- Email: [email protected]
Biological treatment of alkaline industrial waste waters
Process Biochemistry 35 (2000) 595 – 602 www.elsevier.com/locate/procbio
Biological treatment of alkaline industrial waste waters S. Baccella a, G. Cerichelli b, M. Chiarini b, C. Ercole a, E. Fantauzzi a, A. Lepidi a, L. Toro c, F. Veglio` d,* a Dipartimento di Biologia di Base ed Applicata, Uni6ersita` dell’Aquila, 67010 Coppito, L’Aquila, Italy Dipartimento di Chimica, Ingegneria Chimica e Materiali, Uni6ersita` dell’Aquila, 67040 Monteluco di Roio, L’Aquila, Italy c Dipartimento di Chimica, Uni6ersita’ La Sapienza, p.le A. Moro, 5, 00185 Rome, Italy d Dipartimento di Ingegneria Chimica e di Processo, ‘G.B. Bonino’, Uni6ersita’ degli Studi di Geno6a, 6ia Opera Pia, 15, 16145 Genoa, Italy b
Received 5 January 1999; received in revised form 9 August 1999; accepted 14 August 1999
Abstract The biotechnological treatment of alkaline waste waters (AWW) resulting from the production of caprolactam by the SNIA-viscosa process has been studied. The pollutant in the AWW is 80 – 120 g litre − 1 cyclohexanecarboxysulphonate (CECS) sodium salt with a COD up to 325 000 mg litre − 1. Bacterial strains have been isolated which are able to grow on AWW and to degrade the largest possible range of organic compounds. These strains have been screened for their performance in lowering the COD and degrade the sulphonic bonds. Combinations of strains have also been verified. The strains have been compared in cultures both in shake flasks and in a laboratory scale fermenter. The results showed that: (a) a 1/10 dilution of AWW with water permitted microbial growth coupled with decrease in COD and carboxylic concentration (representative of several organic compounds such as cyclohexanecarboxysulphonate, 2-aminocapronic and o-aminocapronic acids); (b) the polymerised caprolactam molecules are exhaustively degraded; (c) very similar results are found both in shake flask tests and in the lab scale fermenter but with different kinetics; and (d) pretreatment of alkaline waters with CaCl2 and lowering the pH with H3PO4, implement the kinetics and yields of the process in terms of degradation of COD and carboxylic compounds. The experiments gave very preliminary results and led to some suggestions for the development of a chemical and biological process to treat this kind of AWW. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Alkaline waste waters; Biological treatment; Bio-recalcitrant compounds
1. Introduction Industrial waste waters contain both organic and mineral contaminants having toxic effects on the microbiological agents of wastewater treatment plants or resisting biological attack and mineralization. This is the case of ‘alkaline waters’ produced by chemical plants for the production of several organic molecules of commercial concern such as caprolactam [1,2]. The properties of the alkaline waters are (a) high or very high alkalinity; (b) concentration of salts at levels approaching or above the bactericidal threshold; (c) very high COD values; and (d) the presence of recalcitrant organic molecules [3,4]. * Corresponding author. Tel.: +39-10-3532583; fax: + 39-103532586. E-mail address: [email protected] (F. Veglio`)
Alkaline waters produced by the caprolactam plants polymerising the fibres of nylon-6 possess every one of these properties so that such waters belong to the group of the industrial waste waters which hinder industrial development, for economic, environmental and social reasons. This problem is very important in southern Italy where about 150 000 tons of this industrial waste has been disposed of in tanks as a result of the absence of a chemical or biological process that could be used to treat it. Thus much effort is being invested in research to resolve this problem. This paper presents preliminary research on the treatment of alkaline waste waters (AWW) by means of a combination of a chemical pretreatment and a biological process with specific microbial strains.
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 1 1 2 - 0
596
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
2. Materials and methods
2.1. Alkaline waste waters The AWW was sampled from a storage stock standing for ca. 6 years in the south of Italy. Their chemical composition and properties, summarised in Table 1 [5], provide (a) the concentration of compounds produced during nylon-6 synthesis, including the Na salts of cycloexanecarboxysulphonate isomers and derivatives [2] plus the Na salt of
Table 1 Composition of the alkaline waste waters (AWW)a Minimum pH Density Dry weight at 105°C (%) Water (%) Cyclohexanecarboxylic sodium salt (%)* N-Hexahydro benzoyl-5-aminocaproic acid sodium salt (%)* 2-Aminocaproic acid sodium salt (%)** Cyclohexanecarboxysulphonate (CECS) bisodium salt (%)** Caprolactame*** Benzoic acid sodium salt (%)* N-Hexahydrobenzoic 5-amino valeric acid sodium salt (%)* Adipic acid sodium salt (%)* 1-Cyclohexancarboxylic acid sodium salt (%)* Cyclometilhexancarboxylic acid sodium salt (%)* 5-Ethyl di-hydro 2 (3H) furanone (%)*** 5-Methyl valero lactone (%)***
Maximum
10 1.1 22 66 0.2
12.5 1.2 34 78 2
0.1
1.8
0.3
4.0
8.0
12.5
0.02 0.01 0.01
1.6 0.5 0.3
0.01 0.02
0.25 0.25
0.03
0.25
0.01
0.25
0.01
0.25
Other organic prodocts (excluded PBC-dioxines-pesticides-hy0.005 drocarbons aromatics polycycles) (%) Sodium sulphate (%) 0.7 Sodium chloride (%) 2.0 Ammonia (%) 0.02 Total alkalinity expressed by Na2CO3 2.5 (%) Total phenols (mg litre−1) 60 Monochloro phenol (mg litre−1) 1 2,4-Dichlorophenol (mg litre−1) 0.01 2,4,6-Trichlorophenol (mg litre−1) 0.01 Toluene (mg litre−1) 0.05 Benzene (mg litre−1) 0.01 N-Hexane (mg litre−1) 0.01 Nitrites (mg litre−1) 50 Nitrates (mg litre−1) 150 COD (mg litre−1) 325 000
0.02
1.5 4.5 0.15 9.0 150 20 2 1 10 0.5 0.5 100 350 350 000
a *Neutral components; **no extractable components; and ***acid components.
