Yeast-insect interactions in southern Africa: Tapping the diversity of yeasts for modern bioprocessing.

Yeast-insect interactions are one of the most interesting long-standing relationships whose research has contributed to our understanding of yeast biodiversity and their industrial applications. Although insect-derived yeast strains are exploited for industrial fermentations, only a limited number of such applications has been documented. The search for novel yeasts from insects is attractive to augment the currently domesticated and commercialized production strains. More specifically, there is potential in tapping the insects native to southern Africa. Southern Africa is home to a disproportionately high fraction of global biodiversity with a cluster of biomes and a broad climate range. This review presents arguments on the roles of the mutualistic relationship between yeasts and insects, the presence of diverse pristine environments and a long history of spontaneous food and beverage fermentations as the potential source of novelty. The review further discusses the recent advances in novelty of industrial strains of insect origin, as well as various ancient and modern-day industries that could be improved by use yeasts from insect origin. The major focus of the review is on the relationship between insects and yeasts in southern African ecosystems as a potential source of novel industrial yeast strains for modern bioprocesses.

Phytostabilization of fly ash from a coalmine in Botswana and biovalorisation of the recovered Napier grass (Pennisetum purpureum Schumach.).

The disposal of fly ash (FA) from coal power plants polluting the air, soil, and groundwater is a major environmental concern. Phytoremediation to rehabilitate fly ash dumpsites is a promising alternative but has practical concerns about the disposal of harvested biomass. This study investigated the effect of supplementing fly ash with fresh sewage sludge (FSS), aged sewage sludge, food waste, and compost (COM) to enhance the phytoremediation potential of Napier grass and its subsequent utilization for ethanol production. The highest removal of Mn (1196.12 g ha-1) and Ni (128.06 g ha-1) from FA could be obtained when Napier is grown in the presence of FSS and inorganic fertilizer (NPK). In addition, the highest bioethanol yield (19.31 g L-1) was obtained from Napier grown in fly ash with COM + NPK, thus providing additional economic benefits aside from the remediation process. Given the significant levels of heavy metals present in the pulp and bio-slurry after ethanol production, further research is required in this area to determine the best ways to utilize this waste such as converting it into biochar.

Using energy crops as a phytoremediation agent for fly ash dumpsites has the potential to remediate heavy metal contamination and provide additional economic benefits. Napier grass was able to tolerate high concentrations of heavy metals and yield high biomass in fly ash in the presence of organic amendments. The harvested biomass was successfully converted into substrate for bioethanol production using heavy metal-tolerant yeast. This is the first report on the production of ethanol from the phytoremediation biomass of Napier grass.

Diversity of dung beetle-associated yeasts from pristine environments of Botswana.

Yeast-insect interactions are increasingly becoming an attractive source of discovery for previously unknown, unique, diverse, and industrially relevant yeast species. Despite a wealth of studies that have recently focused on yeasts in symbiotic association with Hymenopteran insects, yeasts associated with Coleopteran insects, such as lignocellulosic-rich dung-dependent beetles, remain poorly studied. Trends in yeast discovery suggest that species richness and diversity can be attributed to the ecological niche of the insect. Here, we considered the potential of dung beetles inhabiting the extreme environments of Botswana, characterized by desert-like conditions (semi-arid to arid and hot) as well as protected pristine environments, as possible attribute niches that can shape the extremophilic and diverse life history strategies of yeasts. We obtained a total of 97 phylogenetically diverse yeast isolates from six species of dung beetles from Botswana's unexplored environments, representing 19 species belonging to 11 genera. The findings suggest that the guts of dung beetles are a rich niche for non-Saccharomyces yeast species. Meyerozyma and Pichia were the most dominant genera associated with dung beetles, representing 55% (53 out of 97) of the yeast isolates in our study. Trichosporon and Cutaneotrichosporon genera represented 32% (31 out of 97) of the isolates. The remaining isolates belonged to Apiotrichum, Candida, Diutina, Naganishia, Rhodotorula, and Wickerhamiella genera (12 out of 97). We found out that about 62% (60 out of 97) of the isolates were potentially new species because of their low internal transcribed spacer (ITS) sequence similarity when compared to the most recent optimal species delineation threshold. A single isolate was unidentifiable using the ITS sequences. Using an in silico polymerase chain reaction-restriction fragment length polymorphism approach, we revealed that there was genetic diversity within isolates of the same species. Our results contribute to the knowledge and understanding of the diversity of dung beetle-associated yeasts.

Dung beetle-associated yeasts display multiple stress tolerance: a desirable trait of potential industrial strains.

