CC BY
38
UDC 571.27: 581.6 https://doi.org/10.15407/biotech13.04.049
BIOREMOVAL OF TOXIC CHROMIUM(VI) VIA DARK HYDROGEN FERMENTATION OF MULTICOMPONENT ORGANIC WASTE
V. M. Hovorukha
O. A. Havryliuk Zabolotny Institute of Microbiology and Virology
G. V. Gladka of the National Academy of Sciences of Ukraine, Kyiv
O. B. Tashyrev
E-mail: [email protected]
Received 15.05.2020 Revised 02.07.2020 Accepted 31.08.2020
Thermodynamic calculations allow determining optimal metabolic pathways for microbial extraction of toxic soluble hexavalent chromium compounds from contaminated sewage.
The purpose was to predict theoretically and confirm experimentally the possibility of hazardous Cr(VI) removal by hydrogen producing microbiome with simultaneous destruction of multicomponent organic waste and hydrogen synthesis.
The gas composition was determined by the standard gas chromatography method. The redox potential (Eh) and pH of the medium were measured potentiometrically. The Cr(VI) concentration was measured by a photocolorimetric method.
The multicomponent organic waste was effectively destroyed by hydrogen producing microbiome at the absence of chromium. The hydrogen fermentation cycle was not significantly inhibited by addition of Cr(VI). After complete microbial reduction of soluble CrO42- to insoluble Cr(OH)3nH2O^ the metabolic parameters returned to initial values.
The optimal pathway of microbial detoxification of toxic Cr(VI) compounds was thermodynamically predicted and experimentally confirmed. The high efficiency of Cr(VI) removal by strict anaerobic hydrogen producing microbiome via dark hydrogen fermentation of multicomponent organic waste was demonstrated. The obtained results can be used for development of novel environmental biotechnology of chromium-containing sewage purification and simultaneous destruction of environmentally hazardous organic waste as well as obtaining of eco-friendly energy carrier biohydrogen.
Key words: thermodynamic prediction, environmental biotechnologies, hydrogen fermentation,
biohydrogen synthesis, toxic chromium(VI) compounds, microbial reduction of chro-mate, multicomponent organic waste destruction.
Chromium (VI) is one of the hazardous contaminant of the water and soil environment [1]. Human activity has caused the widespread distribution of chromium compounds in natural and man-made ecosystems [1-3]. Chromium is widely applied in the chemical and metallurgic industry, including production of refractory materials, electroplating, stainless steel formation, tanning agents and pigments production [4]. The environmental pollution by toxic chromium compounds is steadily increasing due to the accumulation of huge volumes of chromium-containing industrial sewage.
For example, sewage from electroplating facilities contains toxic forms of hexavalent chromium — chromate and dichromate [5]. Ingestion of toxic sewage to natural ecosystems causes death of living organisms and drastic effects on human health [1, 6]. Chromium(VI) compounds have been found in different industrial sewage. The concentration of chromium(VI) in sewage ranges from 0.2 to 0.65 ppm [7]. The Cr(VI) concentrations were in the range of 0.00190 ± 0.0020 and 0.0010 ± 0.0006 ppm in the influent and effluent sewage samples respectively collected from Nacogdoches
Waste Water Treatment Plant (East Texas, USA) [8].
The toxicity of Cr(VI) for plants, animals, microorganisms as well as human is caused by high oxidizing potential and solubility of chromium, mobility across the membranes in living organisms with effects such as loss of membrane integrity or inhibition of the electron transport chain [1, 9]. Therefore, the development of effective methods for purification of aquatic and soil ecosystems from hexavalent chromium compounds is of a great interest to modern science and industry. Physico-chemical techniques of chromium containing sewage purification such as flotation, chemical precipitation, coagulation-flocculation, ion exchange and membrane filtration are efficient and rapid, but often require considerable economic costs and are environmentally hazardous [10]. The biotechnological techniques are industrially promising, cost-effective and environmentally friendly alternative ones. They are based on the microbial reduction of soluble chromium(VI) to non-toxic insoluble chromium(III) in the form of Cr(OH)3.nH2O^ [9]. For the first time, the possibility of microbial reduction of hexavalent chromium was shown for Pseudomonas spp. by Romanenko and Korenkov over 42 years ago [11]. It is known that microorganisms isolated from polluted industrial sites are able to reduce Cr(VI). The strain Cellulosi microbium cellulans DQ-4 (EU816697) isolated from metallurgical plants of Bagnoli (Naples, Italy) reduced Cr(VI) with high efficiency (92%) at the initial concentration of 50 ppm and was resistant to 150-250 ppm Cr(VI) [9]. Despite the considerable interest to the microbial methods of chromium extraction, scientists have not yet been able to develop biotechnology to completely purify contaminated sewage at high chromium(VI) concentration.
The accumulation of huge amount of organic waste is another global environmental problem today. First of all, they are hazardous due to the accumulation of toxic products of decay (hydrogen sulfide, mercaptans, NH3, fatty acids) and proliferation of pathogenic microorganisms. Physico-chemical methods such as incineration, pelletisation, deposition at the landfill sites [12] are not capable to destroy enormous volumes of organic waste [13], since their rate of accumulation far exceeds the duration of their decomposition.