2-aminocapronic acid. The concentration of these compounds is expressed as carboxylic group concentration (CGC) ranging from 0.40–0.87 M; (b) the pH value over 11; (c) the total salt content over 280 g litre1; and (d) the COD exceeding 300 000 mg litre − 1.
2.2. Micro-organisms: isolation and maintenance Microbial strains were sub-cultured five-fold in a rotatory shaker at 200 rpm and at 37°C on a liquid medium AYM (AWW/tap water 1/10, yeast extract 5 g 1itre − 1, MgCl2 0.1 g litre − 1, pH adjusted at 7.0 by H3PO4 85%). Isolation of pure colonies was effected on solid medium (AYM supplemented with Bacto Agar 2%) by serial dilution (from 10 − 4 to 10 − 7) of the enrichment cultures. Isolated colonies were verified for the capability to grow on AYM with high growth rates and lowering of COD. These strains were chosen as promising candidates for the present purpose. Both pure cultures and the cocktails of strains have been maintained by sub-culturing on AYM. The associations of strains have been used considering that the capability to compete with other bacterial populations is a relevant property for bacterial cultures which are expected to operate in a common wastewater treatment plant. Bacterial strains used in this work [5] were either: (a) associations of more than one strain and/or (b) monospecific pure cultures. Group (a) includes the strains nicknamed Co 27 and Mix 6. Co 27, taken from enrichment cultures of water samples from a sulphurous spring around L’Aquila, is constituted of an association of three strains which, after isolation in pure culture, have been named Co 27-1, Co 27-2 and Pa 7 (see below). Mix 6 is the entire pool of strains obtained through the enrichment cultures of samples taken from a wastewater treatment plant of a chemical industry. In group (b), the following pure cultures are included: Co 27 -1: this strain grows on solid medium plates with flat colonies bordered with a brown halo; the cells are short rods 0.9–1.1 mm in diameter, gram negative; Co 27 -2: this strain grows on solid medium plates with rough powdered colonies, irregularly bordered; the cells are rods 0.8–1.8 mm in diameter, gram negative; Pa 7 : this strain grows on solid medium plates with round microcolonies of milky appearance; the cells are very short rods, almost cocci, 0.8–0.9 mm in diameter, gram negative. According to the morphophysiological characterisation, performed also with BioMerieux tunnels, it has been tentatively attributed to the genus Pseudomonas.
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
597
late a lab-scale fermenter (Biostat B, Braun Biotech International) containing 2 litres of sterile liquid AYM prepared with untreated AWW. The following culture conditions were adopted: 37°C, stirring 300 rpm, pH= 7 (automatically monitored and maintained by adding either H3PO4 or 1N KOH), pO2 = 10% of saturation. Samples of 20 ml were taken out for chemical and biological analysis.
2.6. Bacterial biomass, COD and CGC analyses
Fig. 1. A typical titration result for carboxylic group concentration (CGC).
2.3. Pre-treatment of AWW Some pre-treatment schemes have been verified for AWW, with the aim of (a) lowering the pH; and (b) reducing the salt concentration. The pre-treatment protocols take into consideration technical and economical constraints. In the scheme finally adopted, CaCl2 was added to the final concentration of about 65 g litre − 1; neutrality was attained by adding H3PO4 (85%). The addition of these chemicals produces a black precipitate constituted mainly of organic and inorganic compounds (such as CaSO4). The organic compounds are constituted mainly of a monomer, dimer etc. of caprolactam. The black slurries were removed through sedimentation and filtration. The water phase is recovered and diluted 1/10 with tap water.
2.4. Shaken flask batch cultures Batch cultures were performed in 500 ml Erlenmeyer flasks containing 100 ml of liquid AYM prepared with both native and pre-treated AWW. The flasks were incubated on a rotatory shaker. The inoculation of bacteria was performed with a 48-h culture in liquid AYM of each one of the individual strains and strain cocktails described above. Uninoculated flasks were adopted as blank. Samples (50 ml each) were taken at various times, as reported in the results.
Bacterial biomass was measured as the dry weight of the pellets sedimented by centrifugation for 20 min at 3500 g and washed twice with distilled water. COD was measured as absorbance at 620 nm of the clear centrifugation supernatants after reaction with K2Cr2O7. An analytical method to measure the results of the chemical and biological treatments has been developed for this process. The following protocol has been used to measure the concentration of carboxylic groups (noted as CGC) because they are representative of the two major organic compounds present in the AWW: Na salts of cycloexanecarboxysulphonate isomers and derivatives plus the Na salt of 2-aminocapronic acid. Measurements of CGC were performed with the following protocol (for AWW pre-treated and not): clear supernatants, prepared by centrifuging the samples at 5000 g for 20 min, were extracted three times with ethyl ether; the organic phase containing the neutral components (see Table 1) was discarded or analysed by gas chromatography; the water phase, added with 5 N HCl to reach pH= 2.0, was centrifuged and the pellet was discarded; the three-fold extraction with ethyl ether was repeated, again discarding the organic phase. The organic phase contains extractable acid components (see Table 1); the acids not extractable with organic solvent, mostly consisting of cycloexanecarboxysulphonate acid isomers and 2-aminocapronic acid and minor quantities of o-aminocapronic acid present in the water phase were measured by titration with 1 N NaOH. This titration permits evaluation of carboxylic group concentration (CGC) and it was considered as a measure of the major organic compounds present in the AWW. An example of the titration is reported in Fig. 1.
