A constructed alkaline consortium and its dynamics in treating alkaline black liquor with very high pollution load

Abstract

Background: Paper pulp wastewater resulting from alkaline extraction of wheat straw, known as black liquor, is very difficult to be treated and causes serious environmental problems due to its high pH value and chemical oxygen demand (COD) pollution load. Lignin, semicellulose and cellulose are the main contributors to the high COD values in black liquor. Very few microorganisms can survive in such harsh environments of the alkaline wheat straw black liquor. A naturally developed microbial community was found accidentally in a black liquor storing pool in a paper pulp mill of China. The community was effective in pH decreasing, color and COD removing from the high alkaline and high COD black liquor.

Findings: Thirty-eight strains of bacteria were isolated from the black liquor storing pool, and were grouped as eleven operational taxonomy units (OTUs) using random amplified polymorphic DNA-PCR profiles (RAPD). Eleven representative strains of each OTU, which were identified as genera of Halomonas and Bacillus, were used to construct a consortium to treat black liquor with a high pH value of 11.0 and very high COD pollution load of 142,600 mg l(-1). After treatment by the constructed consortium, about 35.4% of color and 39,000 mg l(-1) (27.3%) COD(cr) were removed and the pH decreased to 7.8. 16S rRNA gene polymerase chain reaction denaturant gradient gel electrophoresis (PCR-DGGE) and gas chromatography/mass spectrometry (GC/MS) analysis suggested a two-stage treatment mechanism to elucidate the interspecies collaboration: Halomonas isolates were important in the first stage to produce organic acids that contributed to the pH decline, while Bacillus isolates were involved in the degradation of lignin derivatives in the second stage under lower pH conditions.

Conclusions/significance: Tolerance to the high alkaline environment and good controllability of the simple consortium suggested that the constructed consortium has good potential for black liquor treatment. Facilitating the treatment process by the constructed consortium would provide a promising opportunity to reduce the pollution, as well as to save forest resources and add value to a waste product.

Figure 1. RAPD profiles of the representative strains of the constructed consortium.

Lane M: DNA size marker (λDNA/HindIII); 17-3, 17-4, Y5, Y6, Y4, 19-D, 19-A, 17-5, 17-1, 19-B and Y2 represent the isolates.

Figure 2. Phylogenetic tree of eleven isolates and their closest sequences based on the analysis of the 16S rRNA gene sequences.

Partial sequences of approximately 600 bp in the 3′ end of full length 16S rDNA were used for constructing the phylogenetic tree.

Figure 3. Degradation curves of black liquor by the constructed consortium.

Symbols: A, pH of the treated black liquor (○); Color of control black liquor (pH 7.6) (□); Color of the treated black liquor (▪); COD_cr of the treated black liquor (▵); B, Lactic acid concentration in treated black liquor (▴); Formic acid concentration in treated black liquor (⋄); Acetic acid concentration in treated black liquor (♦).

Figure 4. DGGE profiles for V3 region of 16S rDNA fragment showing shifts in the constructed consortium during batch treatment.

Lane 1, constructed consortium before treatment; lane 2, consortium after 44-hour treatment; lane 3, consortium after 68-hour treatment; lane 4, consortium after 92-hour treatment; lane 5, consortium after 116-hour treatment; lane 6, consortium after 140-hour treatment; lane 7, consortium after 180-hour treatment.

Figure 5. Compositional changes of the black liquor in two-stage bio-treatment by GC/MS analysis.

A: Control; B: 2-day treatment by the four Halomonas isolates; C: 3-day treatment after supplied with the seven Bacillus isolates; D: sterilized and treated for 3 days after supplied with the seven Bacillus isolates; E: 5-day treatment by the four Halomonas isolates.

Figure 6. Semi-batch black liquor treatment by the constructed consortium.

Symbols: for pH (○); Color removal (▵); COD reduction (▴) and Feed volume (•).

Figure 7. Vanillin degradation by the constructed consortium and metabolites identification by GC/MS.

Control was the sample without inoculation; Treated was the sample with inoculation.