Herein, we propose the novel methodological approach for rapid and effective microbial purification of chromium-containing sewage and simultaneous destruction of multicomponent organic waste (MOW) with obtaining of high-energy carrier — biohydrogen. Biohydrogen is defined as molecular hydrogen obtained via the fermentation of organics.
Thus, the purpose of our study was to predict theoretically and confirm experimentally the possibility of hazardous chromium(VI) removal by hydrogen producing microbiome via dark anaerobic fermentation of multicomponent organic waste.
Materials and Methods
Thermodynamic prediction as a universal method to justify the possibility of microbial interaction with metals. Thermodynamic prediction was applied as the theoretical background to justify the possibility of Cr(VI)removal from sewage by hydrogen-producing anaerobic microbiome. It allows substantiating the most effective mechanisms of soluble toxic chromium(VI) ions detoxification by microbial reduction to insoluble chromium(III) in the form Cr(OH)3nH2O^. The Pourbaix diagram (the diagram of the stability of the elements as aqueous electrochemical system in "pH-Eh" coordinates) was used as a basis for thermodynamic prediction of microbial interaction with chromium(VI) compounds [14].
Bioremoval of 50 ppm Cr(VI) during fermentation of model multicomponent organic waste. Effectiveness of chromium(VI) removal during hydrogen fermentation of MOW was determined using glass bioreactor (0.5 L). We used a spore-forming anaerobic hydrogen-producing microbiome (HPM) as the low-potential donor system. For this purpose, MOW were prepared as follows: 25 g of potato peels, 5 g of tomato tops, 5 g of apple peels, 10 g of rotten apricots, 5 g of meat (Fig. 1) waste and 10 g of granular microbial preparation (GMP) were mixed in a sterile bioreactor (0.5 L).
The GMP was developed as granular preparation containing selected from the digested sludge of methane tank community of spore-forming anaerobic hydrogen-producing microorganisms as well as nutrients and regulators of microbial metabolism.
Fig. 1. Mixture of model multicomponent organic waste for hydrogen fermentation:
А — the potato peels; B — the tomato tops; C — the apple peels; D — the rotten apricots; E — the meat; F — the glass bioreactor after waste loading
All components of model waste were also heated up for 20 minutes on water bath at 90 °C to eliminate of non-spore forming microorganisms and mixed with 250 ml of boiled tap water. Bioreactors were sealed by rubber stoppers with metal securers and cultivated at 30 °C during four days. The gas synthesized in the fermentation process was collected in the gas-holder. For this purpose, we used the gas-holder with rubber pipe and a needle. The rubber stopper of the bottle was pierced by the needle and the gas was transferred to gas-holder through the pipe, connected to the needle (Fig. 2).
Air was the initial gas phase in the bottles. Solution of NaHCO3 was added to the reactor to provide values of pH close to pH = 7.0 for optimal pathway of fermentation of MOW.
Chromium(VI) solution was added to the glass bioreactor (500 ml) when microorganisms reached the final stage of metabolic activity. It was estimated by the decrease of hydrogen yield (concentration of H2 in the gas phase of bioreactor decreased from 36% to 19%) and the redox potential began to rise. After that chromium solution was added to the bioreactor to the final concentration 50 ppm Cr(VI) in the form of K2CrO4 salt. The experiment was
carried out in triplicate. Statistical analysis was carried out using Excel and Origin 8.5.1. software.
Bioremoval of 100 ppm Cr(VI) from liquid phase during final stage of fermentation of MOW. To investigate the possibility of microorganisms to remove 100 ppm Cr(VI) (in form of K2CrO4 salt) the liquid phase of anaerobic batch bioreactor (240 L) was collected [15]. The metabolically active culture liquid containing fermented MOW was sampled in the final phase (80 hours) of fermentation and used to investigate the dynamics of Cr(VI) removal.
For this purpose, 300 ml of culture liquid with low metabolic activity (the Eh increased to -120 mV, the H2 concentration in the gas phase of bioreactor decreased) was sampled from the bioreactor (240 L) and transferred to the laboratory glass bioreactor (0.5 L). The gas phase (0.2 L) of the bioreactor was replaced by argon. The bioreactor was hermetically closed and cultivated at 30 °C during 12 hours to provide strict anaerobic conditions for optimal growth of hydrogen producing microorganisms. After that, chromium solution was added to the cultivation system to the final concentration 100 ppm Cr(VI).
Fig. 2. The measurement of the gas volume:
А — the bioreactor; B — the gas-holder; C — the water seal
The preparation of chromium solution and determination of Cr(VI) concentration. The solution of 30 000 ppm Cr(VI) was used as a stock. It was prepared by K2CrO4 salt dissolution in distilled water in the volumetric flask. The Cr(VI) concentration was determined by a colorimetric method with 1,5-diphenylcarbazide (DPC) in the range of 1.0-25.0 ppm Cr(VI). The method is based on the formation of colored violet complex caused by the interaction of chromium(III) compounds with DFCat the pH < 1.0 [16].
The control of metabolic parameters of fermentation process. The concentration of hydrogen in the gas phase was analyzed by the standard gas chromatography method [17]. The chromatograph equipped with two steel columns — one (I) for the analysis of N2, O2, N2 and CH4, the second (II) — analysis of CO2.