2.7. Gas chromatographic and NMR analysis 2.5. Lab-scale fermenter batch cultures Lag phase batch cultures (150 ml) of the best strains (selected after the shake flask tests) were used to inocu-
The separation and identification of the extractable compounds (see Table 1) through gas chromatography after extraction using ethyl ether was performed with a
598
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
Carlo Erba HRGC 5300 gas chromatograph equipped with a FID detector system using a 15 m SPB-20 0.53 mm Wide Bore Supelco column. The GC operating conditions were: helium flow 9 ml s − 1, oven temperature program for each run initial temperature 30°C, initial time 3 min, rate 5°C min − 1, final temperature 240°C final time 100 min. The NMR spectra were recorded by using a Bruker AC300P spectrometer, operating at 300.130 and 75.465 for 1H and 13C nuclei respectively. Typically sweep width of 4 kHz and 20 kHz have been used for 1H and 13 C nuclei respectively. The flip angle was of 20 – 30° for both the nuclei. 1H FID were processed without applying any mathematical function, while in the case of 13C FID an exponential multiplication of 1 Hz was typically applied. All the spectra are referred to TMS directly or through a calibrated signal. 3. Results To study biodegradability of the AWW, biological tests were carried out by adding the selected microbial strains to a 1:10 dilution of pre-treated and not pretreated AWW. To verify the effect of the microbes on the substrate, COD removal and CGC concentration were systematically monitored during the biological process.
Fig. 3. CGC decreases measured in pre-treated AWW after incubation in shaken flasks with different bacterial strains.
3.1. Shaken flask tests with pre-treated AWW
Fig. 2. COD decreases measured in pre-treated AWW after incubation in shaken flasks with different bacterial strains.
The values of COD and the content of CGC were lowered by pre-treatment with CaCl2. The average COD fell from 38 300 to 22 000 mg litre − 1 whilst the concentrations of CGC decreased from 0.101 to 0.048 M. Decreases of both COD and the CGC were measured after cultivation of different bacterial strains in the AWW submitted to precipitation with CaCl2 and then diluted with water 1/10 (Fig. 2 and Fig. 3). Differences were evident in the decrease of COD according to the type of microbial inoculum (Fig. 2). The best degradation rates were obtained by using Mix 6 and Pa 7 strains: average degradation rates of 1200 and 240 mg litre − 1 per day have been obtained after 7 and 28 days, respectively. With these microbial strains the COD removal after 28 days of bio-treatment was about 60%. Similar behaviour was monitored considering the CGC during the time (Fig. 3). The other strains produced a lower COD decrease (about 35% after 28 days) with respect to Mix 6 and Pa 7 strains (see Table 2). The degradation of both cyclohexanecarboxysulphonate and o-amminocapronic acids (Fig. 2) show some similarities and some differences in comparison with the COD reduction. The rates of CGC degrada-
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
599
tion (during the first 7 days) ranged from 3.7 10 − 3 M per day (3.9% per day) when the inoculum is made with Mix 6 to 8.2 10 − 4 M per day (1.7% per day) when the strain Co 27 was used. When the inoculum was made with Pa 7, a decrease of 2.5 10 − 3 M per day (6.6% per day) was observed after 7 days of incubation. Owing to this performance, Pa 7 was able to degrade over 70% of the initial content of CGC. In this manner this preliminary study was able to select the best microbial strains to perform a microbiological treatment, although no environmentally acceptable levels have been obtained at this stage and a dilution of the waste water has been employed (this last condition may be a limit of this treatment).
Table 2 Final yields of CGC and COD removal after 28 days of shaken flask batch culture with different bacterial strains Removal (%)
CGC
CGC
COD
COD
Pre-treatment Co 27 Co 27-1 Co 27-2 Mix 6 Pa 7
Yes 37 32 43 44 71
No 16 17 24 33 33
Yes 34 34 42 57 60
No 49 28 43 52 43
Fig. 5. CGC decreases measured in native AWW after incubation in shaken flasks with different bacterial strains.
3.2. Shake flask tests with nati6e AWW
Fig. 4. COD decreases measured in native AWW after incubation in shaken flasks with different bacterial strains.
When the native AWW (diluted also in this case 1:10 by tap water) was incubated with the different bacterial strains, the inoculum with Mix 6 behaved as the most active during the first 7 days and it was able to remove as much as 2050 mg litre − 1 per day COD (5.3% per day) and 4.4% per day of CGC. The other strains required a long lag phase for better performance (Fig. 4), so that the highest effectiveness was expressed during the period from 7 to 14 days when the COD removals were as follows: 1780 mg litre − 1 per day (5.5% per day) by Pa 7 strain; 1685 mg litre − 1 per day (5.0% per day) by Co 27 strain; 1257 mg litre − 1 per day (3.7% per day) by Co 27-1 strain; 1514 mg litre − 1 per day (4.6% per day) Co 27-2 strain. Fig. 5 shows the removal of CGC from native AWW after batch incubation with the different bacteria. In this case the best CGC reduction was obtained by using Pa 7 and Mix 6 strains: the best COD removal from native AWW after 28 days of incubation was given by the Mix 6 accounting for more than 50% of the initial value; for CGC removal the best result at 28 days was given by Pa 7 and Mix 6 with around 1/3 of the total initial concentration. A comparison between the bio-treatment of AWW with and without pre-treatment by CaCl2 showed little
600
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
difference in terms of COD removal rate (about 1300 mg litre − 1 per day in the first 7 days of treatment). Some differences have been obtained in terms of: final COD removal (60% with pre-treatment and about 50% without); initial and final COD values (initial COD values of 37 500 mg litre − 1 without pre-treatment and 22 000 mg litre − 1 with pre-treatment); This last result indicates how a pre-treatment process can be used to lower the COD before the biological process although a problem of solid disposal must be considered for the formation of precipitates: however, this precipitation takes place also by adding only H3PO4. Acid addition is used to reach physiological pH and support the microbial growth in terms of phosphorous content in the media.
3.3. Lab scale fermenter tests Considering the experimental results obtained in shake flask tests, native AWW has been used to grow the Mix 6 and Pa 7 strains (as the best responding microbes in the shaken flask tests) in lab-scale fermenter tests. The results, summarised in Figs. 6 and 7, indicate that:
Fig. 7. Kinetic of COD degradation and biomass trend both measured in native AWW after incubation in a lab scale reactor with Mix 6.
