Recent developments in the biology and biotechnological applications of halotolerant yeasts.

Natural hypersaline environments are inhabited by an abundance of prokaryotic and eukaryotic microorganisms capable of thriving under extreme saline conditions. Yeasts represent a substantial fraction of halotolerant eukaryotic microbiomes and are frequently isolated as food contaminants and from solar salterns. During the last years, a handful of new species has been discovered in moderate saline environments, including estuarine and deep-sea waters. Although Saccharomyces cerevisiae is considered the primary osmoadaptation model system for studies of hyperosmotic stress conditions, our increasing understanding of the physiology and molecular biology of halotolerant yeasts provides new insights into their distinct metabolic traits and provides novel and innovative opportunities for genome mining of biotechnologically relevant genes. Yeast species such as Debaryomyces hansenii, Zygosaccharomyces rouxii, Hortaea werneckii and Wallemia ichthyophaga show unique properties, which make them attractive for biotechnological applications. Select halotolerant yeasts are used in food processing and contribute to aromas and taste, while certain gene clusters are used in second generation biofuel production. Finally, both pharmaceutical and chemical industries benefit from applications of halotolerant yeasts as biocatalysts. This comprehensive review summarizes the most recent findings related to the biology of industrially-important halotolerant yeasts and provides a detailed and up-to-date description of modern halotolerant yeast-based biotechnological applications.

Charting Shifts in Saccharomyces cerevisiae Gene Expression across Asynchronous Time Trajectories with Diffusion Maps.

During fermentation, Saccharomyces cerevisiae metabolizes sugars and other nutrients to obtain energy for growth and survival, while also modulating these activities in response to cell-environment interactions. Here, differences in S. cerevisiae gene expression were explored over a time course of fermentation and used to differentiate fermentations, using Pinot noir grapes from 15 unique sites. Data analysis was complicated by the fact that the fermentations proceeded at different rates, making a direct comparison of time series gene expression data difficult with conventional differential expression tools. This led to the development of a novel approach combining diffusion mapping with continuous differential expression analysis (termed DMap-DE). Using this method, site-specific deviations in gene expression were identified, including changes in gene expression correlated with the non-Saccharomyces yeast Hanseniaspora uvarum, as well as initial nitrogen concentrations in grape musts. These results highlight novel relationships between site-specific variables and Saccharomyces cerevisiae gene expression that are linked to repeated fermentation outcomes. It was also demonstrated that DMap-DE can extract biologically relevant gene expression patterns from other contexts (e.g., hypoxic response of Saccharomyces cerevisiae) and offers advantages over other data dimensionality reduction approaches, indicating that DMap-DE offers a robust method for investigating asynchronous time series gene expression data. IMPORTANCE In this work, Saccharomyces cerevisiae gene expression was used as a biosensor to capture differences across and between fermentations of Pinot noir grapes from 15 unique sites representing eight American Viticultural Areas. This required development of a novel analysis method, DMap-DE, for investigation of asynchronous gene expression data. It was demonstrated that DMap-DE reveals biologically relevant shifts in gene expression related to cell-environment interactions in the context of hypoxia and fermentation. Using these data, it was discovered that gene expression by non-Saccharomyces yeasts and initial nitrogen content in grape musts are correlated with differences in gene expression among fermentations. These findings highlight important relationships between site-specific variables and gene expression that may be used to understand why foods and beverages, including wine, possess sensory characteristics associated with or derived from their place of origin.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Overlapping responses between salt and oxidative stress in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.

Biocontrol of Penicillium griseofulvum to reduce cyclopiazonic acid contamination in dry-fermented sausages.

Dry-fermented sausages are very appreciated by consumers. The environmental conditions during its ripening favor colonization of their surface by toxigenic molds. These molds contribute to the development of sensory characteristics; however, some of them could produce mycotoxins such as cyclopiazonic acid (CPA). CPA is mainly produced by Penicillium commune and Penicillium griseofulvum which have been found in dry-cured meat products. Thus, strategies to prevent the CPA contamination in dry-fermented sausages are needed. The objective of this work was to evaluate the ability of P. griseofulvum to produce CPA in dry-fermented sausage during its ripening as well as to test different strategies to prevent CPA production. The ability of PgAFP antifungal protein-producing Penicillium chrysogenum, Debaryomyces hansenii and Pediococcus acidilactici for inhibiting CPA production by P. griseofulvum was tested on dry-fermented sausage-based medium. Only P. chrysogenum inhibited the CPA production, so this mold was co-inoculated with P. griseofulvum on sausages whose ripening was performed at low temperature. CPA reached around 800 ng/g in the control batch, being reduced to 20 ng/g by the presence of P. chrysogenum. This work demonstrates the risk posed by CPA on dry-fermented sausages, and provides a successful strategy to prevent this hazard.