BACKGROUND: Stress-tolerant yeasts are highly desirable for cost-effective bioprocessing. Several strategies have been documented to develop robust yeasts, such as genetic and metabolic engineering, artificial selection, and natural selection strategies, among others. However, the significant drawbacks of such techniques have motivated the exploration of naturally occurring stress-tolerant yeasts. We previously explored the biodiversity of non-conventional dung beetle-associated yeasts from extremophilic and pristine environments in Botswana (Nwaefuna AE et.al., Yeast, 2023). Here, we assessed their tolerance to industrially relevant stressors individually, such as elevated concentrations of osmolytes, organic acids, ethanol, and oxidizing agents, as well as elevated temperatures. RESULTS: Our findings suggest that these dung beetle-associated yeasts tolerate various stresses comparable to those of the robust bioethanol yeast strain, Saccharomyces cerevisiae (Ethanol Red™). Fifty-six percent of the yeast isolates were tolerant of temperatures up to 42 °C, 12.4% of them could tolerate ethanol concentrations up to 9% (v/v), 43.2% of them were tolerant to formic acid concentrations up to 20 mM, 22.7% were tolerant to acetic acid concentrations up to 45 mM, 34.0% of them could tolerate hydrogen peroxide up to 7 mM, and 44.3% of the yeasts could tolerate osmotic stress up to 1.5 M. CONCLUSION: The ability to tolerate multiple stresses is a desirable trait in the selection of novel production strains for diverse biotechnological applications, such as bioethanol production. Our study shows that the exploration of natural diversity in the search for stress-tolerant yeasts is an appealing approach for the development of robust yeasts.

Do microbes evade domestication? - Evaluating potential ferality among diastatic Saccharomyces cerevisiae.

Certain lineages of the wine, beer and bread yeast Saccharomyces cerevisiae have diastatic activity. They contain the chimeric gene STA1 that codes for an extracellular glucoamylase which enables the strains to degrade starch and dextrins. Beer contaminations by diastatic yeasts can be dangerous because they can cause super-attenuation due to the consumption of otherwise non-fermentable oligosaccharides, gushing and off-flavours. Given that diastatic yeasts can be used for beer fermentation it is important to understand the relationship between production and contaminant strains, their natural reservoirs and entry routes into the brewery. Here, we analyze real cases of contamination in a Portuguese craft brewery over a period of 18 months. By analyzing with whole genome sequencing several contaminants, we show that recurrent contaminations by diastatic yeasts are caused by environmental strains. Moreover, some beer contaminants were closely related to diastatic environmental strains isolated in Botswana. We observed the widespread presence of domestication signatures in diastatic strains. Moreover, the combined phylogeny of STA1 and its ancestor, SGA1, suggested a single STA1 origin, as ancient as the entire lineage of diastatic yeasts. Together, our results suggest that diastatic yeasts isolated in natural settings could be escaping from domestication settings and becoming feral.

Evolutionary engineering to improve Wickerhamomyces subpelliculosus and Kazachstania gamospora for baking.

The conventional baker's yeast, Saccharomyces cerevisiae, is the indispensable baking yeast of all times. Its monopoly coupled to its major drawbacks, such as streamlined carbon substrate utilisation base and a poor ability to withstand a number of baking associated stresses, prompt the need to search for alternative yeasts to leaven bread in the era of increasingly complex consumer lifestyles. Our previous work identified the inefficient baking attributes of Wickerhamomyces subpelliculosus and Kazachstania gamospora as well as preliminarily observations of improving the fermentative capacity of these potential alternative baker's yeasts using evolutionary engineering. Here we report on the characterisation and improvement in baking traits in five out of six independently evolved lines incubated for longer time and passaged for at least 60 passages relative to their parental strains as well as the conventional baker's yeast. In addition, the evolved clones produced bread with a higher loaf volume when compared to bread baked with either the ancestral strain or the control conventional baker's yeast. Remarkably, our approach improved the yeasts' ability to withstand baking associated stresses, a key baking trait exhibited poorly in both the conventional baker's yeast and their ancestral strains. W. subpelliculosus evolved the best characteristics attractive for alternative baker's yeasts as compared to the evolved K. gamospora strains. These results demonstrate the robustness of evolutionary engineering in development of alternative baker's yeasts.

Experimental evolution: its principles and applications in developing stress-tolerant yeasts.