Fig. 6. Kinetic of COD degradation and biomass trend both measured in native AWW after incubation in a lab scale reactor with the strain Pa 7.
microbial growth was accomplished within the first 19 h of incubation. The biomass concentration measured per litre reached 2.80 g for Pa 7 and 3.26 g for Mix 6; the biomass/COD consumed ratio were about 0.3 and 0.5 g/g using Pa 7 and Mix 6 respectively; the average COD removal rate were about 3800 and 5000 mg litre − 1 per day using Pa 7 and Mix 6, respectively. These results were larger with respect to the COD removal rates obtained in shaken flask tests (about 1200 mg litre − 1 day): the increase of COD removal was then related to the larger oxygen mass transfer obtained in the lab-scale bio-reactor with respect the shaken flask system; after 19 h the measured amounts of biomass remained constant or decreased (probably for the cellular lysis) but the COD still decreased at a reliable rate. The pH of the process remained constant as a result of the pH-controller by a relative large acid consumption: the amount of acid (as phosphoric acid) which has been required to maintain the pH at 7.0 during the bio-process was in general as high as 160 g litre − 1 after 40 h. The rise of pH in connection with bacterial growth on AWW both native and pre-treated was already noticed in the batch cultures where the pH restoration at 7.0 was performed at every sampling.
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
3.4. Gas chromatography and NMR analyses To determine which organic compounds were preferentially attacked by microbial strains, gas-chromatography and NMR analyses were performed on the organic phase obtained after solvent extraction by using suitable solvents before and after the biological process. The ethyl ether extracts (containing neutral extractable compounds), obtained by mixing the organic solvent with the aqueous phase for 1 h (obtained from the initial media or after the biological process), was analysed by gas chromatography. The experimental results indicate that: in the case of native AWW, a large number of peaks related to low molecular weight compounds were observed (data not shown here) in the sample not biologically treated. Such molecules are likely to belong to the byproducts of the SNIA-Viscosa process eventually transformed during the storage; some signals in that part of the chromatogram, where molecules at high molecular weight are expected, were characterised by the presence of caprolactam polymerisation compounds (dimer and so on); after the microbial attack in batch cultures, a number of low molecular weight compounds and capro-
601
lactam polymers disappeared. This last result shows that the biological process degrades the major part of caprolactam and related polymeric compounds. NMR analyses were also performed to give qualitative information on the biodegradation. A very large number of components in the 1H 13C-NMR group are found by NMR analyses (see Figs. 8 and 9). The interpretation of these spectra indicate that the most representative compounds are the ones related to caprolactam and its polymer Nylon-6 whose first species of polymerisation (dimer etc.) were also detected by gas chromatography. Nylon-6 residues with a higher degree of polymerisation are found in the precipitate after AWW acidification. Figs. 8 and 9 show the NMR analysis before and after biological treatment. From the analysis of these results it was possible to observe how the biological process removed o-caprolactam and related polymers, 6-aminocaproic acid and similar compounds until acetic acid. The only compounds not attacked are the cycloexanecarboxysulphonic acids (CECS), that were (as expected) bio-recalcitrant. These results are in agreement with the CGC in which it was possible to confirm that the residual CGC were due mainly to the cycloexanecarboxysulphonic acids whereas the disappearance of CGC may be as a result of the 6-aminocaproic acids.
Fig. 8. NMR results before biological treatment: AWW 1/10 diluted by tap water.
602
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
Fig. 9. NMR results after biological treatment: AWW 1/10 diluted by tap water.
4. Conclusions From analyses of the results obtained it was possible to establish that: suitable microbial strains (Mix 6 and Pa 7) able to lower both CGC content and COD of the AWW can be found and isolated from the environment. These microbial strains can survive, grow and express their activities on both native and pre-treated AWW when suitable conditions are provided. The usual laboratory techniques of study and physiological implementation can be applied to these strains. The better aerobic conditions obtained in the lab-scale fermenter made it possible to obtain faster kinetics of bio-degradation with respect to shaken flask tests; some of the pollutants present in the AWW can be individually degraded as a consequence of the microbial actions. The kinetics and yields of such degradation are connected with the growth and number of microbial cells. Complex molecules produced during the storage (even for very long times) by the molecules initially present in AWW can be treated as well with significant success; pre-treatment with CaCl2, which both lowers pH and reduces the sulphate content, seems to be a significant step ameliorating AWW susceptibility to microbial treatment. The pre-treatment with CaCl2 can be used to reduce the COD of the waste, but it is necessary to consider a disposal problem of the sludge produced during the pre-treatment. further increase of the biotechnological effectiveness for AWW treatment derives from the pollutant concentration by dilution. In our hands, the 1/10 ratio (AWW in water) has resulted in almost undisturbed microbial growth and activity. Such a dilution rate
seems to be not easy to manage in order to develop a treatment process. The data required for process development are very rough and preliminary. Although interesting results have been obtained in terms of kinetic and COD degradation, the final values of COD remain too large, considering the environmental constraints. Moreover cycloexanecarboxysulphonic acids (the major polluting compound in the waste) seems to be resistent to the biological process. Further studies are thus in progress in which the biological process, carried out by the selected microbial strains, are coupled with chemical treatment [5]. Acknowledgements The authors would like to thank Ing. S. Bartolini of Carbochemicals S.p.A for his precious provision of the supply waste. References [1] Donati G, Sioli M, Taverna M. Caprolattame da toluene: il processo SNIA-Viscosa, La Chimica e L’Industria 1968;50(9): 997 – 1001. [2] Tempesti L, Giuffre’ G, Buzzi F, Serena E, Montomeri. An investigation in to the kinetics of reaction between cyclohexanecarboxylic acid and oleum, La Chimica e l’Industria 1976;58(4):247– 51. [3] Hashim J, Kulundai RS, Hassan. Biodegradability of branched alkylbenzene sulphonates, J Chem Tech Biotechnol 1992;54:207– 14. [4] Chain, Farr DR. Metabolism of arylsulphonates by microrganism, Biochem J 1968;106:859. [5] Fantauzzi E, Baccella S, Lepidi AA, Veglio` F, Chiarini M, Cerichelli G, et al. Development of a chemical and biological treatment of alkaline industrial waste waters: a preliminary study. Fresenius Environ Bull 1998;7:934 – 50.
