Sonia M. Tiquia-Arashiro

Ammonia-oxidizing bacteria and archaea in sediments of the Gulf of Mexico.

The diversity (richness and community composition) of ammonia-oxidizing archaea (AOA) and bacteria (AOB) within sediments of the Gulf of Mexico was examined. Using polymerase chain reaction primers designed to specifically target the archaeal ammonia monooxygenase-subunit (amoA) gene and bacterial amoA gene, we found AOA and AOB to be present in all three sampling sites. Archaeal amoA libraries were dominated by a few widely distributed Nitrosopumilus-like sequence types, whereas AOB diversity showed significant variation in both richness and community composition. Majority of the bacterial amoA sequences recovered belong to Betaproteobacteria and very few belong to Gammaproteobacteria. Results suggest that water depth and nutrient availability were identified as potential drivers that affected the selection of the AOA and AOB communities. Besides influencing the abundance of individual taxa, these environmental factors also had an impact on the overall richness of the overall AOA and AOB communities. The richness and diversity of AOA and AOB genes were higher at the shallowest sediments (100 m depth) and the deepest sediments (1300 m depth). The reduced diversity in the deepest sediments could be explained by much lower nutrient availability.

Sustainable technologies: bioenergy and biofuel from biowaste and biomass

The combustion of petroleum-based fossil fuels has become a concern with respect to global climate change due to accelerated carbon emissions. Reliance on the burning of fossil fuels has also created a concern for unstable and uncertain petroleum sources, as well as, rising cost of fuels. These concerns have shifted global efforts to utilize renewable resources for the production of greener energy replacement that can also meet the high-energy demand of both the developed and developing countries. Currently, the USA and Brazil are leaders in the production of starch-based fuel from corn and sugarcane crops, respectively. This is the production of first-generation fuel from food crop sugars using conventional technologies; however, starch raw materials will not be sufficient enough to meet increasing demand and are a controversial resource for bioconversion. In a time when a foreseeable complete transformation from a petroleum-based economy to a bio-based global economy finds itself in its early infancy, biowastes (e.g. agricultural wastes, municipal solid wastes, sludge, wastewater and foodwastes), currently seen as low-valued materials, are beginning to be recognized as resources for the production of a variety of eco-friendly and sustainable products, with second-generation liquid biofuels being the leading ones. Agricultural wastes, for instance, contain high levels of cellulose, hemicellulose, starch, proteins, as well as lipids. As such, they constitute inexpensive candidates for the biotechnological production of liquid biofuels (e.g. bioethanol, biodiesel, dimethyl ether and dimethyl furan) without competing directly with the ever-growing need for world food supply. As biowastes are generated in large scales, in the range of billions of kilograms per year, thus largely available and rather inexpensive, these materials are seriously considered to be potential sources for the production of biofuels. Much more consideration is also given to replacement products that stem from microbial metabolism. Ethanol has already become a dominant fuel in Brazil. Butanol has even superior qualities and represents an important bulk compound for the chemical industry. Biodiesel can substitute for diesel fuel. Methane and hydrogen are valuable resources for energy and electricity, with potential for transportation as well. The variety of topics in this Special Issue is manifold, which is typical for the bioenergy sector and is essential for finding concepts with a holistic background. Nevertheless, the problems to be solved need highly sophisticated approaches. Hence, it is the aim of this issue to compile current technological developments in the area of producing biofuels from biowastes and biomass and to attract the awareness of scientists, engineers and industrial and political decision makers to the extent microbes can and will play in securing the world’s energy needs. This Special Issue called for scientific contributions that present recent developments and industrial applications in this field. It features 12 review articles and 38 original research articles on the state-of-the-art production of bioethanol, bio-butanol, biodiesel, biohydrogen, and bioelectricity from biowastes and biomass. These papers are published in two parts: Part 1 highlights the challenges and developments; and Part 2 covers the research and technological advances in the field. The biofuels industry often starts with starches, such as that found in corn, because it is easy to hydrolyse using bacterial amylases. Plant cellulose is often complexed as lignocellulose and/or hemicellulose. Dien et al. found that ammonia pretreatment is an effective way to prepare switchgrass for biochemical conversion to sugars and ethanol. Notable was the absence of a lag phase for fermentation of unconditioned switchgrass hydrolysate, and increases in glucose and ethanol yields. Pretreatment of biomass frequently produces a product that microorganisms are incapable of fermenting because of the presence of inhibitory chemicals. Pretreated biomass is either washed or extensively conditioned prior to fermentation, adding to the process costs. However, ammonia pretreatment was found to be relatively benign compared with other pretreatments because carbohydrates are largely preserved. The future will see a technical process using lignocellulosic hydrolysates (Plecha et al.). However, as the enzymatic hydrolysis reaction of cellulose is about two orders of magnitude slower than the average ethanol fermentation rate, there is a theoretical gap between simultaneous saccharification of cellulosic biomass and ethanol fermentation. While ethanol has a long lineage as a bio-based fuel, butanol fermentation also builds on a rich history. Despite the lack of biobutanol economy, research and development in universities in Europe, New Zealand, South Africa and USA has continued. The traditional clostridial fermentation of butanol and acetone suffers from difficulties of switching from acidogenic fermentation state to the solventogenic stage and, thus, a discontinuous production mode from common phage infections, the rising substrate costs and the effort required for downstream processing. Schiel-Bengelsdorf et al. detail how modern biotechnology research, including strain engineering and process design substrates, has addressed these challenges and allowed

Screening for novel bacteria from the bioenergy feedstock switchgrass (Panicum virgatum L.).

