Study on community structure of microbial consortium for the degradation of viscose fiber wastewater

Background Enrichment culture was applied to obtain microbial consortium from activated sludge samples collected from biodegradation system, a chemical fiber plant in Hebei Province, China. Bacterial composition and community dynamic variation were assessed employing denaturing gradient gel electrophoresis fingerprinting technology based on amplified 16S rRNA genes in the entire process of enrichment culture for viscose fiber wastewater. Results Four bacteria named as VF01, VF02, VF03, and VF04 were isolated from the microbial consortium adopting the spray-plate method. The DNA bands of these four bacteria were corresponded to the predominant DNA bands in the electrophoresis pattern. VF01, VF02, VF03, and VF04 were phylogenetically closed to Bacillus licheniformis, Bacillus subtilis, Paracoccus tibetensis, and Pseudomonas sp. by sequence analysis, respectively. The degradation effects for CODCr of single isolated strain, mixed strains, and microbial consortium (VF) originally screened from viscose fiber wastewater were determined. The degradation ability was as follows: microbial consortium (VF) > mixed strains > single isolated strain. Microbial consortium (VF) showed the optimum degradation rate of CODCr of 87% on 14th day. Degradation of pollutants sped up by bio-augmentation of four strains. The molecular weight distribution of organic matter showed that viscose fiber wastewater contained a certain amount of large molecular organic matter, which could be decomposed into smaller molecular substances by microbial consortium (VF). Conclusions The microbial consortium (VF) obtained from enrichment culture exhibited great potential for CODCr degradation. The screened strains had bio-augmentation functions and the addition of a mixture of four bacteria could speed up the degradation rate of pollutants.


Background
Viscose fiber exhibits good physical, mechanical, and servicing qualities, and its status is next to polyester in chemical fiber industry (Lin 2000). Nowadays, a huge volume of wastewater has been produced in chemical industry which is responsible for serious environmental problems. Viscose fiber wastewater contains acid and alkaline wastewater. Acid wastewater is characterized by foul-smelling, high zinc ion concentration, high temperature, high salinity, sulfide, complex composition, and toxic substances. Alkaline wastewater is characterized by caustic soda and cellulose with low polymerization degree (Liang et al. 2009;Wang et al. 2013). The contaminants, as cellulose and lignin, in the wastewater are hard to be biodegraded and cause serious environment pollution, therefore it is very difficult to effectively deal with industrial wastewater (Kang and Yi 2005). These are the main reasons why the viscose fiber production has been kept stagnated over the past 20 years. Thus, it is of great social and environmental benefits to develop wastewater treatment technology.
The mixed wastewater of viscose fiber is mainly used in the industrial degradation. A large number of cellulose precipitate was formed after acid and alkaline wastewater were mixed (pH = 2-3) in the industrial degradation. At the same time, sulfide is turned into hydrogen sulfide and carbon disulfide in recycling (Liang et al. 2009). Zinc ions are turned into zinc hydroxide precipitation when the pH value was 8-9. Several physical and chemical methods have been used to treat wastewater and improve the degradation efficiency of COD Cr (Hamaguchi et al. 2013;Kang et al. 2012). However, the cost is too high and the equipment covers a large area. In contrast, biological processes are the most environmental friendly and economical (Miao et al. 2009). However, no microorganism has been reported that can directly degrade viscose fiber wastewater, due to the complex composition of viscose fiber wastewater, most of which are cellulose, lignin, organic pollutants, and other large molecular substances (Liang et al. 2009;Vikman et al. 2002). It has been observed that few microorganisms can degrade macromolecular substance under high salinity condition .
Previous studies indicated that small molecular substances could be degraded by individual strain or single species Head and Oleszkiewicz 2004). In contrast, large molecular substances should be degraded by diverse species of microorganisms, because the degradation could not be completed by individual type and the cooperation of diverse species is necessary . To study the degradation of viscose fiber wastewater by microorganisms, organic matter is converted through a range of metabolic reactions by assembling of microorganisms in wastewater environment.
It is possible to describe bacterial diversity in real environment by combining molecular biology technology based on 16S rRNA gene analysis and culture-dependent approaches as there have been a number of advances in microbial ecology. PCR-DGGE is a useful and powerful tool for describing the structure changes of microbial community in complex wastewater environments and enrichment cultures. PCR-DGGE has been widely used to identify the bacterial composition of different ecological niches, as these approaches are able to detect microorganisms which are not detected by culture-based methods (Muyzer and Smalla 1998;Brito et al. 2006). Molecular biology technology has contributed to further conduct the studies involving microbial composition, phylogeny, and metabolic mechanism during the whole wastewater treatment process (Al-Thukair et al. 2007;Widada et al. 2002).
The purpose of this paper was to (1) apply the viscose fiber wastewater enrichment strategy to establish viscose fiber wastewater degrading communities to enhance biodegradation of viscose fiber wastewater; (2) analyze the structure changes of bacterial community in viscose fiber wastewater by PCR-DGGE; (3) detect the biodegradability of bacterial consortium gathered from viscose fiber wastewater.