Biological treatment of alkaline industrial waste waters S. Baccella a, G. Cerichelli b, M. Chiarini b, C. Ercole a, E. Fantauzzi a, A. Lepidi a, L. Toro c, F. Veglio` d,* a Dipartimento di Biologia di Base ed Applicata, Uni6ersita` dell’Aquila, 67010 Coppito, L’Aquila, Italy Dipartimento di Chimica, Ingegneria Chimica e Materiali, Uni6ersita` dell’Aquila, 67040 Monteluco di Roio, L’Aquila, Italy c Dipartimento di Chimica, Uni6ersita’ La Sapienza, p.le A. Moro, 5, 00185 Rome, Italy d Dipartimento di Ingegneria Chimica e di Processo, ‘G.B. Bonino’, Uni6ersita’ degli Studi di Geno6a, 6ia Opera Pia, 15, 16145 Genoa, Italy b
Received 5 January 1999; received in revised form 9 August 1999; accepted 14 August 1999
Abstract The biotechnological treatment of alkaline waste waters (AWW) resulting from the production of caprolactam by the SNIA-viscosa process has been studied. The pollutant in the AWW is 80 – 120 g litre − 1 cyclohexanecarboxysulphonate (CECS) sodium salt with a COD up to 325 000 mg litre − 1. Bacterial strains have been isolated which are able to grow on AWW and to degrade the largest possible range of organic compounds. These strains have been screened for their performance in lowering the COD and degrade the sulphonic bonds. Combinations of strains have also been verified. The strains have been compared in cultures both in shake flasks and in a laboratory scale fermenter. The results showed that: (a) a 1/10 dilution of AWW with water permitted microbial growth coupled with decrease in COD and carboxylic concentration (representative of several organic compounds such as cyclohexanecarboxysulphonate, 2-aminocapronic and o-aminocapronic acids); (b) the polymerised caprolactam molecules are exhaustively degraded; (c) very similar results are found both in shake flask tests and in the lab scale fermenter but with different kinetics; and (d) pretreatment of alkaline waters with CaCl2 and lowering the pH with H3PO4, implement the kinetics and yields of the process in terms of degradation of COD and carboxylic compounds. The experiments gave very preliminary results and led to some suggestions for the development of a chemical and biological process to treat this kind of AWW. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Alkaline waste waters; Biological treatment; Bio-recalcitrant compounds
1. Introduction Industrial waste waters contain both organic and mineral contaminants having toxic effects on the microbiological agents of wastewater treatment plants or resisting biological attack and mineralization. This is the case of ‘alkaline waters’ produced by chemical plants for the production of several organic molecules of commercial concern such as caprolactam [1,2]. The properties of the alkaline waters are (a) high or very high alkalinity; (b) concentration of salts at levels approaching or above the bactericidal threshold; (c) very high COD values; and (d) the presence of recalcitrant organic molecules [3,4]. * Corresponding author. Tel.: +39-10-3532583; fax: + 39-103532586. E-mail address: [email protected] (F. Veglio`)
Alkaline waters produced by the caprolactam plants polymerising the fibres of nylon-6 possess every one of these properties so that such waters belong to the group of the industrial waste waters which hinder industrial development, for economic, environmental and social reasons. This problem is very important in southern Italy where about 150 000 tons of this industrial waste has been disposed of in tanks as a result of the absence of a chemical or biological process that could be used to treat it. Thus much effort is being invested in research to resolve this problem. This paper presents preliminary research on the treatment of alkaline waste waters (AWW) by means of a combination of a chemical pretreatment and a biological process with specific microbial strains.
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 1 1 2 - 0
596
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
2. Materials and methods
2.1. Alkaline waste waters The AWW was sampled from a storage stock standing for ca. 6 years in the south of Italy. Their chemical composition and properties, summarised in Table 1 [5], provide (a) the concentration of compounds produced during nylon-6 synthesis, including the Na salts of cycloexanecarboxysulphonate isomers and derivatives [2] plus the Na salt of
Table 1 Composition of the alkaline waste waters (AWW)a Minimum pH Density Dry weight at 105°C (%) Water (%) Cyclohexanecarboxylic sodium salt (%)* N-Hexahydro benzoyl-5-aminocaproic acid sodium salt (%)* 2-Aminocaproic acid sodium salt (%)** Cyclohexanecarboxysulphonate (CECS) bisodium salt (%)** Caprolactame*** Benzoic acid sodium salt (%)* N-Hexahydrobenzoic 5-amino valeric acid sodium salt (%)* Adipic acid sodium salt (%)* 1-Cyclohexancarboxylic acid sodium salt (%)* Cyclometilhexancarboxylic acid sodium salt (%)* 5-Ethyl di-hydro 2 (3H) furanone (%)*** 5-Methyl valero lactone (%)***
Maximum
10 1.1 22 66 0.2
12.5 1.2 34 78 2
0.1
1.8
0.3
4.0
8.0
12.5
0.02 0.01 0.01
1.6 0.5 0.3
0.01 0.02
0.25 0.25
0.03
0.25
0.01
0.25
0.01
0.25
Other organic prodocts (excluded PBC-dioxines-pesticides-hy0.005 drocarbons aromatics polycycles) (%) Sodium sulphate (%) 0.7 Sodium chloride (%) 2.0 Ammonia (%) 0.02 Total alkalinity expressed by Na2CO3 2.5 (%) Total phenols (mg litre−1) 60 Monochloro phenol (mg litre−1) 1 2,4-Dichlorophenol (mg litre−1) 0.01 2,4,6-Trichlorophenol (mg litre−1) 0.01 Toluene (mg litre−1) 0.05 Benzene (mg litre−1) 0.01 N-Hexane (mg litre−1) 0.01 Nitrites (mg litre−1) 50 Nitrates (mg litre−1) 150 COD (mg litre−1) 325 000
0.02
1.5 4.5 0.15 9.0 150 20 2 1 10 0.5 0.5 100 350 350 000
a *Neutral components; **no extractable components; and ***acid components.