Switchgrass is considered as a good candidate for biofuel, especially ethanol production due to its huge biomass output and high cellulose content. In a search for novel microorganisms capable of using and degrading switchgrass to produce sugars and ethanol, enrichment experiments were established to screen for microorganisms from soil samples obtained at the University of Tennessee Agricultural Research Station, Jackson, Tennessee. Three enrichments were prepared and incubated at different pH and temperatures: (1) 30°C, pH 5, (2) 30°C, pH 8 and (3) 60°C, pH5. Bulk community DNA was directly extracted from the enrichments. Microbial community structures were determined by phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment cultures containing switchgrass as the carbon source. The mesophilic enrichments were dominated by Sarcina, Anaerobacter, and Clostrium, which were not found in the thermophilic enrichment. The thermophilic enrichment selected for two types of bacteria belonging to the class Bacilli (Geobacillus and Saccharococcus). The thermophilic enrichments were dominated by the Geobacillus spp. (Firmicutes, class Bacilli), and Saccharococcus (Firmicutes, class Bacilli); both containing thermophilic microorganisms with some cellulolytic members. Enzymatic assays detected the presence of enzymes involved in cellulose (β-glucosidase and cellobiohydrolase) and hemicellulose degradations (β-xylosidase); and the activity tends to be higher in the enrichments incubated at 30°C.

Isolation and screening of carboxydotrophs isolated from composts and their potential for butanol synthesis

Carboxydotrophs are known for their ability to convert carbon monoxide (CO) to butanol through fermentation. Such a platform offers a promising alternative approach to biofuel production from synthesis gas feedstocks. In this study, carboxydotrophs were isolated from various manure compost. Out of 500 isolates, only 11 carboxydotrophs (7 mesophiles and 4 thermophiles) were found to utilize CO as the sole source of carbon and energy. To assess the biochemical basis for their ability to produce biofuel (butanol), the level of activities of CO dehydrogenase (CODH), hydrogenase and butanol dehydrogenase (BDH) enzymes for these isolates against the known carboxydotroph, Butyribacterium methylotrophicum was assessed. All isolates showed evidence of enzyme activities (0.16–2.20 μmol min−1), with the majority exhibiting higher activities compared with the known carboxydotroph, B. methylotrophicum (0.33–0.71 μmol min−1). The level of activities for CODH and BDH ranged from 0.163–3.59 μmol min−1 and 0.19–2.2 μmol min−1, respectively. Three isolates (M7-1, T2-22, and T3-14) demonstrated enzymatic activity three to seven times higher than B. methylotrophicum. Of these, T2-22 exhibited the highest BDH activity and shows great promise in the conversion of toxic CO into butanol more so than other carboxytotrophs known thus far. This study revealed some biochemical basis for butanol production from CO by carboxydotrophs. However, more research is needed to discover a direct biological route for butanol production from CO to strengthen their potential for synthesis gas bioprocessing. Follow-up work will focus on whole-genome sequencing of the promising isolate T2-22 to provide system-level insights into how carboxydotrophs utilize and regulate their molecular machineries for butanol production.

Application of Nanoparticles

In this chapter we discuss the applications of bionanoparticles (BNP) in biomedical and environmental fields. In the biomedical field, these nanoparticles have been investigated for antimicrobial applications, biosensing, imaging, and drug delivery. In the environmental field, nanoparticles have been investigated for applications in bioremediation of diverse contaminants, water treatment, and production of clean energy. Overall, the BNP have attracted the attention of diverse researchers because their syntheses are more environmentally friendly, produces more homogeneously distributed nanoparticles and some of them can be easily biodegradable. Although there are several studies investigating the application of BNP, these nanomaterials are still way less studied than synthetic nanoparticles, since researchers are still identifying the microbiological synthetic pathways of these BNP. It is expected that with the advancement of the understanding of BNP synthesis pathways, the application of BNP will expand to many more fields than biomedical and environmental and will be potentially applied in diverse nanotechnological industries.

Halophiles in Nanotechnology

Halophiles are salt loving organisms that flourish in saline environments such as marine and estuarine environments, solar salterns, salt lakes, brines and saline soils. They offer potential applications in various fields of biotechnology. They can be used as a source of metabolites, compatible solutes and other compounds of industrial value. The biodegradation of organic pollutants in hypersaline environments and treatment of saline effluents contaminated with organic by halophiles have been investigated. Some halophiles are a potential source of extracellular hydrolases like proteases with a wide array of industrial applications. These enzymes exhibit stability over a range of saline conditions and harsh conditions of pH or/and ionic strength. Recently, they are being explored as potential sources of metal tolerant microorganisms with the ability to synthesize metallic nanoparticles. This chapter covers the various halophilic organisms and their by-products that have been exploited for nanomaterial synthesis, the mechanisms that may be involved in the nanomaterial fabrication and the possible applications of the fabricated nanoparticles.

