Advances in ethanol production from hardwood spent sulphite liquors

Hardwood spent sulphite liquors (HSSLs) are by-products from the pulping industry rich in pentoses, which are not yet fully exploited for bioprocessing, namely for the production of bioethanol. The sustainable fermentation of pentoses into bioethanol is a challenge to overcome. Besides sugars, HSSLs contains inhibitors that decrease the possibility of bioprocessing of these by-products. Nevertheless, recent studies have brought new insights in using HSSLs for bioethanol production. This paper reviews the results of relevant studies carried out with HSSLs towards bioprocessing to bioethanol. The composition of SSLs was compared and related with the wood origin stressing specificity of microbial inhibitors from HSSL and their anti-microbial effect. The different fermentative processes, the microorganisms used, and the strategies to improve yield and productivity used so far were also reviewed. This review allowed concluding that research is still needed in several areas, including optimization of detoxification processes, fermentation strategies and selection of suitable microbial strains in order to achieve the integration of the different steps needed for HSSLs bioconversion into ethanol thus contributing for sustainability of pulping mills within biorefinary concept.     

Fermentation to ethanol of pentose-containing spent sulphite liquor

Ethanolic fermentation of spent sulphite liquor with ordinary bakers' yeast is incomplete because this yeast cannot ferment the pentose sugars in the liquor. This results in poor alcohol yields, and a residual effluent problem By using the yeast Candida shehatae (R) for fermentation of the spent sulphite liquor from a large Canadian alcohol-producing sulphite pulp and paper mill, pentoses as well as hexoses were fermented nearly completely, alcohol yields were raised by 33%, and sugar removal increased by 46%. Inhibitors were removed prior to fermentation by steam stripping. Major benefits were obtained by careful recycling of this yeast, which was shown to be tolerant both of high sugar concentrations and high alcohol concentrations. When sugar concentrations over 250 g/L (glucose: xylose 70:30) were fermented, ethanol became an inhibitor when its concentration reached 90 g/L. However, when the ethanol was removed by low-temperature vacuum distillation, fermentation continued and resulted in a yield of 0.50 g ethanol/g sugar consumed. Further improvement was achieved by combining enzyme saccharification of sugar oligomers with fermentation. This yeast is able to ferment both hexoses and pentoses simultaneously, efficiently, and rapidly. Present indications are that it is well suited to industrial operations wherever hexoses and pentoses are both to be fermented to ethanol, for example, in wood hydrolysates.     

Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Identification of dimethyl disulfide-forming bacteria isolated from activated sludge

Twenty-four strains with high dimethyl disulfide (DMDS)-forming ability were isolated from activated sludge and identified to the genus level. These bacteria were classified into four groups (A, B, C, and D) by the API ZYM System (API System S.A., Montalieu, France). Group A (three strains) was identified as genus Lactobacillus by the API 20B System, by the method of Cowan and Steel, and by production of lactic acid as confirmed by gas-liquid chromatography. Group B (eight strains) was identified as genus Corynebacterium by API 20B and the Cowan and Steel method. Group C (one strain) was suggested to belong to genus Corynebacterium by the API 20B System. Group D (12 strains) was identified as genus Pseudomonas or Alcaligenes by the API 20B System, as genus Alcaligenes by the Cowan and Steel method, and as Achromobacter group Vd by the API 20NE System. However, on the basis of guanine-plus-cytosine contents in DNA and form of flagella, these strains were identified as genus Pseudomonas. Formation of DMDS from DL-methionine and S-methyl-L-cysteine was tested. DMDS-forming bacteria isolated from activated sludge formed DMDS from both precursors. In genus Pseudomonas, P. aeruginosa could not form DMDS from either precursor, but P. acidovorans, P. alcaligenes, P. pseudoalcaligenes, and P. testosteroni formed DMDS. In genus Alcaligenes, A. denitrificans subsp. xylosoxydans, A. denitrificans subsp. denitrificans, A. faecalis, and A. odorans formed DMDS from both precursors. Achromobacter group Vd formed DMDS from S-methyl-L-cysteine, but could not from DL-methionine.     

Xylanase production by fungal strains on spent sulphite liquor

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml⁻¹, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322 U ml⁻¹ was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.     