2-aminocapronic acid. The concentration of these compounds is expressed as carboxylic group concentration (CGC) ranging from 0.40–0.87 M; (b) the pH value over 11; (c) the total salt content over 280 g litre1; and (d) the COD exceeding 300 000 mg litre − 1.
2.2. Micro-organisms: isolation and maintenance Microbial strains were sub-cultured five-fold in a rotatory shaker at 200 rpm and at 37°C on a liquid medium AYM (AWW/tap water 1/10, yeast extract 5 g 1itre − 1, MgCl2 0.1 g litre − 1, pH adjusted at 7.0 by H3PO4 85%). Isolation of pure colonies was effected on solid medium (AYM supplemented with Bacto Agar 2%) by serial dilution (from 10 − 4 to 10 − 7) of the enrichment cultures. Isolated colonies were verified for the capability to grow on AYM with high growth rates and lowering of COD. These strains were chosen as promising candidates for the present purpose. Both pure cultures and the cocktails of strains have been maintained by sub-culturing on AYM. The associations of strains have been used considering that the capability to compete with other bacterial populations is a relevant property for bacterial cultures which are expected to operate in a common wastewater treatment plant. Bacterial strains used in this work [5] were either: (a) associations of more than one strain and/or (b) monospecific pure cultures. Group (a) includes the strains nicknamed Co 27 and Mix 6. Co 27, taken from enrichment cultures of water samples from a sulphurous spring around L’Aquila, is constituted of an association of three strains which, after isolation in pure culture, have been named Co 27-1, Co 27-2 and Pa 7 (see below). Mix 6 is the entire pool of strains obtained through the enrichment cultures of samples taken from a wastewater treatment plant of a chemical industry. In group (b), the following pure cultures are included: Co 27 -1: this strain grows on solid medium plates with flat colonies bordered with a brown halo; the cells are short rods 0.9–1.1 mm in diameter, gram negative; Co 27 -2: this strain grows on solid medium plates with rough powdered colonies, irregularly bordered; the cells are rods 0.8–1.8 mm in diameter, gram negative; Pa 7 : this strain grows on solid medium plates with round microcolonies of milky appearance; the cells are very short rods, almost cocci, 0.8–0.9 mm in diameter, gram negative. According to the morphophysiological characterisation, performed also with BioMerieux tunnels, it has been tentatively attributed to the genus Pseudomonas.
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
597
late a lab-scale fermenter (Biostat B, Braun Biotech International) containing 2 litres of sterile liquid AYM prepared with untreated AWW. The following culture conditions were adopted: 37°C, stirring 300 rpm, pH= 7 (automatically monitored and maintained by adding either H3PO4 or 1N KOH), pO2 = 10% of saturation. Samples of 20 ml were taken out for chemical and biological analysis.
2.6. Bacterial biomass, COD and CGC analyses
Fig. 1. A typical titration result for carboxylic group concentration (CGC).
2.3. Pre-treatment of AWW Some pre-treatment schemes have been verified for AWW, with the aim of (a) lowering the pH; and (b) reducing the salt concentration. The pre-treatment protocols take into consideration technical and economical constraints. In the scheme finally adopted, CaCl2 was added to the final concentration of about 65 g litre − 1; neutrality was attained by adding H3PO4 (85%). The addition of these chemicals produces a black precipitate constituted mainly of organic and inorganic compounds (such as CaSO4). The organic compounds are constituted mainly of a monomer, dimer etc. of caprolactam. The black slurries were removed through sedimentation and filtration. The water phase is recovered and diluted 1/10 with tap water.
2.4. Shaken flask batch cultures Batch cultures were performed in 500 ml Erlenmeyer flasks containing 100 ml of liquid AYM prepared with both native and pre-treated AWW. The flasks were incubated on a rotatory shaker. The inoculation of bacteria was performed with a 48-h culture in liquid AYM of each one of the individual strains and strain cocktails described above. Uninoculated flasks were adopted as blank. Samples (50 ml each) were taken at various times, as reported in the results.
Bacterial biomass was measured as the dry weight of the pellets sedimented by centrifugation for 20 min at 3500 g and washed twice with distilled water. COD was measured as absorbance at 620 nm of the clear centrifugation supernatants after reaction with K2Cr2O7. An analytical method to measure the results of the chemical and biological treatments has been developed for this process. The following protocol has been used to measure the concentration of carboxylic groups (noted as CGC) because they are representative of the two major organic compounds present in the AWW: Na salts of cycloexanecarboxysulphonate isomers and derivatives plus the Na salt of 2-aminocapronic acid. Measurements of CGC were performed with the following protocol (for AWW pre-treated and not): clear supernatants, prepared by centrifuging the samples at 5000 g for 20 min, were extracted three times with ethyl ether; the organic phase containing the neutral components (see Table 1) was discarded or analysed by gas chromatography; the water phase, added with 5 N HCl to reach pH= 2.0, was centrifuged and the pellet was discarded; the three-fold extraction with ethyl ether was repeated, again discarding the organic phase. The organic phase contains extractable acid components (see Table 1); the acids not extractable with organic solvent, mostly consisting of cycloexanecarboxysulphonate acid isomers and 2-aminocapronic acid and minor quantities of o-aminocapronic acid present in the water phase were measured by titration with 1 N NaOH. This titration permits evaluation of carboxylic group concentration (CGC) and it was considered as a measure of the major organic compounds present in the AWW. An example of the titration is reported in Fig. 1.
