liquid phase) was added to each vial. A solution of copper(II) and chromium(VI) was added to the vial in the active phase of culture growth (19 h of fermentation) to final concentrations of 50, 100, and 200 mg L −1 . Thus, the effect of the concentration gradients of Cu(II) and Cr(VI) (50, 100, and 200 mg L −1 ) on the growth of the culture in a mineral medium with potato was studied. The efficiency of metals bioremoval by the Clostridium butyricum strain 92 was determined by decreasing the concentration of soluble Cu(II) and Cr(VI) in the culture fluid. The initial gas phase in the vials was argon. The determination of gas composition was performed by the standard gas chromatography method [33]. The following metabolic parameters were determined as following [34]: pH, redox potential (Eh), volume, and composition of the gas phase (H 2 , O 2 , N 2 , and CO 2 ), as well as the concentrations of total carbon and Cu(II) and Cr(VI) in the culture fluid. The concentration of dissolved organic carbon (DOC) was determined by a permanganate method [35]. A medium without metals was used as a control variant of the experiment. The coefficient of Processes 2022, 10, 170 5 of 16 potato degradation (reduction of dry weight after degradation) and the efficiency of the fermentation process (yields of H 2 and CO 2 from 1 L kg −1 VS potato ) were also determined.

Data Analysis
All experimental measurements were performed in triplicate. Mean values (AV) and standard deviations (SDs) were determined with a 95% confidence level. Each value is presented as the mean ± SD. The significance of the influence of metals on the effectiveness of dark hydrogen fermentation were determined by the one-way ANOVA test with the post-hoc test using the statistical platform of Microsoft Excel. The graphs were constructed using OriginLab software. The phylogenetic three was constructed using MEGAX program.

Characteristic of the Strict Hydrogen-Synthesizing Anaerobic Strain
The strictly anaerobic hydrogen-synthesizing strain was isolated from the culture fluid containing a different species of spore-forming bacteria. The strain withstood heating at 90-100 • C for 20 min and grew only in low-potential conditions (−250 mV). The strain was also able to grow in the presence of dextrose, starch, and potatoes as the only sources of carbon and energy. The strain 92 was identified as Clostridium butyricum species. The phylogenetic dendrogram of the species affiliation of the strain is presented in Figure 1.   According to the dendrogram (Figure 1), the strain of anaerobic microorganisms 92 was in a common cluster and was highly similar to strains Clostridium butyricum JCM 1391 (NR_113244.1), Clostridium butyricum VPI3266 (NR_042144.1) тa Clostridium butyricum ATCC 19398 (NR_112170.1), the similarities to which were 100%, 99.81%, and 99.03%, respectively. The obtained results allowed carrying the studied isolate 92 to the Clostridium butyricum species. The GenBank accession number for strain nucleotide sequence is OL744406.

The Influence of Metals on the Gas Synthesis and the Efficiency of Potato Fermentation Process by Clostridium butyricum Strain 92
The main criteria for the efficiency of the anaerobic fermentation process were the concentration of biohydrogen in the gas phase, the volume of gas synthesized by microorganisms, as well as the decrease of the potato weight. All vials contained argon in the gas phase at the beginning of the experiment. The gas phase did not change and consisted only of argon throughout the experiment in the control vials without inoculum. The changes in the composition of the gas phase were not observed throughout the experiment. The values of pH and the redox potential, as well as the concentration of DOC, also did not change throughout the experiment in the control vials without inoculum. Microorganisms synthesized only H 2 and CO 2 under experimental conditions. Hydrogen synthesis took place from the first hours of cultivation and for 5 h the hydrogen concentration was 18.4 ± 3.9 vol.% under control conditions (Figure 2a,b). The H 2 concentration was 36.5 ± 1. The inhibition of H 2 synthesis was also observed in the presence of chromium compounds. Thus, the maximum concentration of H 2 in the control was 38.4 ± 1.9 vol.% for 48 h of fermentation and 34.2 ± 2.5, 33.2 ± 6.2, and 32.3 ± 3.1 vol.% in the presence of 50, 100, and 200 mg L −1 Cr(VI), respectively (Figure 2b). The concentration of CO 2 also increased slightly in the presence of metals compared to in the control (Figure 2d). Increasing the concentration of metals led to a decrease of the efficiency of the fermentation process, namely the reduction of the gas yield and K d ( Table 1). As can be seen from Table 1, metals affected not only the concentration of hydrogen, but also the cumulative H2 production (Figure 3). Obviously, there was no statistically significant difference between the synthesis of hydrogen under control conditions in the presence of both 50 mg L −1 Cu(II) and Cr(VI). In this case, no inhibition of biohydrogen yield was observed (Figure 3a   As can be seen from Table 1, metals affected not only the concentration of hydrogen, but also the cumulative H 2 production (Figure 3). Within each row, the means (±SD, n = 3) with different letters (a-f) show the difference between the treatments based on the post-hoc test at p ≤ 0.05, * Maximum H2 concentration after metals insertion.