Thermophilic bacteria from spent liquor of pulp mills

A thermophilic bacterial mixed culture was isolated from spent liquor of pulp mills, using a chemostat under aerobic conditions. It consists of two components belonging to genus Bacillus. The thermophilic Bacilli were cultivated aerobically and continuously at 67–70°C and pH 6.8–7, rising the percentage of spent liquor in stages. The dilution rate ranged from 0.2 to 0.33 h^?1 during the experiments. The composition of the cell mass produced was analyzed.     

Degradation of substituted thiophenes by bacteria isolated from activated sludge

Actinomycetes were isolated from activated sludge acclimated to thiophene-2-carboxylic acid (T2C) or 5-methyl-thiophene-2-carboxylic acid (T5M2C). These isolates were apparently identical and were identified as strains ofRhodococcus. The strains could grow on T2C, T5M2C, or thiophene-2-acetic acid as sole sources of carbon and energy, but could not use thiophene, methyl thiophenes, several other substituted thiophenes, dibenzothiophene, dimethyl sulfide, or pyrrole-2-carboxylic acid. T2C was degraded quantitatively to sulfate, and its carbon was converted almost entirely to cell biomass and carbon dioxide. Growth yields indicated about 25% conversion of T2C-carbon to cell-carbon. Growth was not supported by thiosulfate or methionine, nor were these compounds oxidized.Rhodococcus strain TTD-1 grown on T2C oxidized both T2C and T5M2C with an apparent K_m of 1.3×10^–5 M. Sulfide was also oxidized by T2C-grown organisms. This is the first demonstration of an actinomycete capable of the complete degradation of thiophene derivatives and of their use by it as sole substrates for growth.     

Anaerobic biodegradation of spent sulphite liquor in a UASB reactor

Anaerobic biodegradation of fermented spent sulphite liquor, SSL, which is produced during the manufacture of sulphite pulp, was investigated. SSL contains a high concentration of lignin products in addition to hemicellulose and has a very high COD load (173 g COD l(-1)). Batch experiments with diluted SSL and pretreated SSL indicated a potential of 12-22 l methane per litre SSL, which corresponds to 0.13-0.22 l methane (g VS)(-1) and COD removal of up to 37%. COD removal in a mesophilic upflow anaerobic sludge blanket, UASB. reactor ranged from 10% to 31% at an organic loading rate, OLR, of 10-51 g (1 d)(-1) and hydraulic retention time from 3.7 to 1.5 days. The biogas productivity was 3 1 (l(reactor d)(-1), with a yield of 0.05 l gas (g VS)(-1). These results suggest that anaerobic digestion in UASB reactors may provide a new alternative for the treatment of SSL to other treatment strategies such as incineration. Although the total COD reduction achieved is limited, bioenergy is produced and readily biodegradable matter is removed causing less load on post-treatment installations.     

Biodegradation of ammonium sulphite spent liquor by pure bacterial cultures

Summary Several bacterial strains able to grow on ammonium sulphite spent liquor (ASSL) were isolated by an enrichment culture technique and identified. The capacities of these bacteria to degrade ASSL in pure culture was compared with the modification of the methoxyl groups of the lignosulphonates. A rapid demethylation followed by remethylation, observed in some species, showed a complex biodegradation mechanism.Attempts to correlate the ability to degrade the substrate studied with that of the micro-organism selected to grow upon aromatic carbon sources were made. The isolate might have numerous activities regarding ASSL.     

Influence of sulphur dioxide on the fermentation of sulphite spent liquor

In fodder yeast production from sulphite spent liquor, sulphur dioxide is well-known as a yeast poison. The present paper deals with the detailed influence of sulphur dioxide. The experimental work was done in a laboratory fermenter under technical conditions. A decrease in biomass yield, and an increase in oxygen consumption, caused by increasing carbon dioxide production, were found. During the fermentation, there is no destruction of the carbonyl-bisulphite adduct to be observed. Sulphur dioxide has no influence on the crude protein content of yeast cells, and there is also no influence on the morphology of cells.     