Stress tolerance and resistance in industrial yeast strains are important attributes for cost-effective bioprocessing. The source of stress-tolerant yeasts ranges from extremophilic environments to laboratory engineered strains. However, industrial stress-tolerant yeasts are very rare in nature as the natural environment forces them to evolve traits that optimize survival and reproduction and not the ability to withstand harsh habitat-irrelevant industrial conditions. Experimental evolution is a frequent method used to uncover the mechanisms of evolution and microbial adaption towards environmental stresses. It optimizes biological systems by means of adaptation to environmental stresses and thus has immense power of development of robust stress-tolerant yeasts. This mini-review briefly outlines the basics and implications of evolution experiments and their applications to industrial biotechnology. This work is meant to serve as an introduction to those new to the field of experimental evolution, and as a guide to biologists working in the field of yeast stress response. Future perspectives of experimental evolution for potential biotechnological applications have also been elucidated.

Improvement of thermotolerance in Lachancea thermotolerans using a bacterial selection pressure.

The use of thermotolerant yeast strains is an important attribute for a cost-effective high temperature biofermentation processes. However, the availability of thermotolerant yeast strains remains a major challenge. Isolation of temperature resistant strains from extreme environments or the improvements of current strains are two major strategies known to date. We hypothesised that bacteria are potential "hurdles" in the life cycle of yeasts, which could influence the evolution of extreme phenotypes, such as thermotolerance. We subjected a wild-type yeast, Lachancea thermotolerans to six species of bacteria sequentially for several generations. After coevolution, we observed that three replicate lines of yeasts grown in the presence of bacteria grew up to 37 °C whereas the controls run in parallel without bacteria could only grow poorly at 35 °C retaining the ancestral mesophilic trait. In addition to improvement of thermotolerance, our results show that the fermentative ability was also elevated, making the strains more ideal for the alcoholic fermentation process because the overall productivity and ethanol titers per unit volume of substrate consumed during the fermentation process was increased. Our unique method is attractive for the development of thermotolerant strains or to augment the available strain development approaches for high temperature industrial biofermentation.

Biodiesel's trash is a biorefineries' treasure: the use of "dirty" glycerol as an industrial fermentation substrate.

"Dirty" glycerol from biodiesel production is having a considerable environmental impact since its disposal is expensive and difficult. The increased biodiesel production in the last two decades has forced glycerol prices down, thereby making it now unprofitable for chemical companies to produce. The problem lies with the impurities of the biodiesel conversion process usually ending up within the crude glycerol fraction. These impurities are often too costly to purify with current processes, particularly for small scale producers. A wide variety of industries, including the paint, tobacco, food and pharmaceutical industries, utilize glycerol as part of their technology or products. However, the crude glycerol from biodiesel production is not of a high enough grade to be used in these industries. Biodiesel-produced crude glycerol is therefore cheap, readily available and presents itself as an attractive carbon source for industrial microbial production systems synthesizing value-added products. This mini-review will look at (a) microbial production processes which use crude glycerol to produce high-value products (product-driven research) and (b) genetic engineering of microbes which is aimed at improving microbial "dirty" glycerol utilization (substrate driven research).

Streptomyces albulus yields ε-poly-L-lysine and other products from salt-contaminated glycerol waste.

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-L-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-L-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.

Yeast-bacteria competition induced new metabolic traits through large-scale genomic rearrangements in Lachancea kluyveri.

Large-scale chromosomal rearrangements are an important source of evolutionary novelty that may have reshaped the genomes of existing yeast species. They dramatically alter genome organization and gene expression fueling a phenotypic leap in response to environmental constraints. Although the emergence of such signatures of genetic diversity is thought to be associated with human exploitation of yeasts, less is known about the driving forces operating in natural habitats. Here we hypothesize that an ecological battlefield characteristic of every autumn when fruits ripen accounts for the genomic innovations in natural populations. We described a long-term cross-kingdom competition experiment between Lachancea kluyveri and five species of bacteria. Now, we report how we further subjected the same yeast to a sixth species of bacteria, Pseudomonas fluorescens, resulting in the appearance of a fixed and stably inherited large-scale genomic rearrangement in two out of three parallel evolution lines. The 'extra-banded' karyotype, characterized by a higher fitness and an elevated fermentative capacity, conferred the emergence of new metabolic traits in most carbon sources and osmolytes. We tracked down the event to a duplication and translocation event involving a 261-kb segment. Such an experimental setup described here is an attractive method for developing industrial strains without genetic engineering strategies.

Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.

Use of non-conventional yeast improves the wine aroma profile of Ribolla Gialla.