Salinity and high pH affect energy pathways and growth in Debaryomyces hansenii.

The physiological behavior of Debaryomyces hansenii in response to saline stress and elevated pH was studied. The combination of 1 M NaCl salt and pH 8.0 was required to produce significant changes in the lag phase of growth and a consequent effect on viability. pH 8.0 in the absence or presence of 1 M NaCl produced changes in physiological functions such as respiration, acidification, rubidium transport, transmembrane potential, and fermentation. Our data indicated a stimulation of the H+-ATPase of the plasma membrane at pH 8.0, which increased the transmembrane potential and favored the entry of Na+; this effect was intensified in the presence of NaCl, so the increased energy expenditure resulting from H+ pumping and the extrusion of excess Na+ affected viability. The gene expression pattern studied by microarrays of cells incubated under saline conditions and high pH revealed a down-regulation in genes related to energy-producing pathways and in some genes involved in the cell cycle and DNA transcription, confirming our experimental hypothesis. Although D. hansenii can tolerate high pH and high salt concentrations, its physiological behavior, is better at pH 6.0 and in the absence of sodium; thus, it is an alkali-halotolerant yeast and not a halophilic yeast as previously proposed by other authors.

Inventory of ABC proteins and their putative role in salt and drug tolerance in Debaryomyces hansenii.

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance. Our bioinformatics analysis identified a total of 30 putative ABC protein-coding genes whose expression at transcript level was confirmed by qRT-PCR. Our comparative phylogenetic analysis of nucleotide binding domains of D. hansenii and topology prediction categorized these proteins into six subfamilies; ABCB/MDR, ABCC/MRP, ABCD/ALDP, ABCF/YEF3, ABCE/RLI, and ABCG/PDR based on the nomenclature adopted by the Human Genome Organization (HUGO). Further, our transmembrane domain (TMD) predictions suggest that out of 30 ABC proteins, only 22 proteins possess either two or one TMD and hence are considered as membrane localized ABC proteins. Notably, our transcriptional dynamics of ABC proteins encoding genes following D. hansenii cells treatment with different salts and drugs concentrations illustrated variable transcriptional response of some of the genes, pointing to their role in salt and drug tolerance. This study first time provides a comprehensive inventory of the ABC proteins of a haploid D. hansenii which will be helpful for exploring their functional relevance.

Functional analysis of Debaryomyces hansenii Rpn4 on a genetic background of Saccharomyces cerevisiae.

The transcription factor ScRpn4 coordinates the expression of Saccharomyces cerevisiae proteasomal genes. ScRpn4 orthologues are found in a number of other Saccharomycetes yeasts. Their functions, however, have not yet been characterised experimentally in vivo . We expressed the Debaryomyces hansenii DEHA2D12848 gene encoding an ScRpn4 orthologue (DhRpn4), in an S. cerevisiae strain lacking RPN4 . We showed that DhRpn4 activates transcription of proteasomal genes using ScRpn4 binding site and provides resistance to various stresses. The 43-238 aa segment of DhRpn4 contains an unique portable transactivation domain. Similar to the ScRpn4 N-terminus, this domain lacks a compact structure Moreover, upon overexpression in D. hansenii , DhRpn4 upregulates protesomal genes. Thus, we show that DhRpn4 is the activator for proteasomal genes.

Genomic expression program of Saccharomyces cerevisiae along a mixed-culture wine fermentation with Hanseniaspora guilliermondii.

BACKGROUND: The introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S. cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniaspora guilliermondii, this being the first time that this relevant yeast-yeast interaction is examined at a genomic scale. RESULTS: Co-inoculation with H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR < 0.05) in response to the presence of H. guilliermondii in the fermentation medium. Genes involved in biosynthesis of vitamins were enriched among those up-regulated in the mixed-culture fermentation, while genes related with the uptake and biosynthesis of amino acids were enriched among those more expressed in the single-culture. The differences in the aromatic profiles of wines obtained in the single and in the mixed-fermentations correlated with the differential expression of S. cerevisiae genes encoding enzymes required for formation of aroma compounds. CONCLUSIONS: By integrating results obtained in the transcriptomic analysis performed with physiological data our study provided, for the first time, an integrated view into the adaptive responses of S. cerevisiae to the challenging environment of mixed culture fermentation. The availability of nutrients, in particular, of nitrogen and vitamins, stands out as a factor that may determine population dynamics, fermentative activity and by-product formation.