Ethanol production from hardwood spent sulfite liquor using an adapted strain of Pichia stipitis

Conditions have been optimized for fermentation of pretreated hardwood spent sulfite liquor (HSSL) using an adapted strain of Pichia stipitis. The pretreatments, consisting of boiling and overliming with Ca(OH)₂ of HSSL, to partially remove inhibitors, and adaptation of the yeast strain to HSSL, were both critical for a successful fermentation. Ethanol concentration was increased from 6.7 to 20.2 g l⁻¹ using adapted P. stipitis (A) and pretreated HSSL. The maximum ethanol yield (Y _p/s) and productivity (Q _p) were 0.41 g g⁻¹ and 0.44 g l⁻¹ h⁻¹, respectively, at an oxygen transfer rate of 2.0 mmol O₂ l⁻¹ h⁻¹. The optimized results with this strain were compared to those of other xylose-fermenting yeasts and Saccharomyces cerevisiae (SSL-acclimatized) currently used at an industrial plant for the fermentation of spent sulfite liquor. Journal of Industrial Microbiology & Biotechnology (2001) 26, 145–150. Received 23 June 2000/ Accepted in revised form 21 October 2000     

Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid

A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.     

Isolation of rhodoquinone-containing chemoorganotrophic bacteria from activated sludge

Abstract Several strains of Gram-negative aerobic chemoheterotrophic bacteria which produced rhodoquinone were isolated from activated sludge. All isolates contained a rhodoquinone with eight isoprene units as a major component in addition to the corresponding homologue of ubiquinone. The isolates seemed to make up a single taxon because of their homogeneity in phenotypic and chemotaxonomic properties, but could not be allocated to any known taxa of Gram-negative bacteria.     

Relationship of protozoan biomass to phosphate and nitrate removal from activated sludge mixed liquor

The relationship between protozoan biomass concentration and phosphate and nitrate removal was investigated in mixed liquor using three different carbon sources as supplements. The study was carried out using three respective initial biomass concentrations in a shaking flask environment. Samples were taken every 24 h to determine phosphate, nitrate, dissolved oxygen and chemical oxygen demand. The results revealed a direct relationship between decreases in nutrient concentrations and increases in cell densities of the isolates. Between 24 and 96 h, the increases in the protozoan density corresponded to a phosphate decreases from initial ranges of 55.42–57.36 mg/L, 50.27–51.17 mg/L and 50.01–50.83 mg/L to final ranges of 2.46–11.90 mg/L, 0.61–11.80 mg/L and 1.29–13.89 mg/L, in the presence of Aspidisca, Trachelophyllum and Peranema, respectively. Nitrate concentrations were observed to decrease from initial ranges of 23.84–25.90 mg/L, 23.94–25.84 mg/L and 26.12–26.54 mg/L to final ranges of 0.11–6.32 mg/L, 0.16–5.60 mg/L and 0.24–9.04 mg/L, respectively. The study had revealed that an increase in cell density of the test isolates produces a corresponding increase in phosphate and nitrate removal.     

Stalked bacteria in activated sludge

Summary A laboratory-scale activated sludge unit was fed continuously with a simulated industrial wastewater, consisting of a dilute solution of inorganic salts, at a rate giving a mean retention time of about 3 days. The system produced a well-settling sludge which on examination by electron microscopy was found to contain considerable numbers of stalked bacteria. These were identified as Caulobacter, which have the ability to attach to surfaces and other organisms by means of a prosthecal holdfast and to flourish in waters with a low content of organic nutrients, and whose occurrence in activated sludge has not apparently been previously recorded. Conditions advantageous to Caulobacter generally prevail in activated sludge systems when these operate in growth phases tending to produce well-settling sludge. Since their holdfast gives Caulobacter the ability to initiate and enlarge microbial clusters by attachment, it is suggested that Caulobacter contribute to microbial floc-formation in activated sludge.     

Glyphosate degradation byAgrobacterium radiobacter isolated from activated sludge

Summary Two species of bacteria capable of growth onN-phosphonomethylglycine (glyphosate) were isolated from a bench scale sequencing batch reactor degrading a waste stream containing glyphosate. The enrichment and isolation medium contained defined salts and glyphosate as the sole carbon and energy source. Glyphosate was stoichiometrically degraded to aminomethylphosphonic acid (AMPA). The bacteria have been identified asAgrobacterium radiobacter andAchromobacter Group V D.