The Biosynthesis of Liquid Fuels and Other Value-Added Products Based on Waste Glycerol—A Comprehensive Review and Bibliometric Analysis

Abstract

Waste glycerol can be subjected to various processing operations, including purification and refining, to obtain glycerol of an appropriate purity. Alternative methods for utilising waste glycerol are also being sought, e.g., by converting it into other valuable chemical products or biofuels. Therefore, various technologies are being developed to ensure effective and sustainable utilisation of this type of waste. The production of value-added products from waste glycerol strongly determines the improvement of the economic viability of biofuel production and corresponds to the model of a waste-free and emission-free circular economy. This paper characterises the mechanisms and evaluates the efficiency of existing methods for microbiological utilisation of waste glycerol into liquid biofuels, including biodiesel, bioethanol and biobutanol, and identifies further production avenues of value-added products. In addition, it presents the results of a bibliographical analysis of publications related to the production of liquid fuels and economically valuable products from glycerol, assesses the progress of research and application work and, finally, identifies areas for future research.

1. Introduction

Glycerol, also known as glycerine, is a three-carbon alcohol that is a by-product of many chemical processes, including the production of biodiesel, bioethanol and soap [1]. Waste glycerol is produced during the transesterification of vegetable oils or animal fats with alcohols, such as methanol or ethanol; during the saponification of fats or oils with a base (e.g., sodium hydroxide (NaOH) or potassium hydroxide (KOH)); and during alcoholic fermentation, especially when carried out by bacteria or yeasts [2]. Glycerol is also a by-product of some petrochemical processes, especially in the production of lubricants, oils, fats and in the refining of crude oil [3]. Waste glycerol can be subjected to various processing operations, including purification and refining, to obtain glycerol of an appropriate purity [4]. Alternative methods for its utilisation are also being sought, such as conversion into other valuable chemical products or biofuels [5]. Therefore, various technologies are currently being developed to enable the effective and sustainable utilisation of this type of waste [6].

An increasing trend may currently be observed in waste glycerol production. Forecasts for the next decade also indicate that the market supply for this waste will systematically increase [7]. The main factor behind this phenomenon is the dynamic growth in demand for bio-components for conventional fuels, which in turn is being fuelled by the environmental and climate policies of many countries [8]. The European Union is a pioneer and leader in this respect, with its ambitious plans contained in the Fit for 55 package [9]. According to Organisation for Economic Co-operation Development and the Food and Agricultural Organization (OECD-FAO) estimates, global biodiesel production currently stands at around 47.1 billion litres, and waste glycerol accounts for 12% of the total esters produced [10]. Waste glycerol is also produced during alcoholic fermentation and accounts for about 10% of the total sugar used to produce bioethanol [11]. A significant oversupply of crude glycerol from the biofuel industry is causing glycerol prices to fall from USD 480/t in 2002 to USD 110/t in 2021. In many cases, it becomes waste that needs to be recovered, managed and neutralised in a stable way [2].

Biodiesel is mainly obtained by chemical or enzymatic transesterification of vegetable oils and animal fats. It features low toxicity and high biodegradability, as it consists of alkyl esters of free fatty acids [12]. First- and second-generation biodiesel, is made by using edible and non-edible oils from plant biomass [13]. However, the extraction of this substrate for the transesterification process requires developing large agricultural areas. This fact has a direct impact on the competition of this type of specialised energy crop with the production of biomass for food and feed purposes [14]. It has also been proven that the production of oil crops requires labour-intensive and costly agrotechnical practices, including the use of herbicides and pesticides. This leads to higher production costs, adversely effects on the environment and increased greenhouse gas emissions [15]. The synthesis of third- and fourth-generation biofuels is based on the use of lipid-storing microalgae biomass and the cultivation of genetically modified terrestrial plant species [16]. As in the case of first- and second-generation biodiesel, production costs are very high compared to petroleum diesel and the production process requires significant technological advances [17]. For this reason, the use of waste substrates for biodiesel production, including mainly waste fats and cooking oils, appears to be a competitive alternative to the sources of fatty substances presented above [18]. In favour of these substrates are the low raw material costs, no competition with food and the compliance with the assumptions of circular economy and recycling through the recovery of waste oils [19]. The use of food waste fats also reduces the formation and emission of pollutants into the environment. Therefore, the use of this substrate is justified from environmental, economic and technological points of view [20]. In practise, the quality of biodiesel depends on the raw material used and the production process applied.

