Exopolysaccharide: a novel important factor in the microbial dissolution of tricalcium phosphate

Four strains (Enterobacter sp. EnHy-401, Arthrobacter sp.ArHy-505, Azotobacter sp.AzHy-510 and Enterobacter sp.EnHy-402) which have the ability to solubilize tricalcium phosphate (TCP) were used to study the mechanism of P-solubilization. It was found that three phosphate solubilizing bacteria (EnHy-401, ArHy-505 and AzHy-510) producing exopolysaccharide (EPS) have a stronger ability for P-solubilization than isolate EnHy-402 without EPS production, of those, the strain EnHy-401 with the highest EPS production and efficient organic acids on P-solubilization had a stronger capacity for P-solubilization than the others. Further studies demonstrated that addition of EPS into medium could increase the amount of phosphorus solubilized by organic acid, but failed to release phosphorus from TCP alone. The synergistic effects of EPS and organic acid on TCP solubilization varied with the origin and the concentration of EPS in medium. EPS produced by EnHy-401 was most effective in promoting phosphorus release at an optimal concentration in medium. The increase of P-solubilization brought by EPS attributed to the participation of EPS led to the change in homeostasis of P-solubilization, pushing it towards P dissolved by holding free phosphorus in the medium, consequently resulting in greater phosphorus released from insoluble phosphate. We therefore suggest that EPS with ability of phosphorus-holding may be a novel important factor in the microbial dissolution of TCP except for organic acid.     

Microbial conversion of glycerol: present status and future prospects

Biodiesel has emerged as a potential alternate renewable liquid fuel in the past two decades. Total annual production of biodiesel stands at 6.96 million tons and 11.2 million tons in USA and Europe, respectively. In other countries, Asia and Latin America, biodiesel production has increased at unprecedented rate. Despite this, the economy of biodiesel is not attractive. An obvious solution for boosting the economy of the biodiesel industry is to look for markets for side products of the transesterification process of biodiesel synthesis. The main by-product is glycerol. However, this glycerol is contaminated with alkali/acid catalyst and alcohol, and thus, is not useful for conventional applications such as in toothpaste, drugs, paints and cosmetics. Conversion of this glycerol to value-added product is a viable solution for effective and economic utilization, which would also generate additional revenue for the biodiesel industry. Intensive research has taken place in area of conversion of glycerol to numerous products. The conventional catalytic route of glycerol transformation employs prohibitively harsh conditions of temperature and pressure, and thus, has slim potential for large-scale implementation. In addition, the selectivity of the process is rather small with formation of many undesired side products. The bioconversion processes, on the other hand, are highly selective although with slower kinetics. In this review, we have given an assessment and overview of the literature on bioconversion of glycerol. We have assessed as many as 23 products from glycerol bioconversion, and have reviewed the literature in terms of microorganism used, mode of fermentation, type of fermentor, yield and productivity of the process and recovery/purification of the products. The metabolic pathway of conversion of glycerol to various products has been discussed. We have also pondered over economic and engineering issues of large-scale implementation of process and have outlined the constraints and limitations of the process. We hope that this review will be a useful source of information for biochemists, biotechnologists, microbiologists and chemical engineers working in the area of glycerol bioconversion.     

Microbial conversion of glycerol: present status and future prospects

Biodiesel has emerged as a potential alternate renewable liquid fuel in the past two decades. Total annual production of biodiesel stands at 6.96 million tons and 11.2 million tons in USA and Europe, respectively. In other countries, Asia and Latin America, biodiesel production has increased at unprecedented rate. Despite this, the economy of biodiesel is not attractive. An obvious solution for boosting the economy of the biodiesel industry is to look for markets for side products of the transesterification process of biodiesel synthesis. The main by-product is glycerol. However, this glycerol is contaminated with alkali/acid catalyst and alcohol, and thus, is not useful for conventional applications such as in toothpaste, drugs, paints and cosmetics. Conversion of this glycerol to value-added product is a viable solution for effective and economic utilization, which would also generate additional revenue for the biodiesel industry. Intensive research has taken place in area of conversion of glycerol to numerous products. The conventional catalytic route of glycerol transformation employs prohibitively harsh conditions of temperature and pressure, and thus, has slim potential for large-scale implementation. In addition, the selectivity of the process is rather small with formation of many undesired side products. The bioconversion processes, on the other hand, are highly selective although with slower kinetics. In this review, we have given an assessment and overview of the literature on bioconversion of glycerol. We have assessed as many as 23 products from glycerol bioconversion, and have reviewed the literature in terms of microorganism used, mode of fermentation, type of fermentor, yield and productivity of the process and recovery/purification of the products. The metabolic pathway of conversion of glycerol to various products has been discussed. We have also pondered over economic and engineering issues of large-scale implementation of process and have outlined the constraints and limitations of the process. We hope that this review will be a useful source of information for biochemists, biotechnologists, microbiologists and chemical engineers working in the area of glycerol bioconversion.     