DGGE analysis of community structure
Ten bands were observed during enrichment process, indicating that the dynamic variation of bacterial community was complex. Figure 1 shows that the intensity of bands 2 and 6 were relatively high in the first lane (the first enrichment), while bands 2 and 6 were not found in the same position of other lanes. These results indicated that these two strains showed low degradation activities to contaminants in viscose fiber wastewater and were unaccommodated to high pressure. While the other strains showed high degradation activities to contaminants in viscose fiber wastewater and were accommodated to high pressure. During enrichment culture, bands 1 and 10 gradually turned bright, demonstrating that these two strains were the predominant microorganisms and showed strong abilities in degradation. Bands 3, 4, and 7 gradually turned dark and band 4 even could not be found in the later enrichment culture, demonstrating that the number of population tended to reduce during enrichment culture; however, some strains were still predominant for degradation. Bands 8 and 9 remained to be bright in all lanes during enrichment culture, suggesting that these strains might utilize organic matter in wastewater as C/N source.

DGGE analysis of mixed culture
Four strains, VF01, VF02, VF03, and VF04, were isolated from the 5th enrichment culture. Bands in VF01, VF02, VF03, and VF04 were well correlated with the corresponding bands of the mixed culture in the PCR-DGGE (Fig. 2). In theory, DNA fragments corresponding to bands on the same horizontal line have identical sequence, the electrophoretic mobility of band 7 was identical with strain VF01; band 8 with strain VF02; band 9 with strain VF03; band 10 with strain VF04. These four bands existed in the whole enrichment cultures, confirming that these four isolate strains exhibited advantage in removing contaminants from wastewater compared with other isolated strains.

Phylogenetic analysis
Bands 1, 2, 7, VF 01, VF 02, VF 03, and VF 04 were excised from gels following by identification of strains and analysis of phylogeny based on the 16S rRNA sequence ( Phylogenetic tree for these strains was developed (Fig. 3). VF01 and VF02 belonged to Bacillus sp., which was the common microorganism used for biodegradation and purification of industrial wastewater (e.g., Bacillus pumilus). Bacillus sp. had apparent advantage in degrading organic pollutants, such as organic phosphorus pesticides, petroleum pollutants, polycyclic aromatic hydrocarbons, phenol, and nitrobenzene (Devaraja et al. 2002;Oliveira et al. 2009;Gopinath et al. 2009;Singh et al. 2008;Wen et al. 2011). Bacillus sp. are widely distributed in the natural environment, some of which can grow well under high acid, high alkali, high temperature, and cold condition, making it possible to decompose complex polysaccharide, protein, and soluble organics.
VF03 was 98% identical with P. tibetensis sp., which is a kind of denitrification bacteria and can be separated from the wastewater plant. P. tibetensis sp. is mainly used in sewage treatment, such as landscape water treatment, urban river governance, especially aquaculture wastewater treatment.
VF04 was 99% identical with the Pseudomonas sp., which is widely distributed in the nature and has the ability to metabolize a series of compounds, such as polycyclic aromatic hydrocarbons, toluene, cyanide, carbazole, simple aromatic compounds, organic solvent in organic compounds, and chlorinated hydrocarbons (Reineke 1998).