Parameters of column: I — l = 3 m, d = 3 mm, molecular sieve 13X (NaX); II — l = 2 m, d = 3 mm, with carrier Porapak-Q; column temperature — 60 °C, the temperature of evaporator — +75 °C, detector (katharometer) — +60 °C, the current detector — 50 mA. Carrier gas — argon, gas duct velocity — 30 cm3/min.Volume of gas samples was: the first column — 2.5 cm3, the second — 1 cm3.
The redox potential (Eh) and pH of the medium were measured potentiometrically. For this purpose, the determination of pH and Eh was performed using an EZODO MP-103 universal ionomer with remote
electrodes and a temperature sensor. To measure pH and Eh, we used the combined electrodes with BNC connectors — PY41 and PO50 models, respectively. Before the measurement the electrodes were tested by convention standard buffer solutions. For pH check conventional pH-buffer solutions were used: solution of KHC2O4H2C2O42H2O (pH = 1.68); mixture of NaH2PO4 and K2HPO4 (pH = 6.86); Na2B4O710H2O (pH = 9.18). Standart pH-buffers were prepared according to the producer's manual (OJSC "Kyiv plant RIAP"). For the validation of Eh measurement three buffer solutions were used. The first one was ferricyanide, with Eh = +273 mV (13.5 g/l K3[Fe(CN)6] and 3.8 g/l K4[Fe(CN)6].3H20), The first one was Fe(II) citrate (10.0 g/l with Eh = -150 mV and the third one was Ti(III) citrate (15.0 g/l) with Eh = -440 mV [18].
The following metabolic parameters of MOW fermentation were controlled:
1) duration of fermentation period (T, days);
2) biohydrogen yield (L); 3) coefficient of waste destruction (Kd) meaning the ratio of initial and final weight of waste [19].
Results and Discussion
The following theoretical positions are the basis for the development of novel biotechnologies of chromium containing sewage purification. All metabolic oxidation-reduction processes are carried out by microorganisms in
the field of thermodynamic stability of water (Fig. 3). In neutral conditions (pH = 7.0) water is stable in the range of standard values of the redox potential E0' from -414 to +814 mV. The lower limit of water stability is determined by the reversible reaction of proton reduction to molecular hydrogen: 2H+ + 2e = H2; E0'= -414 mV. The upper limit of water stability is determined by the reversible oxidation reaction of water oxygen to molecular oxygen: 2H2O = O2 + 4H+ + 4e; E0' = +814 mV (Fig. 3, a). In case when the redox potential of the aqueous solution is higher than +814 mV, the oxygen of the water acts as a reducing agent. It is oxidized to O2, and the Eh returns to the initial value +814 mV. In case if the potential is lower than -414 mV, the proton of water acts as an oxidizing agent. It is reduced to H2 (Fig. 3, b). Water acts as a binary redox buffer that is stable in the range of the standard redox
potentials from -414 to +814 mV [14, 16]. In accordance with our hypothesis microbial growth in the presence of the oxidized forms of toxic metals and interaction with them are theoretically permissible if the redox potential of the system formed by the metal and its reduced form is inside of the field of thermodynamic stability of water [16].
Herein, our methodological approach is based on the thermodynamic justification of the optimal microbial metabolic pathway to reduce high potential CrO42-(Eo' = +555 mV) to insoluble non-toxic Cr(OH)3nH2O^ and low potential (Eo' = -414 mV) anaerobic destruction of organic waste with obtaining high-energy carrier — biohydrogen.
The oxidation levels of soluble form of chromium are +6, +3 and +2 (Fig. 3). Chromium in the +2 level is the reducing agent. It reduces H+ of water to molecular H2
Eh, mV
+1600 +1400
+1200
+1000 -
+800 -
+600
+400
-600
-1200
HCrOj- + 7H + 3e 4H;0 (1
|
Cr-.07:"+ 8H++ 6e = = 2Ct'(OH|s + H,0 (3) z — — —
= ( V
v — ** / zr MH -i-4e
j - , JZ ^'=+814 nf
/ / *
/ (31 z - — a
_
Cr-iO-r1" + 4H++ 6e = _ T « T _
M ! = + 7H .n n\ — 1 , .J 1 —
L— 1 A i, mV
_ _ _ _ _ / —— r. - y us
r'l-n 2- -L in llH rtx «tr-1-J- 3 a —
_ _ __ Z ± W 1 ^ IJ - 1J111 1 Lj±l = Cr(OH)3*nHiO (4 z _
- > V E ' = +555 mV
L
- ^ __
_ __ _ zt 2e
- -- - 1
1 1 V T + -, —+— £o '= -4 14 m V, _
— 1 b
c pH=7,0 s - -
C V, s -
r- + ze = \ z zt
----- 1 _
U)
Cr(OH , + 3lT+ 3e = Cr + 3H, Of/) —-
1 1 i ~T
2 4 6 8 10 1 2
pH
Fig. 3. The Pourbaix Diagram showing thermodynamic stability of chromium redox-forms in solutions: upper and lower limits of stability are determined by reactions a and b
(Fig. 3, No. 5). In the +6 state it is a powerful oxidizing agent, because the redox potential of hexavalent chromium compounds is very high (Fig. 3, reactions No. 1-4) [20]. For example, the standard redox potential (E0') of the reduction of Cr(VI) to Cr(III) is +555 mV (Fig. 3, reaction No. 4).