Bioconversion of crude glycerol by fungi

The production of synthetic glycerol from petrochemical feedstocks has been decreasing in recent years. This is largely due to increasing supplies of crude glycerol derived as a co-product from the oleochemical industry, especially biodiesel production. The price of glycerol is at historic lows, and the supply of crude glycerol is projected to grow faster than its industrial uses. This oversupply is driving the transition from glycerol as a product to glycerol as a precursor for new industrial applications, including its use as a substrate for bioconversion. This article reviews the use of fungi for the bioconversion of crude glycerol to the value-added products 1,2-propanediol, ethanol, single cell oil, specialty polyunsaturated fatty acids, biosurfactants, and organic acids. Information on the impurities of crude glycerol from different industrial processes is also included.     

The protons of gluconic acid are the major factor responsible for the dissolution of tricalcium phosphate by Burkholderia cepacia CC-Al74

Burkholderia cepacia CC-Al74 with a high ability for solubilizing tricalcium phosphate (TCP) was used to study the P-solubilization mechanism. We collected filtrates able to solubilize TCP from the cultures of strain CC-Al74 and demonstrated that the P-solubilization increased from 0 microg ml(-1) to 200 microg ml(-1) during exponential growth, when the pH decreased from 8 to 3. HPLC-analysis revealed that the solubilization of TCP was mainly caused by the release of 16.3 mM gluconic acid. At this concentration, gluconic acid was capable of solubilizing 376 microg ml(-1) of TCP whereas water at pH 3 only solubilized 35 microg ml(-1). The difference is due to the final H+ concentrations which were 13.5 mM and 1 mM in 16.3 mM gluconic acid and deionized water, respectively at pH 3.     

Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry

Although biofuels such as biodiesel and bioethanol represent a secure, renewable and environmentally safe alternative to fossil fuels, their economic viability is a major concern. The implementation of biorefineries that co-produce higher value products along with biofuels has been proposed as a solution to this problem. The biorefinery model would be especially advantageous if the conversion of byproducts or waste streams generated during biofuel production were considered. Glycerol-rich streams generated in large amounts by the biofuels industry, especially during the production of biodiesel, present an excellent opportunity to establish biorefineries. Once considered a valuable 'co-product', crude glycerol is rapidly becoming a 'waste product' with a disposal cost attributed to it. Given the highly reduced nature of carbon in glycerol and the cost advantage of anaerobic processes, fermentative metabolism of glycerol is of special interest. This review covers the anaerobic fermentation of glycerol in microbes and the harnessing of this metabolic process to convert abundant and low-priced glycerol streams into higher value products, thus creating a path to viability for the biofuels industry. Special attention is given to products whose synthesis from glycerol would be advantageous when compared with their production from common sugars.     

Microbial utilization of crude glycerol for the production of value-added products

Energy fuels for transportation and electricity generation are mainly derived from finite and declining reserves of fossil hydrocarbons. Fossil hydrocarbons are also used to produce a wide range of organic carbon-based chemical products. The current global dependency on fossil hydrocarbons will not be environmentally or economically sustainable in the long term. Given the future pessimistic prospects regarding the complete dependency on fossil fuels, political and economic incentives to develop carbon neutral and sustainable alternatives to fossil fuels have been increasing throughout the world. For example, interest in biodiesel has undergone a revival in recent times. However, the disposal of crude glycerol contaminated with methanol, salts, and free fatty acids as a by-product of biodiesel production presents an environmental and economic challenge. Although pure glycerol can be utilized in the cosmetics, tobacco, pharmaceutical, and food industries (among others), the industrial purification of crude glycerol is not economically viable. However, crude glycerol could be used as an organic carbon substrate for the production of high-value chemicals such as 1,3-propanediol, organic acids, or polyols. Microorganisms have been employed to produce such high-value chemicals and the objective of this article is to provide an overview of studies on the utilization of crude glycerol by microorganisms for the production of economically valuable products. Glycerol as a by-product of biodiesel production could be used a feedstock for the manufacture of many products that are currently produced by the petroleum-based chemical industry.     