The Influence of Metals on Metabolic Parameters of the Potato Fermentation Process
The dynamics of pH, Eh, and the DOC were studied during the fermentation of potato by Clostridium butyricum 92 strain both in control conditions without metals and in the presence of Cu(II) and Cr(VI) (Figure 4). Therefore, the measurements of metabolic parameters were presented in control conditions during this time. The pH decreased from 7.32 ± 0.4 (beginning of the cultivation) to 5.1 ± 0.25 (168 h) under control conditions (Figure 4a,b).
The redox potential also decreased intensively and, at two days (48 h), was −281 ± 23 mV (Figure 4b,c). The initial concentration of DOC under control conditions was 2681 ± 334 mg L −1 and, within three days (72 h), decreased to 920 ± 246 mg L −1 .
The intensive degradation of potatoes was observed after the 72 h of cultivation, as evidenced by the increase in the concentration of soluble organic compounds to 1250 ± 162.5 and 1550 ± 177.5 mg L −1 for 96 and 120 h of cultivation, respectively. It should be noted that the rate of degradation of the substrate and its accumulation may correlate in the active phase of growth. Therefore, an increase in the concentration of DOC on the third day (72 h) may indicate a slowdown in the growth of anaerobic bacteria.

The Influence of Metals on Metabolic Parameters of the Potato Fermentation Process
The dynamics of pH, Eh, and the DOC were studied during the fermentation of potato by Clostridium butyricum 92 strain both in control conditions without metals and in the presence of Cu(II) and Cr(VI) (Figure 4). Therefore, the measurements of metabolic parameters were presented in control conditions during this time. The pH decreased from 7.32 ± 0.4 (beginning of the cultivation) to 5.1 ± 0.25 (168 h) under control conditions (Figure 4a,b).
The redox potential also decreased intensively and, at two days (48 h), was −281 ± 23 mV (Figure 4b,c). The initial concentration of DOC under control conditions was 2681 ± 334 mg L −1 and, within three days (72 h), decreased to 920 ± 246 mg L −1 .
The intensive degradation of potatoes was observed after the 72 h of cultivation, as evidenced by the increase in the concentration of soluble organic compounds to 1250 ± 162.5 and 1550 ± 177.5 mg L −1 for 96 and 120 h of cultivation, respectively. It should be noted that the rate of degradation of the substrate and its accumulation may correlate in the active phase of growth. Therefore, an increase in the concentration of DOC on the third day (72 h) may indicate a slowdown in the growth of anaerobic bacteria.   The values of metabolic parameters of potato fermentation differed significantly in the presence of a gradient of metal concentration. Solutions of copper and chromium were inserted to the culture fluid in the active phase of fermentation (for 19 h). The Eh of the culture fluid was very low during this phase. The metals had the greatest effect on the value of the redox potential of the culture fluid. The Eh rapidly increased from −268 ± 34 to +160 ± 12 mV immediately after the insertion of Cu(II) solution at a concentration of 50 mg L −1 , as well as from −290 ± 35 to +212 ± 22 mV (Figure 4b) after the insertion of 200 mg L −1 Cu(II), and did not decrease significantly until the end of fermentation. A similar situation was observed after the insertion of a chromate solution. Thus, the E h rapidly increased from −290 ± 34 to +50 ± 12 mV after the insertion of 50 mg L −1 Cr(VI). However, the redox potential stabilized and again amounted to −225 mV after 5 h, which was quite an optimal value for the growth of Clostridium butyricum stain 92. However, a significant increase of the redox potential from −266 ± 35 mV (before insertion) to +321 ± 22 mV (after insertion) at 200 mg L −1 Cr(VI) was observed, which slowed down the fermentation process and substrate consumption. Thus, in contrast to control conditions, the concentration of soluble organic compounds was not decreased in the presence of 200 mg L −1 Cr(VI). The DOC concentration did not change significantly for six days after chromium insertion, because the growth of microorganisms was inhibited by this concentration of chromium. No statistically significant changes in the DOC concentration were observed in the presence of 200 mg L −1 Cu(II). Thus, similar patterns of the influences of both chromium and copper on the degradation of the substrate were observed (Figure 4e,f). The patterns of change in pH during fermentation were similar in all variants of the experiment (Figure 4a,b). However, a less intense decrease in pH was observed in the presence of 200 mg L −1 Cu(II) and Cr(VI). For example, the pH decreased from 7.25 ± 0.4 to 5.2 ± 0.3 in the presence of 50 mg L −1 Cu(II) and from 7.34 ± 0.4 to 5.65 ± 0.3 for 168 h of cultivation in the presence of 200 mg L −1 Cu(II) (Figure 4a).