2.7. Gas chromatographic and NMR analysis 2.5. Lab-scale fermenter batch cultures Lag phase batch cultures (150 ml) of the best strains (selected after the shake flask tests) were used to inocu-
The separation and identification of the extractable compounds (see Table 1) through gas chromatography after extraction using ethyl ether was performed with a
598
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
Carlo Erba HRGC 5300 gas chromatograph equipped with a FID detector system using a 15 m SPB-20 0.53 mm Wide Bore Supelco column. The GC operating conditions were: helium flow 9 ml s − 1, oven temperature program for each run initial temperature 30°C, initial time 3 min, rate 5°C min − 1, final temperature 240°C final time 100 min. The NMR spectra were recorded by using a Bruker AC300P spectrometer, operating at 300.130 and 75.465 for 1H and 13C nuclei respectively. Typically sweep width of 4 kHz and 20 kHz have been used for 1H and 13 C nuclei respectively. The flip angle was of 20 – 30° for both the nuclei. 1H FID were processed without applying any mathematical function, while in the case of 13C FID an exponential multiplication of 1 Hz was typically applied. All the spectra are referred to TMS directly or through a calibrated signal. 3. Results To study biodegradability of the AWW, biological tests were carried out by adding the selected microbial strains to a 1:10 dilution of pre-treated and not pretreated AWW. To verify the effect of the microbes on the substrate, COD removal and CGC concentration were systematically monitored during the biological process.
Fig. 3. CGC decreases measured in pre-treated AWW after incubation in shaken flasks with different bacterial strains.
3.1. Shaken flask tests with pre-treated AWW
Fig. 2. COD decreases measured in pre-treated AWW after incubation in shaken flasks with different bacterial strains.
The values of COD and the content of CGC were lowered by pre-treatment with CaCl2. The average COD fell from 38 300 to 22 000 mg litre − 1 whilst the concentrations of CGC decreased from 0.101 to 0.048 M. Decreases of both COD and the CGC were measured after cultivation of different bacterial strains in the AWW submitted to precipitation with CaCl2 and then diluted with water 1/10 (Fig. 2 and Fig. 3). Differences were evident in the decrease of COD according to the type of microbial inoculum (Fig. 2). The best degradation rates were obtained by using Mix 6 and Pa 7 strains: average degradation rates of 1200 and 240 mg litre − 1 per day have been obtained after 7 and 28 days, respectively. With these microbial strains the COD removal after 28 days of bio-treatment was about 60%. Similar behaviour was monitored considering the CGC during the time (Fig. 3). The other strains produced a lower COD decrease (about 35% after 28 days) with respect to Mix 6 and Pa 7 strains (see Table 2). The degradation of both cyclohexanecarboxysulphonate and o-amminocapronic acids (Fig. 2) show some similarities and some differences in comparison with the COD reduction. The rates of CGC degrada-
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
599
tion (during the first 7 days) ranged from 3.7 10 − 3 M per day (3.9% per day) when the inoculum is made with Mix 6 to 8.2 10 − 4 M per day (1.7% per day) when the strain Co 27 was used. When the inoculum was made with Pa 7, a decrease of 2.5 10 − 3 M per day (6.6% per day) was observed after 7 days of incubation. Owing to this performance, Pa 7 was able to degrade over 70% of the initial content of CGC. In this manner this preliminary study was able to select the best microbial strains to perform a microbiological treatment, although no environmentally acceptable levels have been obtained at this stage and a dilution of the waste water has been employed (this last condition may be a limit of this treatment).
Table 2 Final yields of CGC and COD removal after 28 days of shaken flask batch culture with different bacterial strains Removal (%)
CGC
CGC
COD
COD
Pre-treatment Co 27 Co 27-1 Co 27-2 Mix 6 Pa 7
Yes 37 32 43 44 71
No 16 17 24 33 33
Yes 34 34 42 57 60
No 49 28 43 52 43
Fig. 5. CGC decreases measured in native AWW after incubation in shaken flasks with different bacterial strains.
3.2. Shake flask tests with nati6e AWW
Fig. 4. COD decreases measured in native AWW after incubation in shaken flasks with different bacterial strains.
When the native AWW (diluted also in this case 1:10 by tap water) was incubated with the different bacterial strains, the inoculum with Mix 6 behaved as the most active during the first 7 days and it was able to remove as much as 2050 mg litre − 1 per day COD (5.3% per day) and 4.4% per day of CGC. The other strains required a long lag phase for better performance (Fig. 4), so that the highest effectiveness was expressed during the period from 7 to 14 days when the COD removals were as follows: 1780 mg litre − 1 per day (5.5% per day) by Pa 7 strain; 1685 mg litre − 1 per day (5.0% per day) by Co 27 strain; 1257 mg litre − 1 per day (3.7% per day) by Co 27-1 strain; 1514 mg litre − 1 per day (4.6% per day) Co 27-2 strain. Fig. 5 shows the removal of CGC from native AWW after batch incubation with the different bacteria. In this case the best CGC reduction was obtained by using Pa 7 and Mix 6 strains: the best COD removal from native AWW after 28 days of incubation was given by the Mix 6 accounting for more than 50% of the initial value; for CGC removal the best result at 28 days was given by Pa 7 and Mix 6 with around 1/3 of the total initial concentration. A comparison between the bio-treatment of AWW with and without pre-treatment by CaCl2 showed little
600
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
difference in terms of COD removal rate (about 1300 mg litre − 1 per day in the first 7 days of treatment). Some differences have been obtained in terms of: final COD removal (60% with pre-treatment and about 50% without); initial and final COD values (initial COD values of 37 500 mg litre − 1 without pre-treatment and 22 000 mg litre − 1 with pre-treatment); This last result indicates how a pre-treatment process can be used to lower the COD before the biological process although a problem of solid disposal must be considered for the formation of precipitates: however, this precipitation takes place also by adding only H3PO4. Acid addition is used to reach physiological pH and support the microbial growth in terms of phosphorous content in the media.
3.3. Lab scale fermenter tests Considering the experimental results obtained in shake flask tests, native AWW has been used to grow the Mix 6 and Pa 7 strains (as the best responding microbes in the shaken flask tests) in lab-scale fermenter tests. The results, summarised in Figs. 6 and 7, indicate that:
Fig. 7. Kinetic of COD degradation and biomass trend both measured in native AWW after incubation in a lab scale reactor with Mix 6.
