Hardwood Spent Sulfite Liquor Research Articles

Ethanol production from non-detoxified hardwood spent sulfite liquor in submerged fed-batch culture using advanced yeasts

To improve process feasibility, it is essential to use hardwood spent sulfite liquor (HSSL) as the main feedstock for bioethanol production, without prior detoxification. In addition, operating at large-scale under cost-effective conditions such as a small inoculum size (< 1 g/L), pH 5, using industrially acceptable nutrients, and without sugar addition, will require the use of harsh, concentrated HSSL streams. The potential of non-detoxified HSSL as a feedstock for ethanol production using two recombinant Saccharomyces cerevisiae strains, CelluX™4 and TFA7, was assessed. The inhibitory effect of non-detoxified HSSL was mitigated, and the ethanol titer increased from 4.1 to 7.9 g/L when pulse fed-batch was used instead of batch production, with CelluX™4 performing best. Both strains made use of the xylose isomerase (XI) pathway, with strain TFA7 engineered for increased tolerance against inhibitors. By administering concentrated HSSL in pulses to shake-flask cultures, the ethanol titer could be increased by approximately 50–90% when compared to simple batch cultures supplemented with 20%, 40%, and 60% (v/v) dilutions of HSSL. CelluX™4 was used in non-aerated, non-sterile 5-L bioreactor fermentations with a low cell concentration (< 1 g/L), pH 5, and 5 g/L corn steep liquor (CSL) as the nitrogen source. In comparison, undiluted HSSL was fed continuously to obtain a final 65% (v/v) HSSL supplementation, which corresponded to a total sugar concentration of 70.8–80.8 g/L. Despite the use of harsher, concentrated feedstock and inexpensive process conditions, the reactor fed-batch fermentations obtained ethanol yields of 0.35–0.43 g/g, which, based on a maximum theoretical ethanol yield of 0.51 g/g of hexoses or pentoses, corresponds to yield efficiencies of 68.6 and 84.3%. This illustrates an improvement on the highest titers reported in the literature for non-detoxified HSSL. The use of the advanced industrial S. cerevisiae strain, CelluX™4, combined with a fed-batch strategy, offers an inexpensive and straightforward process with real upscaling potential for industrial HSSL fermentations.

Enrichment of a mixed microbial culture of PHA-storing microorganisms by using fermented hardwood spent sulfite liquor

Pulp and paper factories produce several residues that can be explored and valorized through polyhydroxyalkanoate (PHA) production via a three-step process. The objective of this work was focused on the selection step. Acidified hardwood spent sulfite liquor (HSSL), a fermented waste stream from a pulp and paper factory, was used to select a mixed microbial culture (MMC) in a sequencing batch reactor (SBR) operated for 156 days under different operational conditions. The MMC adapted to the imposed conditions, revealing its robustness whenever the operational parameters were changed. Feast-to-Famine ratio was kept below or equal to 0.2, with constant production of a copolymer of P(3HB-co-3 HV), and with storage contents values over 30 %. Changes in the operational conditions, namely cycle length, and organic load rate (OLR), successfully led to the selection of an MMC with a stable accumulation capacity and an increased biomass concentration. Next Generation Sequencing analysis was performed on samples collected during the SBR operational period. The analysis of the microbial composition of the MMC showed a rise in PHA-accumulating bacteria over time. Acidovorax and Comamonas species were found mainly to drive the PHA storage process during the first two periods of operation. After an increase in the OLR, in the last period, a shift towards Comamonas dominance occurred, suggesting a higher tolerance to the inhibitory compounds of the HSSL for this genus.