The antibiotic resistance arrow of time: efflux pump induction is a general first step in the evolution of mycobacterial drug resistance.

We hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminated Mycobacterium avium-M. intracellulare complex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, in M. avium. By day 7, a subpopulation of M. avium with low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified in Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, and Mycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an "antibiotic resistance arrow of time."

Proteomic changes in response to potassium starvation in the extremophilic yeast Debaryomyces hansenii.

In this work, we performed for the first time a proteomic approach to the processes induced by long-term potassium starvation in the halotolerant yeast Debaryomyces hansenii. The proteomic profile under this ionic stress conditions shows that important changes in gene expression take place as an adaptive response. We found a significant protein expression repression as well as metabolic changes such as the inhibition of the upper part of the glycolysis, the amino acid synthesis, and the Krebs cycle. On the other hand, genes related to stress responses, protein degradation, and sterols synthesis were upregulated in response to potassium deprivation. The findings in this study provide important information about how this particular yeast copes with ionic stress at molecular levels, which might further enrich the global understanding of salt tolerance processes in eukaryal systems and moreover highlighting the importance of the 'omics' approaches as a complement to the classical physiological studies.

Mechanisms involved in reduction of ochratoxin A produced by Aspergillus westerdijkiae using Debaryomyces hansenii CYC 1244.

Aspergillus westerdijkiae is one of the most relevant ochratoxin A (OTA) producing species within the Section Circumdati contaminating a number of agroproducts. The yeast Debaryomyces hansenii CYC 1244 was previously reported to be able to reduce growth and extracellular OTA produced by A. westerdijkiae. In this work, we examined several mechanisms possibly involved in this OTA reduction in in vitro experiments. OTA biosynthesis was evaluated by quantitation of expression levels of pks (polyketide synthase) and p450-B03 (cytochrome p450 monooxygenase) genes using newly developed and specific real time RT-PCR protocols. Both genes showed significant lower levels in presence of D. hansenii CYC 1244 suggesting an effect on regulation of OTA biosynthesis at transcriptional level. High levels of removal of extracellular OTA were observed by adsorption to yeast cell walls, particularly at low pH (98% at pH 3). On the contrary, no evidences were obtained of absorption of OTA into yeast cells or the production of constitutively expressed enzymes that degrade OTA by D. hansenii CYC 1244. These results described the potential of this yeast strain as a safe and efficient biocontrol agent to decrease OTA in A. westerdijkiae and two important mechanisms involved which may permit its application at different points of the food chain.

Monovalent cations regulate expression and activity of the Hak1 potassium transporter in Debaryomyces hansenii.

Debaryomyces hansenii was able to grow in a medium containing residual amounts of K(+), indicating the activity of high affinity K(+) transporters. Transcriptional regulation analysis of the genes encoding the two potassium uptake systems in D. hansenii revealed that while DhTRK1 is not regulated at transcriptional level, expression of DhHAK1 required starvation in the absence of K(+) and Na(+) and was not affected by changes in membrane potential. Rb(+) transport in cells expressing DhHAK1 was activated by external Na(+) or acidic pH and inhibited by high pH. We propose a K(+)-H(+) symporter that, under certain conditions may work as a K(+)-Na(+) transporter, as the mechanism driving K(+) influx mediated by DhHak1p.

Halotolerant and halophilic fungi.

Extreme environments have for long been considered to be populated almost exclusively by prokaryotic organisms and therefore monopolized by bacteriologists. Solar salterns are natural hypersaline environments characterized by extreme concentrations of NaCl, often high concentrations of other ions, high uv irradiation and in some cases extremes in pH. In 2000 fungi were first reported to be active inhabitants of solar salterns. Since then many new species and species previously known only as food contaminants have been discovered in hypersaline environments around the globe. The eukaryotic microorganism most studied for its salt tolerance is Saccharomyces cerevisiae. However, S. cerevisiae is rather salt sensitive and not able to adapt to hypersaline conditions. In contrast, some species like Debaryomyces hansenii, Hortaea werneckii, and Wallemia ichthyophaga have been isolated globally from natural hypersaline environments. We believe that all three are more suitable model organisms to study halotolerance in eukaryotes than S. cerevisiae. Furthermore, they belong to different and distant taxonomic groups and have developed different strategies to cope with the same problems of ion toxicity and loss of water.

Genome-wide expression profiling of the osmoadaptation response of Debaryomyces hansenii.