Consumer wine preferences are changing rapidly towards exotic flavours and tastes. In this work, we tested five non-conventional yeast strains for their potential to improve Ribolla Gialla wine quality. These strains were previously selected from numerous yeasts interesting as food production candidates. Sequential fermentation of Ribolla Gialla grape juice with the addition of the Saccharomyces cerevisiae T73 Lalvin industrial strain was performed. Zygosaccharomyces kombuchaensis CBS8849 and Kazachstania gamospora CBS10400 demonstrated positive organoleptic properties and suitable fermentation dynamics, rapid sugar consumption and industrial strain compatibility. At the same time, Torulaspora microellipsoides CBS6641, Dekkera bruxellensis CBS2796 and Dekkera anomala CBS77 were unsuitable for wine production because of poor fermentation dynamics, inefficient sugar consumption and ethanol production levels and major organoleptic defects. Thus, we selected strains of K. gamospora and Z. kombuchaensis that significantly improved the usually plain taste of Ribolla wine by providing additional aromatic complexity in a controlled and reproducible manner.

Why, when, and how did yeast evolve alcoholic fermentation?

The origin of modern fruits brought to microbial communities an abundant source of rich food based on simple sugars. Yeasts, especially Saccharomyces cerevisiae, usually become the predominant group in these niches. One of the most prominent and unique features and likely a winning trait of these yeasts is their ability to rapidly convert sugars to ethanol at both anaerobic and aerobic conditions. Why, when, and how did yeasts remodel their carbon metabolism to be able to accumulate ethanol under aerobic conditions and at the expense of decreasing biomass production? We hereby review the recent data on the carbon metabolism in Saccharomycetaceae species and attempt to reconstruct the ancient environment, which could promote the evolution of alcoholic fermentation. We speculate that the first step toward the so-called fermentative lifestyle was the exploration of anaerobic niches resulting in an increased metabolic capacity to degrade sugar to ethanol. The strengthened glycolytic flow had in parallel a beneficial effect on the microbial competition outcome and later evolved as a "new" tool promoting the yeast competition ability under aerobic conditions. The basic aerobic alcoholic fermentation ability was subsequently "upgraded" in several lineages by evolving additional regulatory steps, such as glucose repression in the S. cerevisiae clade, to achieve a more precise metabolic control.

The marula and elephant intoxication myth: assessing the biodiversity of fermenting yeasts associated with marula fruits (Sclerocarya birrea).

The inebriation of wild African elephants from eating the ripened and rotting fruit of the marula tree is a persistent myth in Southern Africa. However, the yeasts responsible for alcoholic fermentation to intoxicate the elephants remain poorly documented. In this study, we considered Botswana, a country with the world's largest population of wild elephants, and where the marula tree is indigenous, abundant and protected, to assess the occurrence and biodiversity of yeasts with a potential to ferment and subsequently inebriate the wild elephants. We collected marula fruits from over a stretch of 800 km in Botswana and isolated 106 yeast strains representing 24 yeast species. Over 93% of these isolates, typically known to ferment simple sugars and produce ethanol comprising of high ethanol producers belonging to Saccharomyces, Brettanomyces, and Pichia, and intermediate ethanol producers Wickerhamomyces, Zygotorulaspora, Candida, Hanseniaspora, and Kluyveromyces. Fermentation of marula juice revealed convincing fermentative and aromatic bouquet credentials to suggest the potential to influence foraging behaviour and inebriate elephants in nature. There is insufficient evidence to refute the aforementioned myth. This work serves as the first work towards understanding the biodiversity marula associated yeasts to debunk the myth or approve the facts.

Looking into the world's largest elephant population in search of ligninolytic microorganisms for biorefineries: a mini-review.

Gastrointestinal tracts (GIT) of herbivores are lignin-rich environments with the potential to find ligninolytic microorganisms. The occurrence of the microorganisms in herbivore GIT is a well-documented mutualistic relationship where the former benefits from the provision of nutrients and the latter benefits from the microorganism-assisted digestion of their recalcitrant lignin diets. Elephants are one of the largest herbivores that rely on the microbial anaerobic fermentation of their bulky recalcitrant low-quality forage lignocellulosic diet given their inability to break down major components of plant cells. Tapping the potential of these mutualistic associations in the biggest population of elephants in the whole world found in Botswana is attractive in the valorisation of the bulky recalcitrant lignin waste stream generated from the pulp and paper, biofuel, and agro-industries. Despite the massive potential as a feedstock for industrial fermentations, few microorganisms have been commercialised. This review focuses on the potential of microbiota from the gastrointestinal tract and excreta of the worlds' largest population of elephants of Botswana as a potential source of extremophilic ligninolytic microorganisms. The review further discusses the recalcitrance of lignin, achievements, limitations, and challenges with its biological depolymerisation. Methods of isolation of microorganisms from elephant dung and their improvement as industrial strains are further highlighted.