Bioremoval of Metals from a Medium by Clostridium butyricum Strain 92
Metals were inserted into the potato medium in the active phase of microbial growth at 19 h of cultivation without metals. The efficiency of the bioremoval of metal compounds depended on the concentration of each metal. The bioremoval rate of copper and its efficiency at 50 mg L −1 Cu(II) were very high. The duration of bioremoval was only 5 h, and the concentration of Cu(II) decreased from 48.9 ± 4.4 to 3.5 ± 1.1 mg L −1 (Figure 5a). However, a complete bioremoval of Cu(II) could not be achieved. The bioremoval efficiency was 92.8%. A similar result was also observed with the removal of 50 mg L −1 Cr(VI). Chromium was completely removed by microorganisms during 4 h after its insertion (Figure 5b). The duration of the bioremoval of 100 mg L −1 Cr(VI) was 10 h, and 100% toxic Cr(VI) was removed. However, its concentration decreased from 98 ± 15.2 to 15.3 ± 4.1 mg L −1 already for 1 h of cultivation (Figure 5d). The duration of removal of 100 mg L −1 Cu(II) was 9 h, but the concentration of Cu(II) did not change after 9 h and stayed at 12.5 ± 1.6 mg L −1 (Figure 5c  The duration of bioremoval was seven days (144 h). However, during the first eight hours after application, 89.9% of Cr(VI) was removed (the concentration decreased from 205.7 ± 21.8 to 20.7 ± 7.0 mg L −1 ; Figure 5e). The decrease of the chromium concentration correlated with a decrease of the Eh to permissible value for the growth of anaerobic microorganisms (−100 ÷ −200 mV). The redox potential did not decrease to negative values in the variants with Cu(II) during the bioremoval process ( Figure 5).
The process of potato fermentation in the presence of metals is shown in Figure 6.
Processes 2022, 10, x FOR PEER REVIEW 12 of 16 The process of potato fermentation in the presence of metals is shown in Figure 6. Thus, an unadapted strict anaerobic strain was able to efficiently remove Cu(II) and Cr(VI) compounds from the culture fluid via the dark hydrogen fermentation of the potato.
The chemical detoxification of metals was completely excluded, because in the control versions without inoculum, the concentration of metals remained at a constant level from the moment of metals insertion until the end of the experiment. The metal concentration remained at baseline (about 50, 100, and 200 mg L −1 ) throughout the experiments in the absence of inoculum. Physicochemical changes in the state of copper and chromium compounds in the solution were not observed.

Discussion
Strict anaerobic bacteria are very promising for the biogas production and fermentation of different types of organic waste such as food [36], fruits [37], date seeds [38], and rice straw [39]. Bacteria of the Clostridium species are one of the most common representatives used for the efficient synthesis of biohydrogen [40]. Herein, we also showed a high efficiency of potato fermentation by Clostridium butyricum strain 92. The H2 yield was as high as −91.4 ± 9.4 L kg −1 VSpotato. The presented studies also showed a high yield of hydrogen. For example, the hydrogen yield was 61.0 L kg −1 VSpotato achieved via the hydrogen fermentation of the food waste of a western-style restaurant [41]. An example of super-high hydrogen productivity is the production of 226 L kg −1 by Clostridium thermocellum from sugarcane bagass (SCB) [42]. However, this substrate is high-energy and easier to ferment than polymeric compounds.
In addition, microorganisms are used to detoxify toxic metals in wastewater [43,44]. However, an incomplete removal of metals from solutions is usually achieved. For example, the Cupriavidus gilardii strain CR3 was able to remove only 27% of Cu(II) from Thus, an unadapted strict anaerobic strain was able to efficiently remove Cu(II) and Cr(VI) compounds from the culture fluid via the dark hydrogen fermentation of the potato.
The chemical detoxification of metals was completely excluded, because in the control versions without inoculum, the concentration of metals remained at a constant level from the moment of metals insertion until the end of the experiment. The metal concentration remained at baseline (about 50, 100, and 200 mg L −1 ) throughout the experiments in the absence of inoculum. Physicochemical changes in the state of copper and chromium compounds in the solution were not observed.