Chromium in the +6 level is considerably important due to its carcinogenicity and toxicity to living organisms [9]. In aqueous solutions Cr(VI) can exist in different forms (HCrO4-, Cr2O72-,CrO42-) depending on the value of pH (Fig. 3, reactions No. 1-4). The precipitation of chromium(VI) by microorganisms is impossible, because the insoluble chromium(VI) compounds do not exist. Therefore, the reduction of CrO42- to insoluble Cr(OH)3-nH2OI is theoretically acceptable and suitable microbial pathway of toxic chromium(VI) removal from solutions. The Eh of this reaction is +555 mV and it takes place within the field of water stability. This reaction (Fig. 3, reaction 4) is limited by pH value that should not be lower than 6.5 (Fig. 3) [16]: CrO42- + (n-1)H2O + 5H+ + 3e = Cr(OH)3nH2O^, Eo'(pH > 6,5) = +555 mV (Fig. 3, reactions No. 4).
Thus, the redox potentials of the reaction underlying the microbial removal of chromium(VI) is inside thermodynamic stability of water field (Fig. 3).
As it follows from our theoretical positions, microorganisms can realize only those reactions of interaction with metals which redox potential is inside of the field of thermodynamic stability of water (Fig. 3). Consequently, the reactions No. 1, 2, 3, and 4 are theoretically acceptable, and reactions No. 5, 6, and 7 are inaccessible to microorganisms. Thus, microorganisms can reduce chromium(VI) compounds only to Cr(III). Further reduction of Cr(III) to Cr(II) and Cr(0) by microorganisms is impossible. All compounds of Cr(VI) are the strongest oxidizing agents that explains their high toxicity to microorganisms (Fig. 3, reactions No. 1-4). Detoxification of chromium(VI) compounds is possible only via their removal from aqueous solutions by microorganisms. It is known that the removal of metals is possible both via the formation of insoluble compounds (hydroxides, carbonates, sulfides, etc.) without changing their valence as well as via the reduction to insoluble compounds [16, 21]. However, insoluble chromium(VI) compounds do not exist. All Cr(VI) compounds are stable in
the form of anions HCrO4- (pH = 0.0...3), Cr2O72- (pH = 0.0...6.0), CrO42- (pH = 6.0...12.0) and do not precipitate in the form of sulphides, carbonates in a wide range of pH (0.0... 12.0). This clearly implies that removal of Cr(VI) anions from solutions is possible only via reduction to insoluble compounds, for example, Cr(OH)3nH2O^ (Fig. 3, reaction No. 4). This mechanism of chromate removal is possible only in neutral or slightly alkaline conditions, because under acidic conditions (pH < 4.0), the Cr(VI) compounds will be reduced to soluble Cr3+ cation (Fig. 3, reaction No. 5). It is known that the effectiveness of redox reactions is proportional to the potential difference between the donor and acceptor systems [22]. Obviously, the hydrogen producing strict anaerobic microorganisms create the lowest redox potential (Eh) of the donor system. Thus, microorganisms create AEo'= -414 mV via hydrogen fermentation of carbohydrates (starch, etc.). The redox potential difference between the donor and acceptor systems is maximum (AEo' = 969 mV) in the related redox reaction:
Н2 = 2Н+ + 2е; (E0' = -414 mV) CrO42- + (n-1) H2O + 5H+ + 3e = = Cr(OH)3nH2O (E0' = +555 mV).
According to the thermodynamic calculations dark hydrogen fermentation is shown to be the most effective pathway of soluble Cr(VI) detoxification. Therefore we suggest the removal of toxic CrO42- via fermentation of model multicomponent organic wastes by anaerobic hydrogen producing microbiome as the most effective pathway.
Our model organic waste is multicomponent and simulates real municipal waste because it contains substrates of both plant and animal origin. The microbiome that produces hydrogen includes microorganisms of the genus Clostridium [23], Bacillus, Enterobacter, Enterococcus [24, 25], Escherichia [26-28], etc. The GMP was used as the inoculum, the fermentation of the waste and CrO42-bioremoval occurred by spore-forming microbiome. Most likely, it consisted mainly of two genus of microorganisms — Clostridium and Bacillus.
In our investigation, HPM have carried out rapid and effective MOW fermentation in 0.5 L bioreactor in control conditions (without chromium) with simultaneous regulation of microbial metabolism (Fig. 4, A). This was evidenced by the redox-potential (Eh) decrease from +325 mV to -320 mV
during the first 32 hours of MOW fermentation and high concentration of H2 (36%) in the gas phase of the bioreactor. The pH value was decreased from 7.15 to 5.0 after 24 hours of cycle. Then it was adjusted and stabilized in the range 6.5-7.3 to preserve optimal conditions for MOW destruction and simultaneous hydrogen production (Fig. 4, A). The MOW was effectively destroyed by hydrogen producing microbiome in control conditions at the absence of chromium(VI). The following parameters were obtained: biohydrogen yield was 81 L/kg of solid organic waste, coefficient of waste destruction Kd = 92.
change after addition of 50 ppm Cr(VI). It was in the range 25-26% after complete chromium(VI) bioremoval (Fig. 6, A). Under these conditions, the H2 yield was 75 L/kg of solid food waste, coefficient of waste destruction Kd = 83. Thus, the process of fermentation was not significantly inhibited by 50 ppm Cr(VI) (Fig. 4, B, Fig. 6, A). The H2 yield and efficiency of waste destruction (Kd) were decreased on 7% and 10% consequently. The duration (T) of fermentation cycle in both variants of the experiment was 82 hours (Fig. 4).