Functional Characterization of khadi Yeasts Isolates for Selection of Starter Cultures.

Yeasts play an important role in spontaneous fermentation of traditional alcoholic beverages. Our previous study revealed that a mixed-consortia of both Saccharomyces and non-Saccharomyces yeasts were responsible for fermentation of khadi, a popular, non-standardized traditional beverage with an immense potential for commercialization in Botswana. Functional characterization of isolated fermenting yeasts from mixed consortia is an indispensable step towards the selection of potential starter cultures for commercialization of khadi. In this study, we report the characterization of 13 khadi isolates for the presence of brewing-relevant phenotypes such as their fermentative capacity, ability to utilize a range of carbon sources and their ability to withstand brewing-associated stresses, as a principal step towards selection of starter cultures. Khadi isolates such as Saccharomyces cerevisiae, Saccharomycodes ludwigii and Candida ethanolica showed good brewing credentials but Lachancea fermentati emerged as the isolate with the best brewing attributes with a potential as a starter culture. However, we were then prompted to investigate the potential of L. fermentati to influence the fruity aromatic flavor, characteristic of khadi. The aroma components of 18 khadi samples were extracted using headspace solid phase micro-extraction (HSSPME) and identified using a GC-MS. We detected esters as the majority of volatile compounds in khadi, typical of the aromatic signature of both khadi and L. fermentati associated fermentations. This work shows that L. fermentati has potential for commercial production of khadi.

Mycotoxins in khadi, A Traditional Non-Cereal Based Alcoholic Beverage of Botswana.

Mycotoxin contamination is a major food safety drawback towards the commercialization of food products. The commercialization of khadi, a popular fermented alcoholic beverage of Botswana necessitates the investigation of the presence of mycotoxins. Khadi brewing involves the uncontrolled and unstandardized spontaneous fermentation of sun-dried Grewia flava fruits, which could be a source of mycotoxin-producing filamentous fungi (molds). This study sought to investigate the presence of mycotoxins producing fungi and mycotoxins in 18 samples of khadi collected in Central and Northern Botswana. Periconia thailandica, Cladosporium cladosporioides, Aspergillus ochraceus, Phoma eupyrena, Setosphaeria turcica, Cladosporium sphaerospermum, Chaetomium longiciliata, and Flavodon ambrosius were identified in 10 out of 18 khadi samples. Mycotoxins were detected using the Myco-10 Randox Evidence Investigator biochip kit and confirmed using a UPLC-ESI-MS/MS. Mycotoxins such as paxilline, ochratoxin A, ergot alkaloids, aflatoxin G1/G2, and zearalenone were detected using the Myco-10 Randox Evidence Investigator biochip kit. The Myco-10 results revealed that the mycotoxins in the khadi samples were lower than the regulatory limits set by FDA or European Commission. Confirmation of results using an UPLC-ESI-MS/MS system involved confirming selected mycotoxins (AFB1, DON. ZEA, FB1, FB2, FB3, NIV, and OTA) from selected khadi samples (Palapye 1, Palapye 2, Letlhakane 2, Maun 3, Mmashoro 3, and Tonota 3). The UPLC results demonstrated that the aforementioned mycotoxins in the selected khadi samples were below the detection thresholds. The study shows that while fungal isolates were present, there is no to minimal danger/risk of exposure to toxic mycotoxins after consumption of khadi. Towards commercialization endeavors, the production process would necessitate minimal mycotoxin monitoring and product preservation but no detoxifying steps.

Bioethanolic yeasts from dung beetles: tapping the potential of extremophilic yeasts for improvement of lignocellulolytic feedstock fermentation.

Bioethanol from abundant and inexpensive agricultural and industrial wastes possesses the potential to reduce greenhouse gas emissions. Bioethanol as renewable fuel addresses elevated production costs, as well as food security concerns. Although technical advancements in simultaneous saccharification and fermentation have reduced the cost of production, one major drawback of this technology is that the pre-treatment process creates environmental stressors inhibitory to fermentative yeasts subsequently reducing bioethanol productivity. Robust fermentative yeasts with extreme stress tolerance remain limited. This review presents the potential of dung beetles from pristine and unexplored environments as an attractive source of extremophilic bioethanolic yeasts. Dung beetles survive on a recalcitrant lignocellulose-rich diet suggesting the presence of symbiotic yeasts with a cellulolytic potential. Dung beetles inhabiting extreme stress environments have the potential to harbour yeasts with the ability to withstand inhibitory environmental stresses typically associated with bioethanol production. The review further discusses established methods used to isolate bioethanolic yeasts, from dung beetles.