Two-Stage Aeration Fermentation Strategy to Improve Bioethanol Production by Scheffersomyces stipitis

Hardwood spent sulfite liquor (HSSL) is a by-product from pulp industry with a high concentration of pentose sugars, besides some hexoses suitable for bioethanol production by Scheffersomyces stipitis. The establishment of optimal aeration process conditions that results in specific microaerophilic conditions required by S. stipitis is the main challenge for ethanol production. The present study aimed to improve the ethanol production from HSSL by S. stipitis through a two-stage aeration fermentation. Experiments with controlled dissolved oxygen tension (DOT) in the first stage and oxygen restriction in the second stage were carried out. The best results were obtained with DOT control at 50% in the first stage, where the increase of oxygen availability provided faster growth and higher biomass yield, and no oxygen supply with an agitation rate of 250 rpm, in the second stage allowed a successful induction of ethanol production. Fermentation using 60% of HSSL (v/v) as substrate for S. stipitis provided a maximum specific growth rate of 0.07 h−1, an ethanol productivity of 0.04 g L h−1 and an ethanol yield of 0.39 g g−1, respectively. This work showed a successful two-stage aeration strategy as a promising aeration alternative for bioethanol production from HSSL by S. stipitis.

Impact of Organic Acids Supplementation to Hardwood Spent Sulfite Liquor as Substrate for the Selection of Polyhydroxyalkanoates-Producing Organisms

The effectiveness of polyhydroxyalkanoates (PHAs) production process from a waste stream is determined by the selection of a suitable mixed microbial culture (MMC). In this work, a feedstock from the paper industry, hardwood spent sulfite liquor (HSSL), supplemented with short-chain organic acids (SCOAs) to simulate a fermented effluent, was used as substrate to enrich a MMC in PHA-storing microorganisms. A stable culture was quickly established, and during the accumulation step the selected MMC reached a maximum PHA content of 34.6% (3HB:3HV-76:24). The bacterial community was analyzed through FISH analysis. Bacteria belonging to the four main classes were identified: Betaproteobacteria (44.7 ± 2.7%), Alphaproteobacteria (13.6 ± 1.3%) and Gammaproteobacteria (2.40 ± 1.1%) and Bacteroidetes (9.20 ± 3.8%). Inside the Betaproteobacteria class, Acidovorax (71%) was the dominant genus.

Highly complex substrates lead to dynamic bacterial community for polyhydroxyalkanoates production.

Mixed microbial cultures (MMC) and waste/surplus substrates, as hardwood spent sulfite liquor, are being used to decrease polyhydroxyalkanoates' (PHA) production costs. The process involves two or three steps, being the selection step a crucial one. For the industrial implementation of this strategy, reactor stability in terms of both performance and microbial community presence has to be considered. A long-term operation of a sequencing batch reactor under feast/famine conditions was performed along with microbial community identification/quantification using FISH and DGGE. The community was found to be extremely dynamic, dominated by Alphaproteobacteria, with Paracoccus and Rhodobacter present, both PHA-storing microorganisms. 16S rRNA gene clone library further revealed that side populations' non-PHA accumulators were able to strive (Agrobacterium, Flavobacteria, and Brachymonas). Nevertheless, reactor performance in terms of PHA storage was stable during operation time. The monitoring of the MMC population evolution provided information on the relation between community structure and process operation.

Determinants of tolerance to inhibitors in hardwood spent sulfite liquor in genome shuffled Pachysolen tannophilus strains.

Genome shuffling was used to obtain Pachysolen tannophilus mutants with improved tolerance to inhibitors in hardwood spent sulfite liquor (HW SSL). Genome shuffled strains (GHW301, GHW302 and GHW303) grew at higher concentrations of HW SSL (80 % v/v) compared to the HW SSL UV mutant (70 % v/v) and the wild-type (WT) strain (50 % v/v). In defined media containing acetic acid (0.70-0.90 % w/v), GHW301, GHW302 and GHW303 exhibited a shorter lag compared to the acetic acid UV mutant, while the WT did not grow. Genome shuffled strains produced more ethanol than the WT at higher concentrations of HW SSL and an aspen hydrolysate. To identify the genetic basis of inhibitor tolerance, whole genome sequencing was carried out on GHW301, GHW302 and GHW303 and compared to the WT strain. Sixty single nucleotide variations were identified that were common to all three genome shuffled strains. Of these, 40 were in gene sequences and 20 were within 5 bp-1 kb either up or downstream of protein encoding genes. Based on the mutated gene products, mutations were grouped into functional categories and affected a variety of cellular functions, demonstrating the complexity of inhibitor tolerance in yeast. Sequence analysis of UV mutants (UAA302 and UHW303) from which GHW301, GHW302 and GHW303 were derived, confirmed the success of our cross-mating based genome shuffling strategy. Whole-genome sequencing analysis allowed identification of potential gene targets for tolerance to inhibitors in lignocellulosic hydrolysates.