The active synthesis of gas, the formation of foam, as well as the intensive destruction of
A
B
H2, % 50-1
pH Eh, mV
40-
30-
20'
10-
KMM
ft s 1 , ....... \Y ! —
\\ \ fl pi i
\ '.N \ i * L V T
\ » 1 r ■ . f I 7- T
1 \ \l * •é.
2 \ \ St t î - :
i \ l.h y r ■
\ f.J •r ■
r- —r ->— — — — — —
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
r 4110
- 200
-0
- -2110
1- -400
U 10 20 3U 40 5(1 611 70 SO 'HI Time, hours
H,, % 50-1
40'
50 ppm
30'
20-
10-
■ I'H ; KMM t
\ / » i V J
\ / H 1IM T T 11 S s
1 \ \ T 1 v N Î:
\ S i T* I. -è y' \ i i
\ A i T \
\ \ 1 i m
V'" \_ _ F.h ,1 1-
k ■ -/ 1
: ¥
|)ll l".h. m Y 7.5 p400
7.0
6.5
6.11
5,5
5.11
4.5
4.0
3.5
3.0
201)
-0
- -200
<--400
0 10 20 30 40 50 6(1 70 8<l 9(1 Time, hours
Fig. 4. The efficiency of solid waste fermentation in control conditions without Cr(VI) (A)
and at the presence of 50 ppm Cr(VI) (B)
(RMM — the points indicate when the regulators of microbial metabolism were added)
As it was expected, the addition of 50 ppm of Cr(VI) in the form of K2Cr2O4 at the beginning of the final phase of fermentation (50 hours) caused a rapid increase of the Eh from -320 mV to +311 mV (Fig. 4, B; Fig. 6, A). However, the redox-potential was autoregulated by HPM and decreased from +311 mV to -5 mV after 1.5 hours of 50 ppm Cr(VI) addition (Fig. 6, A). It should be noted that microorganisms overcame the negative effect of high-potential CrO42-. Hydrogen fermentation resumed after Cr(VI) reduction.
The pH increased from 5.7 to 6.5 immediately after the addition 50 ppm of Cr(VI), but the medium was acidified to 5.8 and 5.36 at 62 and 82 hours consequently during further fermentation (Fig. 4, B). The concentration of H2 did not significantly
solid particles of waste in both variants of the experiment (Fig. 5) also indicated the absence of inhibition of the fermentation process by the addition of 50 ppm Cr(VI).
At the same time, the high efficiency of 50 ppm Cr(VI) removal by HPM was demonstrated in our study (Fig. 6, A). The concentration of soluble chromium(VI) was decreased from 50 ppm to 27 ppm after only one hour of fermentation. The efficiency of Cr(VI) bioremoval was 100% after 53 hours of waste fermentation (Fig. 6, A). The removal of Cr(VI) from the solution is suggested to take place due to its reduction. It is evidenced by the appearance of grey precipitate typical for Cr(III) hydroxide, i.e Cr(OH)3nH2O^.
To investigate the effectiveness of Cr(VI) bioremoval during the final phase of hydrogen
fermentation of solid multicomponent organic waste by liquid phase (i.e. culture medium) was important issue of our research. The final parameters of the end fermentation process in large bioreactor (240 L) testified to the decrease of metabolic activity of microorganisms. The concentration of H2 in the gas phase of the bioreactor decreased from 36% to 19%, the value of the Eh increased from -340 mV to -120 mV, as well as the gas volume increase was ended. After that the liquid phase was sampled (after 80 hours of fermentation of MOW) into the glass bioreactor (0.5 L) and cultivated for 12 hours at 30 °C to renew the partial metabolic activity of the HPM. Despite the weak
metabolic activity, the microbiome showed high reductase activity and produced H2. The initial parameters before the addition of Cr(VI) were as follows: Eh = -190 mV, 16% of H2 (Fig. 6, B), the absence of gas volume synthesis as well as the complete destruction of solid particles of waste. The parameters indicated the final phase of dark hydrogen fermentation of MOW. As expected, the addition of 100 ppm of Cr(VI) causes the high potential stress for strict anaerobic hydrogen producing microorganisms (Fig. 6, B). The addition of 100 ppm Cr (VI) caused an increase of the redox potential from -190 to + 325 mV. Then, the concentration of Cr(VI) has decreased to analytical zero (over
Fig. 5. The absence of inhibition of MOW fermentation by toxic hexavalent chromium compounds
(70 hours of fermentation)
Cr(Vi), ppni 60 n
A
B
Eh, raV II,, % Cr(VI), ppm
504030 201 10
di "> KX.......~
p
50 ppm Cr(VI)
i -
F *
> /i > : Z1 4
> 27 ppm Cr(YI)
Eh
i
A
50
-r-
51
—i-1-
52
Time, hours
N O.OppmCr(VI)
500 400 300 200 100 0 -1011 -200 -300 -400 -500
53
r35 -30 -25 -20 -15 -10 5 L0
80
10
100 ppm Cr(VI)
k— X - — 68 ppm Cr(V - V / 1)
- 1 1 F.h'- "1 » » \ \
. >......i . 1 ,7 M, i....... ......... i......•........ f t - X X X • ■
- X X 0.0 ppm Cr(VI) \
Eh, niV H„% r35
1 3
Time, hours
400 300
200 100
-100 -200
-300 -400
-500
1-30 25 -20 -15 -10 5 1-0
Fig. 6. Bioremoval of Cr(VI) during final phase of hydrogen fermentation of multicomponent food waste:
A — addition of 50 ppm Cr(VI) to the glass bioreactor (0.5 L) after 50 hours of hydrogen fermentation; B — addition of 100 ppm Cr(VI) to the liquid phase sampled from bioreactor (240 L) after 80 hours of hydrogen fermentation
4 hours of fermentation). The decrease of Cr(VI) concentration correlated with the decrease of the Eh from +325 to -99 mV (Fig. 6, B). This result is quite natural, because the rate and efficiency of the reaction is determined by the potential difference between the donor and acceptor systems in accordance with the thermodynamic prognosis [16]. The standard redox potential of the chromate reduction reaction is +555 mV, and the potential of the investigated HPM was -190 mV. Microorganisms completely reduced the chromate in just 4 hours due to the high difference in redox potentials (745 mV).