Disassembly of lignin and chemical recovery in supercritical water and p-cresol mixture. Studies on lignin model compounds

The aim of the study was to gain insight into the role of the each unit of lignin in the formation of products. Glycerol, guaiacol, the mixture of glycerol and guaiacol (Gly&Gua), and guaiacylglycerol-beta-guaiacyl ether (GGGE) were used as lignin model compounds to study fragmentation of lignin in an excess of water and p-cresol at 400 degrees C. The products have been analyzed employing gas chromatography (GC)-mass spectrometer (MS) and gas chromatography-frame ionization detector for qualitative and quantitative analysis. GC-MS analysis indicated that phenol, o-cresol, methyl-anisole, di-methyl-phenol, ethyl-methyl-phenol, 2-(hydroxy-benzyl)-4-methyl-phenol (BMP) and 2-(2-hydroxy-5-methyl-benzyl)-4-methyl-phenol were formed. The products were similar to the products by the fragmentation of lignin. The products, except o-cresol, were primarily derived from glycerol and/or guaiacol. We also found that the amount of BMP derived from GGGE, which has glycerol unit and guaiacol unit in its structure, is much more than that derived from Gly&Gua. The increase of the BMP yield by reaction with GGGE compared with (glycerol and/or guaiacol) indicates that guaiacylglycerol unit with linkage of Gly&Gua plays an important role in the formation of BMP and also it is suggested that the BMP formation from GGGE has pathways other than that from Gly&Gua.     

Rapid hydrolysis of diacylglycerol formed during phosphatidate phosphatase assay by lipase activities in rat liver cytosol and microsomes

Side reactions which may affect the determination of phosphatidate phosphatase activity were investigated in rat liver cytosol and microsomes. Incubation of these subcellular fractions with either 14C-labeled phosphatidate bound to microsomal membranes (PAmb) or that coemulsified with microsomal lipids resulted in rapid formation of water-soluble products, most of which were identified as glycerol, in addition to diacylglycerol. Neither lysophosphatidate nor glycerol 3-phosphate accumulated under any of the conditions used and only a minute amount of activity catalyzing hydrolysis of glycerol 3-phosphate could be detected in cytosol and microsomes, suggesting that glycerol was not formed by the deacylation of phosphatidate to glycerol 3-phosphate and subsequent dephosphorylation. On the other hand, pretreatment of cytosol or microsomes with diisopropylfluorophosphate abolished the formation of water-soluble products, indicating that glycerol was formed from diacylglycerol, the product of the phosphatidate phosphatase reaction, by lipase-type activities. Rapid deacylation of diacylglycerol by these subcellular fractions was also observed with an emulsion of phosphatidate, which has been purified from the total lipid extract of PAmb as substrate. The rate of hydrolysis of diacylglycerol was maximum when the concentration of diacylglycerol was less than 20 microM with either cytosol or microsomes. The present results suggest that it is essential to characterize the reaction products before employing specific assay conditions for phosphatidate phosphatase. At least under the conditions we tested, reliable measurement of the enzyme activity in rat liver cytosol and microsomes can be achieved only by determining the release of Pi or that of water-soluble activity from 32P-labeled phosphatidate.     

A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source

Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed‐stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol‐based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4‐transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli.     