Adaptation of Scheffersomyces stipitis to hardwood spent sulfite liquor by evolutionary engineering.

BackgroundHardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol.ResultsA continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C4) was isolated and characterized in 60% undetoxified HSSL. C4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C4.ConclusionS. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C4 may represent a major advantage for the production of bioethanol using HSSL as substrate.

Bioethanol Production: Adaptation of Scheffersomyces stipitis to Hardwood Spent Sulfite Liquor

Bioethanol Production: Adaptation of Scheffersomyces stipitis to Hardwood Spent Sulfite Liquor

PHA production by mixed cultures: A way to valorize wastes from pulp industry

In this work, hardwood spent sulfite liquor (HSSL), a complex feedstock originating from the pulp industry, was tested for the first time as a substrate for polyhydroxyalkanoate (PHA) production by a mixed microbial culture (MMC) under aerobic dynamic feeding (ADF) conditions. A sequencing batch reactor (SBR) fed with HSSL was operated for 67days and the selected MMC reached a maximum PHA content of 67.6%. The MMC demonstrated a differential utilization of the carbon sources present in HSSL. Acetic acid was fully consumed, while xylose and lignosulphonates were partially consumed (30% and 14%, respectively). The selected culture was characterized by Fluorescence in Situ Hybridization (FISH). Bacteria belonging to the three main classes were identified: Alpha- (72.7±4.0%), Beta- (11.1±0.37%) and Gammaproteobacteria (10.3±0.3%). Within Alphaproteobacteria, a small amount of Paracoccus (4.2±0.51%) and Defluvicoccus related to Tetrad Forming Organisms (9.0±0.28%) were detected.

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.

Removal of Acetic Acid from Spent Sulfite Liquor Using Anion Exchange Resin for Effective Xylose Fermentation with Pichia stipitis

Acetic acid is one of the major inhibitors of spent sulfite liquor (SSL) fermentation for ethanol production. The objective of this study was to remove acetic acid from hardwood SSL using anion exchange resin in order to achieve effective fermentation with Pichia stipitis CBS6054. Lignosulfonate, as well as sulfate and sulfite ions in the SSL hindered the removal of acetic acid by anion exchange resins. CaO treatment was an effective method for removing these materials from SSL, which facilitated the removal of acetic acid in the subsequent ion exchange resin treatment. A two-stage strong base ion exchange resin (OH- form) treatment removed approximately 90% of the acetic acid from CaO-treated SSL, which decreased the acetic acid concentration to less than 1 g/L. The combined treatment of CaO and ion exchange resin treatments in a relatively short time achieved the selective removal of acetic acid from SSL and significantly increased the ethanol production from SSL.

Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor

Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars which can be fermented to ethanol. However, fermentative yeasts used for the fermentation of the sugars in SSL are adversely affected by the inhibitory substances in this complex feedstock. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome-shuffling technology based on large-scale population cross mating. Populations of UV-light-induced yeast mutants more tolerant than the wild type to hardwood spent sulfite liquor (HWSSL) were first isolated and then recursively mated and enriched for more-tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and to support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. Our results showed that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. These strains may not be obtainable through classical evolutionary engineering and can serve as a model for further understanding of the mechanism behind simultaneous tolerance to multiple inhibitors.

Mutants of the pentose‐fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor

Mutants of Pichia stipitis NRRL Y-7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars.

Fortifying spent sulfite pulping liquor with hydrolyzed reject knots

A limitation for spent sulfite liquor (SSL) fermentation, particularly hardwood SSL fermentation is the low sugar content. The objective of this work was to increase the sugar content of SSL through the addition and hydrolysis of reject knots from the pulp line. Factorial experiments indicated that hydrolysis yield was significantly affected by cellulase and β-glucosidase loading, concentration of SSL, knot concentration, pH and temperature. Spent sulfite liquor reduced the hydrolysis rate and yield, however this inhibition decreased with time. The resultant hydrolysates were readily fermented alone or in combination with 10% SSL. In 22% SSL, fermentation yields were reduced. Economic analysis indicated that knots would be an economically-viable source of supplemental sugars at a cellulase cost of up to $5 CDN per million FPU.

Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds

Synthetic mixtures of predominant lignocellulosic hexose sugars were supplemented with separate aliquots of three inhibitory compounds (furfural, hydroxymethylfurfural (HMF), and acetic acid) in a series of concentrations and fermented by the spent sulfite liquor (SSL)-adapted yeast strain Tembec T1 and the natural isolate Saccharomyces cerevisiae (S. cerevisiae) Y-1528 to compare tolerance and assess fermentative efficacy. The performance of Y-1528 exceeded that of Tembec T1 by a significant margin, with faster hexose sugar consumption, higher ethanol productivity, and in the case of furfural and HMF, faster inhibitor consumption. Nevertheless, furfural had a dose-proportionate effect on sugar consumption rate and ethanol productivity in both strains, but did not substantially affect ethanol yield. HMF had a similar effect on sugar consumption rate and ethanol productivity, and also lowered ethanol yield. Surprisingly, acetic acid had the least impact on sugar consumption rate and ethanol productivity, and stimulated ethanol yield at moderate concentrations. Sequential iterations of softwood (SW) and hardwood (HW) SSL were subsequently inoculated with the two yeast strains in order to compare adaptation to, and performance in lignocellulosic substrates in a cell recycle batch fermentation (CRBF) regime. Both strains were severely affected by the HW SSL, which was attributed to specific syringyl lignin-derived degradation products and synergistic interactions between inhibitors. Though ethanologenic capacity was preserved, a net loss of performance was evident from both strains, indicating the absence of adaptation to the substrates, regardless of the sequence in which the SSL types were employed.

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 (Yp/s) and productivity (Qp) 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.

Selective removal of acetic acid from hardwood-spent sulfite liquor using a mutant yeast

The hexoses in spent sulfite liquor can be converted to ethanol by yeasts, but conversion to ethanol of the pentose d-xylose in lignocellulosic hydrolysates is inhibited generally by the presence of acetic acid. The feasibility of a yeast-based process for selective removal of the acetic acid in hardwood-spent sulfite liquor was demonstrated. The process depends on the use of a mutant of Saccharomyces cerevisiae that grows on acetic acid but not on d-xylose, d-glucose, d-mannose, or d-fructose. The process could be used to decrease the concentration of acetic acid in hardwood liquor within 24 h to levels that no longer inhibit bioconversion of xylose to ethanol. Indications of the conversion of d-xylose to ethanol in the acetic acid-depleted liquor were the ability of several d-xylose-fermenting yeasts to use essentially all of the sugars and produce as much as 73% of the theoretical amount of ethanol within 24 h. The process might be generally applicable to obviation of acetic acid inhibition effects in ethanol production from hemicellulose hydrolysates.

Development of xylose-fermenting yeasts for ethanol production at high acetic acid concentrations

Mutants resistant to comparatively high levels of acetic acid were isolated from the xylose-fermenting yeastsCandida shehatae andPichia stipitis by adapting these cultures to increasing concentrations of acetic acid grown in shake-flask cultures. These mutants were tested for their ability to ferment xylose in presence of high acetic acid concentrations, in acid hydrolysates of wood, and in hardwood spent sulfite liquor, and compared with their wild-type counterparts and between themselves. TheP. stipitis mutant exhibited faster fermentation times, better tolerance to acid hydrolysates, and tolerance to lower pH.

Acidified and Spray-Dried Spent Sulfite Liquor Powder as Binder for Particleboard

Powdered resins were prepared from softwood or hardwood spent sulfite liquor (SSL) by acidifying and spray-drying, and they were used as binders for particleboard. The strength properties and dimensional stability of the board were evaluated. The results obtained were as follows.(1) The particleboard with SSL powder showed good dimensional stability, but low modulus of rupture (MOR) and low internal bond (IB).(2) The effects of spray-drying temperature of SSL on the properties of the board were little in the range from 120°C to 150°C.(3) The board with softwood SSL powder was better in both strength properties and dimensional stability than that with hardwood SSL powder.