To summarize, the effectiveness of chromium(VI) bioremoval from culture medium of bioreactor was 100% and continued 4 hours. The process of chromium removal correlated with the decrease of the redox potential (from +325 mV to -99 mV) (Fig. 6, B). The concentration of H2 was not changed during the process of chromium(VI) bioremoval (Fig. 6, B). So, the toxic Cr(VI) did not significantly affect on the fermentation process.
Thus, we have experimentally confirmed the theoretical background of effective soluble Cr(VI) removal by hydrogen producing microbiome. The investigation of optimal microbial pathway of chromium-containing sewage purification from soluble hexavalent
chromium compounds is of a great interest to modern science [5, 9, 29-31]. We suggested the novel methodological approach based on thermodynamic calculations to provide complete detoxification of contaminated environments from toxic chromium compounds.
The optimal pathway of microbial detoxification of toxic chromium(VI) compounds was thermodynamically predicted and experimentally confirmed. The high efficiency of Cr(VI) removal by strict anaerobic hydrogen producing microbiome via dark hydrogen fermentation of multicomponent organic waste was demonstrated. The obtained results can be used as a basis for development of novel environmental biotechnology of chromium-containing sewage purification and simultaneous destruction of environmentally hazardous organic waste as well as obtaining of eco-friendly energy carrier — biohydrogen.
The study was funded under the budget theme of Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine 06-80 "Properties of extreme microorganisms and their biotechnological potential". The authors declare no conflict of interests.
REFERENCES
1. Oliveira H. Chromium as an Environmental Pollutant: Insights on Induced Plant Toxicity. J. Bot. 2012, V. 2012, P. 1-8. https://doi. org/10.1155/2012/375843
2. Bhalerao S., Sharma A. Chromium: As an Environmental Pollutant. Int. J. Curr. Microbiol. App. Sci. 2015, 4 (4),732-746.
3. Tchounwou P. B., Yedjou C. G., Patlolla A K., Sutton D. J. Heavy Metal Toxicity and the Environment. In: Luch A. (eds). Molecular, Clinical and Environmental Toxicology. Experientia Supplementum, Springer, Basel. 2012, V. 101, P. 133-164. https://doi. org/10.1007/978-3-7643-8340-4_6
4. Lunk H. J. Discovery, properties and applications of chromium and its compounds. Chem. Texts. 2015, 1 (1), 1-17. https://doi. org/10.1007/s40828-015-0007-z
5. Peng H., Leng Y., Cheng Q., Shang Q., Shu J., Guo J. Efficient removal of hexavalent chromium from wastewater with electro-reduction. Processes. 2019, 7 (1), 41, 1-12. https://doi.org/10.3390/pr7010041
6. Sanyal T, Kaviraj A., Saha S. Deposition of chromium in aquatic ecosystem from effluents of handloom textile industries in Ranaghat-Fulia region of West Bengal, India. J. Adv.
Res. 2015, 6 (6), 995-1002. https://doi. org/10.1016/j.jare.2014.12.002
7. Tang A. N., Jiang D. Q., Jiang Y., Wang S. W, Yan X. P. Cloud point extraction for highperformance liquid chromatographic speciation of Cr(III) and Cr(VI) in aqueous solutions. J. Chromatogr. A. 2004, 1036 (2004), 183-188. https://doi.org/10.1016Zj.chroma.2004.02.065
8. Onchoke K. K., Sasu S. A. Determination of Hexavalent Chromium (Cr(VI)) Concentrations via Ion Chromatography and UV-Vis Spectrophotometry in Samples Collected from Nacogdoches Wastewater Treatment Plant, East Texas (USA). Adv. Environ. Chem. 2016, V. 2016, P. 1-10. https://doi. org/10.1155/2016/3468635
9. Focardi S., Pepi M., Focardi S. E. Microbial Reduction of Hexavalent Chromium as a Mechanism of Detoxification and Possible Bioremediation Applications. Biodegrad. — Life Sci. 2013, P. 321-347. https://doi. org/10.5772/56365
10. Kurniawan T. A., Chan G. Y. S., Lo W. H., Babel S. Physico-chemical treatment techniques for wastewater laden with heavy metals. Chem. Eng. J. 2006, 118 (1-2), 83-98. https://doi. org/10.1016/j.cej.2006.01.015
11. Romanenko V. I., Koren'kov V. N. Pure culture of bacteria using Chromates and bichromates as hydrogen acceptors during development under anaerobic conditions. Mikrobiologiia. 1977, 46 (3), 414-417. (In Russian).