The euryhaline marine yeast Debaromyces hansenii is a model system for the study of processes related to osmoadaptation. In this study, microarray-based gene expression analyses of the entire genome of D. hansenii was used to study its response to osmotic stress. Differential gene expression, compared to control, was examined at three time points (0.5, 3 and 6 h) after exposure of D. hansenii cultures to high salt concentration. Among the 1.72% of genes showing statistically significant differences in expression, only 65 genes displayed at least three-fold increases in mRNA levels after treatment with 2 M NaCl. On the other hand, 44 genes showed three-fold repression. Upregulated as well as the downregulated genes were grouped into functional categories to identify biochemical processes possibly affected by osmotic stress and involved in osmoadaptation. The observation that only a limited number of genes are upregulated in D. hansenii in response to osmotic stress supports the notion that D. hansenii is pre-adapted to survive in extreme saline environments. In addition, since more than one-half of the upregulated genes encode for ribosomal proteins, it is possible that a translational gene regulatory mechanism plays a key role in D. hansenii's osmoregulatory response. Validation studies for ENA1 and for hyphal wall/cell elongation protein genes, using real-time PCR, confirmed patterns of gene expression observed in our microarray experiments. To our knowledge, this study is the first of its kind in this organism and provides the foundation for future molecular studies assessing the significance of the genes identified here in D. hansenii's osmoadaptation.

Impairment of cobalt-induced riboflavin biosynthesis in a Debaryomyces hansenii mutant.

Flavinogenic yeasts such as Debaryomyces hansenii overproduce riboflavin (RF) in the presence of heavy metals. Growth and RF production were compared between wild-type D. hansenii and a RF production-impaired metal-tolerant ura3 mutant in the presence of sublethal cobalt(II) concentrations. Debaryomyces hansenii (wild type) exhibits an extended lag phase with an increase in RF synthesis. Supplementation of exogenous uracil shortened the lag phase at the highest concentration of cobalt(II) used, suggesting that uracil has a possible role in metal acclimation. The D. hansenii ura3 mutant isolated by chemical mutagenesis exhibited a higher level of metal tolerance, no extended lag phase, and no marked increase in RF synthesis. Transformation of the mutant with the URA3 gene isolated from Saccharyomyces cerevisiae or D. hansenii did not restore wild-type characteristics, suggesting a second mutation that impairs RF oversynthesis. Our results demonstrate that growth, metal sensitivity, and RF biosynthesis are linked.

Cloning and characterization of two K+ transporters of Debaryomyces hansenii.

Two genes from the halotolerant yeast Debaryomyces hansenii were cloned, DhTRK1 and DhHAK1. These genes encode K(+) transporters with sequence similarities to the TRK and HAK transporters from Debaryomyces occidentalis and Candida albicans. The DhHAK1p transporter was only expressed in K(+)-starved cells, as shown by Northern blot analysis. Both DhTRK1p and DhHAK1p were expressed in a trk1Delta trk2Delta mutant of Saccharomyces cerevisiae, unable to grow at low K(+). This expression resulted in partial recovery of growth and ability to retain K(+) at low concentrations. In liquid media, 0.5 M NaCl affected growth of these S. cerevisiae transformants as it does in D. hansenii, resulting in a much less deleterious effect than in wild-type S. cerevisiae. Kinetics of Rb(+) uptake in the transformants suggest that DhTRK1p and DhHAK1p code for moderate-affinity K(+) transporters exhibiting a sigmoid response against Rb(+) concentration and presenting a deviation from classic Michaelis-Menten kinetics at low substrate concentrations. Rb(+) uptake by the DhTRK1p transporter was stimulated by millimolar concentrations of Na(+) at pH 4.5. The good performance of DhTRK1p in the presence of NaCl may be a key feature in the halotolerance of D. hansenii.

Characterization of DhKHA1, a gene coding for a putative Na(+) transporter from Debaryomyces hansenii.

The KHA1 gene from Debaryomyces hansenii has been identified and characterized by heterologous expression in Saccharomyces cerevisiae. The gene is orthologous to ScKHA1, previously reported in S. cerevisiae, and on the basis of the deduced amino acid sequence, DhKha1p can be classified as an Na(+)/H(+) transporter. Reverse transcriptase (RT)-PCR experiments indicated that the expression level of DhKHA1 was not dependent on high pH or on the presence of a high salt level in the growth medium. Overexpression of DhKHA1 in a salt-sensitive S. cerevisiae mutant (ena1-4 nha1 kha1) rendered cells specifically more tolerant to Na(+). In addition, internal K(+) and Na(+) measurements and experiments performed with green fluorescence protein (GFP)-tagged DhKha1p indicated the intracellular localization of this protein when expressed in S. cerevisiae.

Proteomic changes in Debaryomyces hansenii upon exposure to NaCl stress.

The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed.

Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation.

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.