Degradation rate of COD Cr
The degradation rate of COD Cr of individual strains, mixed strains, and the enriched consortium (VF) were measured to better understand the metabolic capabilities of consortium. As shown in Fig. 4, after incubated for 14 days, strain VF01 could degrade 10% of COD Cr ; strain VF02 could degrade 7.5% of COD Cr ; and strain VF04 could degrade 14.8% of COD Cr ; however, strain VF03 could not degrade COD Cr in wastewater. The degradation effect of single strain was the worst, which ranged from 0.5 to 14.8%, as the viscose fiber wastewater contained complex macromolecular organic matter, such as cellulose, hemicellulose, and lignin. The degradation process involves in complex biochemical metabolic reaction and requires the synergistic action of different types of microorganisms. Bacillus sp. and Pseudomonas sp. could produce degradation enzymes of lignin and cellulose, making it possible to decompose cellulose, hemicellulose, and lignin in viscose fiber wastewater (Hernández et Kumar et al. 2001). However, the decomposition rate is very slow because the degradation enzymes are intracellular enzyme (Schwarz 2001). P. tibetensis sp. is a kind of denitrification bacteria, which cannot produce degradation enzymes but was able to convert nitrate to nitrogen and provided the nitrogen source for the growth of other organisms. Bacillus sp. can cooperate with Pseudomonas sp. to degrade the large molecules substances in viscose fiber wastewater, such as cellulose and lignin, etc. (Tuomela et al. 2000;Kumar et al. 2001). The mechanism of synergistic degradation has been clear now (Wilson 2004). The interaction of fungi, bacteria, and the microbial community contributed to the complete degradation of large molecules (Hernández et al. 2001), but the degradation of single Bacillus sp. or single Pseudomonas sp. on cellulose, lignin, and other large molecules was limited.
The degradation rate of COD Cr of the mixed strains, the enriched consortium (VF), and the mixture with consortium (VF) were measured to better understand the metabolic capability of the mixed strains. The degradation effect of COD Cr depicted in Fig. 5 was as follows: consortium (VF) > the mixture of four isolates > individual isolates. This is due to the number and types of microorganisms in the consortium (VF) which are more than that of mixed and individual strains. The small molecular substances can be metabolized by individual strains, while the biodegradation of macromolecular substances requires the combined efforts of different strains, especially cellulose and lignin.
After operation for 14 days, the mixed strains degraded 45.4% of COD Cr , which was far below than 87% removal efficiency of consortium (VF), due to that the number and types of microorganisms in the consortium (VF) are more than that of mixture. In contrast, the degradation rate of the mixture and consortium (VF) is faster than that of consortium (VF) from the 1st to 8th day. After 8th day, the degradation rate of the mixture and the consortium (VF) was same with that of consortium (VF), indicating that partial large molecules could be degraded into small molecules by the mixture, such as cellulose and lignin, etc. The addition of the mixture in the consortium (VF) can improve the degradation effect on pollutants. Bacillus sp. and Pseudomonas sp. had bio-augmentation function and could enhance the degradation effect of viscose wastewater.