12. Sharholy M., Ahmad K., Mahmood G., Trive-di R. C. Municipal solid waste management in Indian cities — A review. Waste Manag. 2008, 28 (2), 459-467. https://doi. org/10.1016/j.wasman.2007.02.008
13. Guerrero L. A., Maas G., Hogland W. Solid waste management challenges for cities in developing countries. Waste Manag. 2013, 33 (1), 220-232. https://doi.org/10.1016/j. wasman.2012.09.008
14. Pourbaix M. Atlas of electrochemical equilibria in aqueous solutions. Houston:NACE International. Mater. Sci. Forum. 1974, P. 43-54.
15. Hovorukha V., Tashyrev O., Tashyreva H., Havryliuk O, Bielikova O., Iastremska L. Increase in efficiency of hydrogen production by optimization of food waste fermentation parameters. Energetika. 2019, 65 (1). https://doi.org/httpsV/doi.org/10.6001/ energetika.v65i1.3977
16. Hovorukha V., Havryliuk O., Tashyreva H., Tashyrev O., Sioma I. Thermodynamic Substantiation of Integral Mechanisms of Microbial Interaction With Metals. Ecol. Eng. Environ. Prot. 2018, 55-63. https://doi. org/10.32006/eeep.2018.2.5563
17. Berezkin V. G. Chemical Methods in Gas Chromatography. Amsterdam-Oxford-New Yourk-Tokio: Elsevier B. V. 1983, 311 p.
18. Zehnder A. J. B., Wuhrmann K. Titanium (III) Citrate as a Nontoxic Oxidation-Reduction Buffering System for the Culture of Obligate Anaerobes Vertebrate Central Nervous System : Same Neurons Mediate Both Electrical and Chemical Inhibitions. Science. 1975, 194 (6), 1165-166. https:// doi.org/10.1126/science.793008
19. Tashyrev O., Govorukha V., Havryliuk O. The Effect of Mixing Modes on Biohydrogen Yield and Spatial pH Gradient At Dark Fermentation of Solid Food Waste. Ecol. Eng. Environ. Prot. 2017, V. X, P. 53-62.
20. Shupack S. I. The chemistry of chromium and some resulting analytical problems. Environ. Health Perspect. 1991, 92 (1), 7-11.
21.Ashida J., Higashi N., Kikuchi T. An electronmicroscopic study on copper precipitation by copper-resistant yeast cells. Protoplasma. 1963, 57 (1-4), 27-32. https:// doi.org/10.1289/ehp.91927
22. Lehninger A. L. Principles of biochemistry. 2nd ed. New York: Worth Publishers. 1993, 1013 p.
23. Collet C., Adler N., Schwitzguébel J. P., Péringer P. Hydrogen production by Clostridium thermolacticum during continuous fermentation of lactose. Int. J. Hydrogen Energy. 2004, 29 (14), 1479-1485. https://doi.org/10.1016/j. ijhydene.2004.02.009
24. Eder A. S., Magrini F. E, Spengler A., da Silva J. T., Beal L. L., Paesi S. Comparison of hydrogen and volatile fatty acid production by Bacillus cereus, Enterococcus faecalis and Enterobacter aerogenes singly, in co-cultures or in the bioaugmentation of microbial consortium from sugarcane vinasse. Environ. Technol. Innov. 2020, V. 18. https://doi. org/10.1016/j.eti.2020.100638
25. Mazareli R. C. da S., Sakamoto I. K., Silva E. L., Varesche M. B. A. Bacillus sp. isolated from banana waste and analysis of metabolic pathways in acidogenic systems in hydrogen production. J. Environ. Manage. 2019, V. 247, P. 178-186. https://doi.org/10.1016/j. jenvman.2019.06.040
26. Valle A., Cantero D., Bolívar J. Metabolic engineering for the optimization of hydrogen production in Escherichia coli: A review. Biotechnol. Adv. 2019, 37 (5), 616-633. https://doi.org/10.3389/fbioe.2019.00351
27. Mirzoyan S., Trchounian A., Trchounian K. Hydrogen production by Escherichia coli during anaerobic utilization of mixture of lactose and glycerol: Enhanced rate and yield, prolonged production. Int. J. Hydrogen Energy. 2019, 44 (18), 9272-9281. https:// doi.org/10.1016/j.ijhydene.2019.02.114
28. Poladyan A., Trchounian A. Characterization of Hydrogen Production by Escherichia coli Wild-type and Mutants of Hydrogenases Utilizing Xylose as Fermentation Substrate. Bioenergy Res. 2019, 12 (4), 1033-1341. https://doi.org/10.1007/s12155-019-10035-4
29. Ilias M., Rafiqullah I. M., Debnath B. C., Mannan K. S., Hoq M. M. Isolation and Characterization of Chromium(VI)-Reducing Bacteria from Tannery Effluents. Indian J. Microbiol. 2011, 51 (1), 76-81. https://doi. org/10.1007/s12088-011-0095-4
30. Narayani M., Shetty K. V. Chromium-resistant bacteria and their environmental condition for hexavalent chromium removal: A review. Crit. Rev. Environ. Sci. Technol. 2013, 43 (9), 955-1009. https://doi.org/10. 1080/10643389.2011.627022
31. Verma T., Garg S. K., Ramteke P. W. Genetic correlation between chromium resistance and reduction in Bacillus brevis isolated from tannery effluent. J. Appl. Microbiol. 2009, 107 (5), 1425-1432. https://doi. org/10.1111/j.1365-2672.2009.04326.x
Б1ОВИЛУЧЕННЯ ТОКСИЧНОГО ХРОМУ(У1) В ПРОЦЕС1 ЗБРОДЖУВАННЯ БАГАТОКОМПОНЕНТНИХ ОРГАН1ЧНИХ В1ДХОД1В
В. M. Говоруха, O. A. Гаврилюк, Г. В. Гладка, O. Б. Таширев,
1нститут мшробмлогй i вiрусологil iM. Д.К. Заболотного НАН Укра!ни, Ки!в
E-mail: [email protected]
Термодинамiчнi розрахунки дають змогу визначити оптимальш метаболiчнi шляхи для мiкробного вилучення токсичних розчинних сполук шестивалентного хрому з контамшова-них смчних вод.