The glycerol teichoic acid from the cell wall of Bacillus stearothermophilus B65

1. A glycerol teichoic acid has been extracted from cell walls of Bacillus stearothermophilus B65 and its structure examined. 2. Trichloroacetic acid-extractable teichoic acid accounted for 68% of the total cell-wall phosphorus and residual material could be hydrolysed to a mixture of products including those characteristic of glycerol teichoic acids. 3. The extracted polymer is composed of glycerol, phosphoric acid, d-glucose and d-alanine. 4. Hydrolysis of the polymer with alkali gave glycerol, 1-O-alpha-d-glucopyranosylglycerol and its monophosphates, glycerol mono- and di-phosphate, as well as traces of a glucosyldiglycerol triphosphate and a glucosylglycerol diphosphate. 5. The teichoic acid is a polymer of 18 or 19 glycerol phosphate units having alpha-d-glucopyranosyl residues attached to position 1 of 14 or 15 of the glycerol residues. 6. The glycerol residues are joined by phosphodiester linkages involving positions 2 and 3 in each glycerol. 7. d-Alanine is in ester linkage to the hydroxyl group at position 6 of approximately half of the glucose residues. 8. One in every 13 or 12 polymer molecules bears a phosphomonoester group on position 3 of a glucose residue, the possible significance of which in linkage of the polymer to other wall constituents is discussed.     

Current Prospects on Production of Microbial Lipid and Other Value-Added Products Using Crude Glycerol Obtained from Biodiesel Industries

Production of microbial lipids using crude glycerol from the biodiesel industry is reviewed in this paper. Approximately 10 wt.% of crude glycerol is obtained for every batch of biodiesel. The crude glycerol accumulated contains various impurities and hence cannot be used for any commercial applications without further purification. Its conversion via biological and chemical routes into valuable products has been studied by different researchers. Varieties of fungal, yeasts, and algal species have been used to produce microbial lipids from crude glycerol. However, research focus on screening a robust industrial oleaginous strain capable of doing this is still on-going. Due to its chemical similarity to vegetable oils, microbial lipids are considered a potential renewable feedstock for biodiesel production and for applications in food and pharmaceutical industries. Its conversion to polyols and subsequently to biobased polymers is also being explored. The rising price of vegetable oils, increasing energy demands, growing environmental concerns, and availability of crude glycerol as a cheap carbon substrate result in considerable potential for the application of these processes in the future.     

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis> ATCC 6013

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.     

Spotlight on biodiversity of microbial cell factories for glycerol conversion

Plant oil based industrial oleochemistry leads to a large side stream of crude glycerol, which offers itself as a low price carbon source for microbial chemical production. Compared to sugar, glycerol is more reduced and less microorganisms are able to use it as carbon source. An interesting feature of glycerol conversion is that many organisms cannot use it as carbon source at all, but they readily use it as electron sink under anaerobic conditions. In any case the number of pathways by which glycerol enters the microbial metabolism is quite limited. Having said this, an interesting variety of products of industrial relevance is accumulated by naturally occurring microorganisms which can use glycerol. These chemicals range from fuels and solvents to polymer precursors up to food additives. The limited number of metabolic pathways and the manageable amount of products allow to highlight the importance of tapping the outstanding resource of biodiversity for industrial purposes. The interplay of microbial biodiversity, metabolic engineering and bioprocess engineering is key for economic success in industrial microbiology. In this article we shed light on the biodiversity of naturally glycerol consuming microorganisms and their impact and importance on microbial chemical production.     

An approach to measuring the in vivo transformation of glycerol to a very low density lipoprotein triglyceride.

This paper describes a new method which permits measurement of the steady-state rate of transformation of serum glycerol to a very low density lipoprotein (VLDL) triglyceride in vivo in dogs. Although the turnover of glycerol and the turnover of VLDL triglyceride glycerol have both been previously measured, the rate of transformation of the former into the latter has not. While there is considerable dog-to-dog variation in the absolute turnover and transformation rates, the relationship between the various rates is quite constant. Thus, 13% of the serum glycerol which normal fasting dogs utilize is converted to VLDL triglyceride. The remaining 87% is converted to other products. Also, 28% of VLDL triglyceride glycerol in these dogs is derived from serum glycerol. The balance, 72%, is derived from other sources. The procedure described here can be used to quantitate the contribution of glycerol to VLDL in a number of conditions in which glycerol and (or) VLDL triglyceride metabolism is altered, thereby providing another way to gain insight into the metabolism of VLDL. Even more generally, the principles developed here can be applied to estimate the transformation of other precursors to other products in vivo.     