Dissolved organic matters (DOM) size distribution in viscose fiber wastewater
The molecular weight distribution of organic matter during the degradation process was measured to further analyze the bio-augmentation effect of four strains. As shown in Fig. 6, the molecular weight distribution in 10 samples was asymmetric and DOM was mainly composed of small molecules, e.g., <1 kDa. In control sample, the percentage of DOM (<1 kDa) reached 66.1% and the percentage of DOM (10-3 kDa) reached 20.6%. For large molecular organic matter, the percentage of DOM (>100 kDa, 100-30 kDa, and 30-10 kDa) reached 3.6, 4, and 5.2%, respectively. Viscose fiber wastewater also contained a lot of insoluble large molecular organics, thus leading to the difficulty of further degradation. The molecular weight distribution of VF01, VF02, VF03, and VF04 was similar to that of the control group. This result was consistent with the degradation effect of COD Cr (Fig. 4).
Microbial degradation rate sped up by bio-augmentation of four strains by comparing the distribution of  Biodegradation of COD Cr by mixture of the four isolates, the consortium VF, and mixture + the consortium VF molecular weight. The organic compounds in wastewater were accelerated into small molecules by bio-augmentation of four strains. The proportion of the large molecular organic matter in wastewater became less and less, e.g., the percentage of 100-30 kDa changed from 3.6 to 0.85% and the percentage of 30-10 kDa changed from 4 to 0.25%. However, the fraction of small molecule organic matter became more and more, e.g., the percentage of <1 kDa changed from 66.1 to 92.27%. The molecular weight distribution of DOM in sewage not only reflected the characteristics of organic matter but also closely related to the degradation efficiency for wastewater (Chang et al. 2000).
Bio-augmentation utilizes microorganisms as biodegraders to purify sewage in nature and bioreactors to remove contaminants (Chong et al. 1997;El Fantroussi and Agathos 2005;Head and Oleszkiewicz 2004;Reberto et al. 2003). Bio-augmentation is efficient in degrading target pollutants and removing the refractory organics involved in wastewater by inoculating strains. Previous studies indicated that bio-augmentation was feasible for the treatment of waste streams produced from pharmaceutical factories, coke plants, pulp mills, dye, and other industries (Saravanane et al. 2001;Park et al. 2008;Wang et al. 2002;Yu and Mohn 2001;Chen et al. 2006).

Conclusions
The microbial consortium for wastewater degradation was enriched from samples of viscose fiber wastewater collected from a chemical fiber plant in Hebei, China. The method of PCR-DGGE was employed to investigate the changes in microbial community structure and helped us to determine the predominant species for COD Cr biodegradation throughout the enrichment process. Consortium (VF) exhibited great potential in COD Cr removal and the degradation rate of COD Cr reached 87% until 14th day, indicating that enrichment culture as an isolation method was both feasible and effective. Four strains had biological reinforcement functions and the addition of a mixture of four bacteria could speed up the degradation rate of pollutants and the application of these species to bio-augmentation fields would assist in observing their catabolic behavior in highly polluted environments.

Sampling, enrichment, and isolation of the viscose fiber wastewater degraders
The mixed wastewater of viscose fiber samples were collected from a chemical fiber wastewater plant in Hebei, China. Activated sludge samples were collected from biochemical treatment system. The samples were stored at 4 °C until use. The characteristics of wastewater were listed in Table 1.
Activated sludge samples (2.0 g) were cultured in 250 mL Tri-flask with 100 mL LB media at 30 °C and 150 rpm for 2 days. Hereafter, this culture of 10 mL was shifted to another flask of 250 mL. The amount of viscose fiber wastewater was gradually increased until all wastewater was used as a microbial growth substrate at

Table 1 The characteristics of viscose wastewater sample
The units for the parameters except for pH and salt are in mg/L, TDS is g/L ND no detectable, COD Cr chemical oxygen demand, BOD 5 biochemical oxygen demand after 5 day, SS suspended solids, TN total nitrogen, TP total phosphorus, TDS salt 10 days. The acclimation time for enrichment culture lasted for 40 days. Bacteria were screened from the consortium by tenfold series dilution method. Morphologically distinct individual colonies were further purified on LB agar plates.

DNA samples preparation
DNA was respectively extracted from the isolated strains and the enrichment cultures, i.e., the first day of enrichment culture sample, the 10th day of enrichment culture sample, the 20th day of enrichment culture sample, the 30th day of enrichment culture sample, the 40th day of enrichment culture sample following the manufacturer's protocol (Qiagen, Valencia, CA, USA). The integrity of DNA was detected by agarose gel electrophoresis.
Cycling conditions for 16S rRNA gene amplification were as follows: initial denaturation at 94 °C for 5 min, 30 cycles each involved denaturation at 94 °C for 1 min; anneal at 55 °C for 1 min, extended at 72 °C for 1 min; and another extension at 72 °C for 10 min. 5 μL of PCR products was analyzed by electrophoresis on 2% agarose gel in 1× TAE buffer (20 mM Tris-HCl, 10 mM glacial acetic acid, 0.5 mM EDTA, pH 8.0) at 120 V for 30 min.
Samples of PCR products were loaded onto 8% (w/v) polyacrylamide gel within 30-60% denaturing gradient (100% denaturant consisted of 7 M urea and 40% deionized formamide). The electrophoresis was run in 1× TAE buffer for 10 h, (100 V, 60 °C) in Universal Mutation Detection System apparatus (Bio-Rad, Dcode, USA). Finally, the gels were stained with ethidium bromide solution of 0.1% (v/v) (1× TAE) for 15 min, rinsed in distilled water for 20 min, and then were scanned in a Gel Doc XR documentation system (Bio-Rad).