Fig. 6. Kinetic of COD degradation and biomass trend both measured in native AWW after incubation in a lab scale reactor with the strain Pa 7.
microbial growth was accomplished within the first 19 h of incubation. The biomass concentration measured per litre reached 2.80 g for Pa 7 and 3.26 g for Mix 6; the biomass/COD consumed ratio were about 0.3 and 0.5 g/g using Pa 7 and Mix 6 respectively; the average COD removal rate were about 3800 and 5000 mg litre − 1 per day using Pa 7 and Mix 6, respectively. These results were larger with respect to the COD removal rates obtained in shaken flask tests (about 1200 mg litre − 1 day): the increase of COD removal was then related to the larger oxygen mass transfer obtained in the lab-scale bio-reactor with respect the shaken flask system; after 19 h the measured amounts of biomass remained constant or decreased (probably for the cellular lysis) but the COD still decreased at a reliable rate. The pH of the process remained constant as a result of the pH-controller by a relative large acid consumption: the amount of acid (as phosphoric acid) which has been required to maintain the pH at 7.0 during the bio-process was in general as high as 160 g litre − 1 after 40 h. The rise of pH in connection with bacterial growth on AWW both native and pre-treated was already noticed in the batch cultures where the pH restoration at 7.0 was performed at every sampling.
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
3.4. Gas chromatography and NMR analyses To determine which organic compounds were preferentially attacked by microbial strains, gas-chromatography and NMR analyses were performed on the organic phase obtained after solvent extraction by using suitable solvents before and after the biological process. The ethyl ether extracts (containing neutral extractable compounds), obtained by mixing the organic solvent with the aqueous phase for 1 h (obtained from the initial media or after the biological process), was analysed by gas chromatography. The experimental results indicate that: in the case of native AWW, a large number of peaks related to low molecular weight compounds were observed (data not shown here) in the sample not biologically treated. Such molecules are likely to belong to the byproducts of the SNIA-Viscosa process eventually transformed during the storage; some signals in that part of the chromatogram, where molecules at high molecular weight are expected, were characterised by the presence of caprolactam polymerisation compounds (dimer and so on); after the microbial attack in batch cultures, a number of low molecular weight compounds and capro-
601
lactam polymers disappeared. This last result shows that the biological process degrades the major part of caprolactam and related polymeric compounds. NMR analyses were also performed to give qualitative information on the biodegradation. A very large number of components in the 1H 13C-NMR group are found by NMR analyses (see Figs. 8 and 9). The interpretation of these spectra indicate that the most representative compounds are the ones related to caprolactam and its polymer Nylon-6 whose first species of polymerisation (dimer etc.) were also detected by gas chromatography. Nylon-6 residues with a higher degree of polymerisation are found in the precipitate after AWW acidification. Figs. 8 and 9 show the NMR analysis before and after biological treatment. From the analysis of these results it was possible to observe how the biological process removed o-caprolactam and related polymers, 6-aminocaproic acid and similar compounds until acetic acid. The only compounds not attacked are the cycloexanecarboxysulphonic acids (CECS), that were (as expected) bio-recalcitrant. These results are in agreement with the CGC in which it was possible to confirm that the residual CGC were due mainly to the cycloexanecarboxysulphonic acids whereas the disappearance of CGC may be as a result of the 6-aminocaproic acids.
Fig. 8. NMR results before biological treatment: AWW 1/10 diluted by tap water.
602
S. Baccella et al. / Process Biochemistry 35 (2000) 595–602
Fig. 9. NMR results after biological treatment: AWW 1/10 diluted by tap water.
4. Conclusions From analyses of the results obtained it was possible to establish that: suitable microbial strains (Mix 6 and Pa 7) able to lower both CGC content and COD of the AWW can be found and isolated from the environment. These microbial strains can survive, grow and express their activities on both native and pre-treated AWW when suitable conditions are provided. The usual laboratory techniques of study and physiological implementation can be applied to these strains. The better aerobic conditions obtained in the lab-scale fermenter made it possible to obtain faster kinetics of bio-degradation with respect to shaken flask tests; some of the pollutants present in the AWW can be individually degraded as a consequence of the microbial actions. The kinetics and yields of such degradation are connected with the growth and number of microbial cells. Complex molecules produced during the storage (even for very long times) by the molecules initially present in AWW can be treated as well with significant success; pre-treatment with CaCl2, which both lowers pH and reduces the sulphate content, seems to be a significant step ameliorating AWW susceptibility to microbial treatment. The pre-treatment with CaCl2 can be used to reduce the COD of the waste, but it is necessary to consider a disposal problem of the sludge produced during the pre-treatment. further increase of the biotechnological effectiveness for AWW treatment derives from the pollutant concentration by dilution. In our hands, the 1/10 ratio (AWW in water) has resulted in almost undisturbed microbial growth and activity. Such a dilution rate
seems to be not easy to manage in order to develop a treatment process. The data required for process development are very rough and preliminary. Although interesting results have been obtained in terms of kinetic and COD degradation, the final values of COD remain too large, considering the environmental constraints. Moreover cycloexanecarboxysulphonic acids (the major polluting compound in the waste) seems to be resistent to the biological process. Further studies are thus in progress in which the biological process, carried out by the selected microbial strains, are coupled with chemical treatment [5]. Acknowledgements The authors would like to thank Ing. S. Bartolini of Carbochemicals S.p.A for his precious provision of the supply waste. References [1] Donati G, Sioli M, Taverna M. Caprolattame da toluene: il processo SNIA-Viscosa, La Chimica e L’Industria 1968;50(9): 997 – 1001. [2] Tempesti L, Giuffre’ G, Buzzi F, Serena E, Montomeri. An investigation in to the kinetics of reaction between cyclohexanecarboxylic acid and oleum, La Chimica e l’Industria 1976;58(4):247– 51. [3] Hashim J, Kulundai RS, Hassan. Biodegradability of branched alkylbenzene sulphonates, J Chem Tech Biotechnol 1992;54:207– 14. [4] Chain, Farr DR. Metabolism of arylsulphonates by microrganism, Biochem J 1968;106:859. [5] Fantauzzi E, Baccella S, Lepidi AA, Veglio` F, Chiarini M, Cerichelli G, et al. Development of a chemical and biological treatment of alkaline industrial waste waters: a preliminary study. Fresenius Environ Bull 1998;7:934 – 50.