Effect of impurities in biodiesel-derived waste glycerol on the performance and feasibility of biotechnological processes

The rapid development of biodiesel production technology has led to the generation of tremendous quantities of glycerol wastes, as the main by-product of the process. Stoichiometrically, it has been calculated that for every 100 kg of biodiesel, 10 kg of glycerol are produced. Based on the technology imposed by various biodiesel plants, glycerol wastes may contain numerous kinds of impurities such as methanol, salts, soaps, heavy metals, and residual fatty acids. This fact often renders biodiesel-derived glycerol unprofitable for further purification. Therefore, the utilization of crude glycerol though biotechnological means represents a promising alternative for the effective management of this industrial waste. This review summarizes the effect of various impurities-contaminants that are found in biodiesel-derived crude glycerol upon its conversion by microbial strains in biotechnological processes. Insights are given concerning the technologies that are currently applied in biodiesel production, with emphasis to the impurities that are added in the composition of crude glycerol, through each step of the production process. Moreover, extensive discussion is made in relation with the impact of the nature of impurities upon the performances of prokaryotic and eukaryotic microorganisms, during crude glycerol bioconversions into a variety of high added-value metabolic products. Finally, aspects concerning ways of crude glycerol treatment for the removal of inhibitory contaminants as reported in the literature are given and comprehensively discussed.     

Glycerol as a substrate for Saccharomyces cerevisiae based bioprocesses – Knowledge gaps regarding the central carbon catabolism of this ‘non-fermentable’ carbon source

Glycerol is an interesting alternative carbon source in industrial bioprocesses due to its higher degree of reduction per carbon atom compared to sugars. During the last few years, significant progress has been made in improving the well-known industrial platform organism Saccharomyces cerevisiae with regard to its glycerol utilization capability, particularly in synthetic medium. This provided a basis for future metabolic engineering focusing on the production of valuable chemicals from glycerol. However, profound knowledge about the central carbon catabolism in synthetic glycerol medium is a prerequisite for such incentives. As a matter of fact, the current assumptions about the actual in vivo fluxes active on glycerol as the sole carbon source have mainly been based on omics data collected in complex media or were even deduced from studies with other non-fermentable carbon sources, such as ethanol or acetate. A number of uncertainties have been identified which particularly regard the role of the glyoxylate cycle, the subcellular localization of the respective enzymes, the contributions of mitochondrial transporters and the active anaplerotic reactions under these conditions. The review scrutinizes the current knowledge, highlights the necessity to collect novel experimental data using cells growing in synthetic glycerol medium and summarizes the current state of the art with regard to the production of valuable fermentation products from a carbon source that has been considered so far as ‘non-fermentable’ for the yeast S. cerevisiae.     

Biochemistry,genetics and biotechnology of glycerol utilization in Pseudomonas species

The use of renewable waste feedstocks is an environment-friendly choice contributing to the reduction of waste treatment costs and increasing the economic value of industrial by-products. Glycerol (1,2,3-propanetriol), a simple polyol compound widely distributed in biological systems, constitutes a prime example of a relatively cheap and readily available substrate to be used in bioprocesses. Extensively exploited as an ingredient in the food and pharmaceutical industries, glycerol is also the main by-product of biodiesel production, which has resulted in a progressive drop in substrate price over the years. Consequently, glycerol has become an attractive substrate in biotechnology, and several chemical commodities currently produced from petroleum have been shown to be obtained from this polyol using whole-cell biocatalysts with both wild-type and engineered bacterial strains. Pseudomonas species, endowed with a versatile and rich metabolism, have been adopted for the conversion of glycerol into value-added products (ranging from simple molecules to structurally complex biopolymers, e.g. polyhydroxyalkanoates), and a number of metabolic engineering strategies have been deployed to increase the number of applications of glycerol as a cost-effective substrate. The unique genetic and metabolic features of glycerol-grown Pseudomonas are presented in this review, along with relevant examples of bioprocesses based on this substrate – and the synthetic biology and metabolic engineering strategies implemented in bacteria of this genus aimed at glycerol valorization.     

Fermentation of glycerol and production of valuable chemical and biofuel molecules

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.