Identification of strains and analysis of phylogeny
Pieces of DGGE bands were excised from polyacrylamide gel with sharp blade and then were, respectively, transferred to a new 2.0 mL Eppendorf tubes with 50 μL of sterilized ultra-pure water and incubated at 4 °C for a night to accelerate the diffusion of DNA out of the gel bands. The centrifugation was conducted at 10,000g for 6 min to obtain supernatant from above DNA solution. 1 μL supernatant was used as amplified template with a set of primer 341F and 518R with no GC clamp. Amplified products were further analyzed by the technique of denaturing gradient gel electrophoresis to confirm that they were successfully isolated.
The amplified products were purified from agarose gel and bound with a pMD18-T vector and then transformed into Escherichia coli DH5 and the recombinant clones with a 1.5 kb insert were sent to be sequenced by sequencing company. All generated sequences were compared with nucleotide sequences of known sequences listed in GenBank databases (http://www.ncbi.nlm.nih. gov/) using BLAST (Basic Local Alignment Search Tool). The phylogenetic tree was constructed according to maximum-likelihood analysis implemented in MEGA6 software.

Biodegradation of contaminants
Enrichment culture processes were monitored periodically to isolate the strains capable of removing COD Cr in viscose fiber wastewater. Isolated strains presenting clear colonial morphology on LB agar culture medium were further purified and incubated in LB culture media of 200 mL at 30 °C. The cells were harvested by centrifugation at 12,000 rpm for 10 min when the optical density (600 nm) reached at 0.6, pellets were washed with sterile 0.85% NaCl (w/v) twice and then inoculated into conical flasks containing 300 mL of viscose fiber wastewater and placing on a rotary shaker (180 rpm) at 30 °C. The mixture of isolates, the consortium labeled VF, and the mixture with the consortium were added into viscose fiber wastewater under the same conditions. The culture flask containing 300 mL of wastewater without bacterial consortium (VF) was as control experiment. Flasks of the isolates, mixture, the consortium, and the control were taken out from the rotary shaker on the 0th, 2nd, 4th, 6th, 8th, 10th, 12th, and 14th days. The analysis of COD Cr content in each sample was carried out with Microwave digestion COD analyzer .
The degradation rate of COD Cr was calculated using the formula as follows: where C i and C e are raw water and the sample concentrations in mg L −1 .

The isolation of dissolved organic matters (DOM)
The relative molecular weight was determined by ultrafiltration membrane method to reflect the changes of dissolved organic matter (DOM) during the biodegradation process. Particulate matter in samples was firstly got rid of using 0.45 μm filtering membrane, pretreated samples were added into a continuous flow system consisting of UF following the method of Zhao et al. (2006). And the molecular weight range for each fraction is listed in Table 2.
The quantity of dissolved organic carbon (DOC) corresponding to each size fraction of DOM after filtered with different pore-size UF membranes was calculated from DOC concentration and corresponding volume.
The ultrafilter is Amicon 8200 type ultrafiltration cup which was produced by the United States Millipore, having an effective volume of 180 mL, effective film area of 28.7 cm 2 , maximum withstand pressure of 0.53 MPa, built-in magnetic stirring Pressure-driven high-purity nitrogen (Qiao et al. 2007).

Analytical methods
The pH value was measured by a pH meter (METTLER TOLEDO, FE20K). The content of BOD 5 was measured by incubation method. The content of total nitrogen (TN) and total phosphorus (TP) were analyzed using alkaline potassium persulfate digestion-UV spectrophotometer and molybdenum blue spectrophotometric (Carranzo 2012). Dissolved organic carbon (DOC) in water samples were measured using UV-per-sulfate technique and infrared carbon dioxide analyzer (Phoenix 8000), and calibrated with potassium hydrogen phthalate.