Метою дослщження було теоретично пе-редбачити та експериментально пiдтвердити можлив^ть вилучення небезпечного Cr(VI) за допомогою воденьсинтезувального мiкробiому у процесi одночасно! деструкцй багатокомпо-нентних органiчних вiдходiв та синтезу водню.
Склад газу визначали, використовуючи стандартний метод газово! хроматографы. Ре-докс-потенцiал (Eh) та рН середовища вимiрю-вали потенщометрично. Концентрацiю Cr(VI) визначали фотоколориметричним методом.
Багатокомпонентнi органiчнi вщходи було ефективно зруйновано воденьсинтезувальним мiкробiомом за вiдсутностi хрому. Цикл вод-невого зброджування несуттево iнгiбувався додаванням Cr(VI). Пiсля повного мшробного вiдновлення розчинного Cr042- до нерозчинно-го Cr(0H)3nH20l метаболiчнi параметри повер-нулися до початкового рiвня.
Оптимальний шлях мшробного вилучення сполук токсичного Cr(VI) термодинамiчно об-Грунтовано та експериментально тдтверджено. Була показана висока ефектившсть вилучення Cr(VI) за допомогою облшатно анаеробного воденьсинтезувального мшроб^му у процес1 водневого зброджування багатокомпонентних органiчних вiдходiв. Отримаш результати мож-на використовувати для розроблення новггшх природоохоронних бiотехнологiй очищення хроматвмiсних стiчних вод та одночасно! деструкцй екологiчно небезпечних оргашчних вiдходiв, а також з метою отримання еколопч-но чистого енергоноия — бiоводню.
Ключовi слова: термодинамiчне прогнозуван-ня, природоохороннi б^технологп, водневе зброджування, синтез бмводню, сполуки токсичного хрому^!), мiкробне вiдновлення хромаив, деструкцiя багатокомпонентних органiчних вiдходiв.
БИОИЗВЛЕЧЕНИЕ ТОКСИЧНОГО ХРОМА(У1) В ПРОЦЕССЕ СБРАЖИВАНИЯ МНОГОКОМПОНЕНТНЫХ ОРГАНИЧЕСКИХ ОТХОДОВ
В. M. Говоруха, А. A. Гаврилюк, Г. В. Гладка, А. Б. Таширев
Институт микробиологии и вирусологии им. Д. К. Заболотного НАН Украины, Киев
E-mail: [email protected]
Термодинамические расчеты позволяют определить оптимальные метаболические пути для микробного извлечения токсичных растворимых соединений шестивалентного хрома из контаминированных сточных вод.
Целью исследования было теоретически предсказать и экспериментально подтвердить возможность извлечения опасного Cr(VI) с помощью водородсинтезирующего микробиома в процессе одновременной деструкции многокомпонентных органических отходов и синтеза водорода.
Состав газа определяли, используя стандартный метод газовой хроматографии. Редокс-потенциал (Eh) и рН среды измеряли потенцио-метрически. Концентрацию Cr(VI) определяли фотоколориметрическим методом.
Многокомпонентные органические отходы были эффективно разрушены водородсинтези-рующим микробиомом при отсутствии хрома. Цикл водородного сбраживания несущественно ингибировался внесением Cr(VI). После полного микробного восстановления растворимого Cr04 - до нерастворимого Cr(0H)3-nH2 Ol метаболические параметры вернулись к исходному уровню.
Оптимальный путь микробного извлечения соединений токсичного Cr(VI) был термодинамически обоснован и экспериментально подтвержден. Была показана высокая эффективность извлечения Cr(VI) облигатно анаэробным водородсинтезирующим микробиомом в процессе водородного сбраживания многокомпонентных органических отходов. Полученные результаты могут быть использованы для разработки новейших природоохранных биотехнологий очистки хроматсодержащих сточных вод и одновременной деструкции экологически опасных органических отходов, а также получения экологически чистого энергоносителя — биоводорода.
Ключевые слова: термодинамическое прогнозирование, природоохранные биотехнологии, водородное сбраживание, синтез биоводорода, соединения токсичного хрома^^, микробное восстановление хроматов, деструкция многокомпонентных органических отходов.