Butanol tolerance of carboxydotrophic bacteria isolated from manure composts.

ABSTRACT Carboxydotrophic bacteria (carboxydotrophs) have the ability to uptake carbon monoxide (CO) and synthesize butanol. The aims of this study were to determine the butanol tolerance and biological production of butanol carboxydotrophic strains. In this study, 11 carboxydotrophic strains were exposed to increasing n-butanol concentrations (1–3% vol/vol) to determine their effect on growth. Butanol production by the strains was quantified and the identity of the strains was elucidated using 16S rRNA sequencing. The carboxydotrophic strains possessed inherent tolerance to butanol and tolerated up to 3% n-butanol. Among the 11 strains, T1-16, M2-32 and M3-28 were the most tolerant to butanol. The 16S rRNA gene sequence of these strains was similar (99% nucleotide similarity) to the butanol-tolerant strains Bacillus licheniformis YP1A, Pediococcus acidilacti IMUA20068 and Enterococcus faecium IMAU60169, respectively. The carboxydotrophic strains screened in this study have two distinct features: (1) high tolerance to butanol and (2) natural production of low concentration of butanol from CO, which distinguish them from other screened butanol-tolerant strains. The butanol tolerance of these carboxydotrophic strains makes them ideal for genetic studies, particularly the molecular mechanisms that enable them to survive such hostile environmental conditions and the identification of genes that confer tolerance to butanol.

Biotechnological Applications of Thermophilic Carboxydotrophs

Microorganisms have been used in industry in a number of ways that generally exploit their natural metabolic capabilities. They are used in manufacture of foods and production of antibiotics, probiotics, drugs, vaccines, starter cultures, insecticides, enzymes, fuels, and solvents (Tiquia and Mormile in Environ Technol 31:823, 2010, Environ Technol 34:1637–1638, 2013). In addition, with genetic engineering technology, bacteria can be programmed to make various substances used in food science, agriculture, and medicine.

CO-oxidizing Microorganisms

CO is metabolized by a wide variety of microorganisms. A sharp division exists between aerobic and anaerobic species, as they contain fundamentally different enzyme systems for CO biotransformation. Aerobic CO-oxidizing bacteria may be divided in two groups: metabolic, in which CO oxidation provides energy for growth, and co-metabolic, in which CO is used as pseudo-substrate for the enzyme system, but does not provide a nutritional value (Colby et al. Trends Biotechnol 3:12–17, 1985). The latter is observed during aerobic CO oxidation by methane-oxidizing bacteria employing the methane monooxygenase complex, which is rather unspecific with respect to its substrate (Higgins et al. Nature 286:561–564, 1980; Daniels et al. J Bacteriol 132:118–126, 1977). Aerobic metabolic CO-oxidizing bacteria or aerobic carboxydotrophs use CO as a source of energy, which is oxidized with O2 as terminal electron acceptor. These bacteria contain a specific CO-tolerant cytochrome b1 oxidase and O2-insensitive Mo–Fe-flavin carbon monoxide dehydrogenase. The diversity and ecology of bacteria that grow aerobically with CO has been intensively studied and reviewed (Zavarzin and Nozhevnikova Microb Ecol 3:305–326, 1977; Meyer et al. FEMS Microbiol Rev 87:253–260, 1990; Conrad Microbiol Rev 60:609–640, 1996; King and Webber Nat Rev Microbiol 5:107–118, 2007). Aerobic carboxydotrophs include CO-utilizing microorganisms that belong to α-proteobacteria, Firmicutes, and Actinobacteria (Table 3.1).

Applicability of API ZYM to capture seasonal and spatial variabilities in lake and river sediments

ABSTRACT Waters draining into a lake carry with them much of the suspended sediment that is transported by rivers and streams from the local drainage basin. The organic matter processing in the sediments is executed by heterotrophic microbial communities, whose activities may vary spatially and temporally. Thus, to capture and evaluate some of these variabilities in the sediments, we sampled six sites: three from the St. Clair River and three from Lake St. Clair in spring, summer, fall, and winter of 2016. At all sites and dates, we investigated the spatial and temporal variations in 19 extracellular enzyme activities using API ZYM. Our results indicated that a broad range of enzymes were found to be active in the sediments. Phosphatases, lipases, and esterases were synthesized most intensively by the sediment microbial communities. No consistent difference was found between the lake and sediment samples. Differences were more obvious between sites and seasons. Sites with the highest metabolic (enzyme) diversity reflected the capacity of the sediment microbial communities to breakdown a broader range of substrates and may be linked to differences in river and lake water quality. The seasonal variability of the enzymes activities was governed by the variations of environmental factors caused by anthropogenic and terrestrial inputs, and provides information for a better understanding of the dynamics of sediment organic matter of the river and lake ecosystems. The experimental results suggest that API ZYM is a simple and rapid enzyme assay procedure to evaluate natural processes in ecosystems and their changes. GRAPHICAL ABSTRACT