Discussion
Strict anaerobic bacteria are very promising for the biogas production and fermentation of different types of organic waste such as food [36], fruits [37], date seeds [38], and rice straw [39]. Bacteria of the Clostridium species are one of the most common representatives used for the efficient synthesis of biohydrogen [40]. Herein, we also showed a high efficiency of potato fermentation by Clostridium butyricum strain 92. The H 2 yield was as high as −91.4 ± 9.4 L kg −1 VS potato . The presented studies also showed a high yield of hydrogen. For example, the hydrogen yield was 61.0 L kg −1 VS potato achieved via the hydrogen fermentation of the food waste of a western-style restaurant [41]. An example of super-high hydrogen productivity is the production of 226 L kg −1 by Clostridium thermocellum from sugarcane bagass (SCB) [42]. However, this substrate is high-energy and easier to ferment than polymeric compounds.
In addition, microorganisms are used to detoxify toxic metals in wastewater [43,44]. However, an incomplete removal of metals from solutions is usually achieved. For example, the Cupriavidus gilardii strain CR3 was able to remove only 27% of Cu(II) from the contaminated waste water [45]. Strain Bacillus cereus RC-1 was able to remove 16.7%, 38.3%, 81.4%, and 40.3% Cu(II), Zn 2+ , Cd 2+ , and Pb 2+ , respectively, with an initial concentration of only 10 mg L −1 at pH 7.0 [46]. Thus, most of the presented data relate to the extraction of metals by aerobic or facultative anaerobic microorganisms.
For the first time, we used a pure culture of strict anaerobe to extract copper(II) and chromium(VI) from solutions. We have previously shown that anaerobic hydrogen- [26,31] and methane-synthesizing microbial communities [34] are able to remove copper from solutions. In addition, the possibility of hexavalent chromium reduction in an anaerobic hydrogen-synthesizing bioreactor was shown [47]. The only one research on the use of a mixed culture (Thiobacillus sp. and Clostridium sp.) to remove Cu(II) was found [48]. That is why we first investigated the patterns of copper and chromium compounds bioremoval by Clostridium butyricum strain 92 of strict anaerobes. In addition, we had a clear theoretical approach and assumed that the bioremoval of copper and chromium by obligate anaerobes would be very effective. The removal of metals can be carried out simultaneously by several mechanisms. First, the reduction of Cu 2+ to Cu 2 O (CrO 4 2− to Cr(OH) 3 ) may be due to the significant difference of the redox potentials between the acceptor and donor systems. Really, the acceptor systems (the metals) have very high values of potentials (i.e., E 0 = +486 mV of Cu 2+ and E 0 = +555 mV of CrO 4 2− ), and strict anaerobes create very low redox potentials, such as −200 ÷ −300 mV [26,49]. Second, the accumulation of metals inside a microbial cells also takes place due to the stereochemical analogy with macroelements [50]. The third mechanism by which copper can be removed is the precipitation of copper carbonate CuCO 3 by the CO 3 2− anion [31]. Several mechanisms of metals bioremoval can be implemented simultaneously during the growth of the strict anaerobes in the presence of oxidizing metal. We showed a 100% bioremoval efficiency for 50, 100, and 200 mg L −1 Cr(VI) and a more than 90% bioremoval efficiency for 100 and 200 mg L −1 Cu(II) as a result of potato fermentation by strict anaerobe Clostridium butyricum strain 92.

Conclusions
A technologically promising strictly anaerobic strain was isolated, capable of the degradation of organic waste, biohydrogen production, and the detoxification of metalcontaining waste. A pure culture of strict anaerobes Clostridium butyricum strain 92 was used for the first time for the detoxification of copper and chromium. The presented results confirmed the possibility of this strain application for the bioremediation of contaminated by dangerous organic compouns sites and industry H 2 production. The presented results change the idea of the current approach for the microbial detoxification of metals and concentrate on the study of anaerobic metabolism of fermentative bacteria for this purpose.