Biodiesel and bioethanol are generally considered to be clean energy sources whose use is directly in line with the assumptions of the circular economy and responds to the challenges related to reducing greenhouse gas emissions into the atmosphere. It is estimated that powering internal combustion engines with liquid biofuels would reduce greenhouse gas emissions by about 35% compared to the use of diesel oil [21]. However, it is noteworthy that, despite the development of biofuel technologies, the cost of producing this type of alternative fuel is still higher than that of fuels produced from fossil fuels, mainly petroleum [22]. Nevertheless, the amount of biofuels produced and used is gradually increasing, which is related to the growing ecological awareness and the legal and market regulations that support the development of this economic sector. This increase is also directly reflected in the increase in the market supply of waste glycerine and the development of technologies for its management and utilisation [23]. A diagram of the technological processes leading to waste glycerine production is shown in Figure 1 [24].

The search for cost-effective and environmentally friendly solutions that enable waste glycerol processing into valuable and economically utilisable products is a necessary prerequisite for the economic competitiveness of the biofuel market. Due to its properties, poor quality and the presence of impurities, crude glycerine from the biofuel sector cannot be used for most conventional applications in the food and pharmaceutical industries [25]. Physicochemical refining is also a common method for processing waste glycerol [26]. Only after purification can glycerol be used in the pharmaceutical and cosmetic industry to produce creams, ointments, syrups, alcohol extracts and e-cigarette liquids. Because it is non-toxic, it can also be used in the food industry, e.g., as a solvent for colourings and flavourings and as a sweetener [27]. In addition, it is also subjected to thermochemical conversion into polypropylene glycol [28] or acetol [29]. Purified glycerol also undergoes a number of chemical modifications, which make it a viable raw material for the production of dihydroxyacetone, epichlorohydrin, acrylic acid and glycerol esters and ethers [30]. Dihydroxyacetone, which is produced during glycerol oxidation, is a non-toxic sugar that is involved in the Maillard reactions. It reacts with the amino acids of the creatine structures and leads to the formation of water pigments that impart a brown colour to the skin; hence, it is used in self-tanning creams [31]. Epichlorohydrin is an important component in the production of paints, epoxy resins, rubber, greases and polyvinyl chloride (PVC) additives [32]. In turn, glycerol dehydration produces acrolein, which in turn is a raw material for the production of acrylic acid, an intermediate product of the production of polymers that are used as absorbents in nappies, pads and sanitary towels [33].

The purified glycerine fraction is also used in the feeding of cows with high milk yields. Its addition to the feed provides the necessary energy after calving, increases the efficiency of milk production, increases the protein content of the milk, improves the palatability of the feed, and improves the health condition of animals [34]. The waste glycerol fraction can also be used as a feed additive in pig and poultry farming [35]. Crude glycerol is disposed of by methods such as incineration [36] or composting [37]. Glycerol can also serve as a source of fuel energy. Its treatment at temperatures of 650–800 °C produces a gas consisting mainly of carbon monoxide, hydrogen, methane and ethane [38]. The catalytic decomposition of glycerol leads to the production of hydrogen [39]. However, this process is very expensive as it requires the use of platinum–aluminium catalysts and high temperatures. Another example of glycerine fraction use is the production of inhibitors for the metalworking industry. This fraction is suitable for use in coolants and lubricating fluids, as well as for the production of non-flammable hydraulic oils, polishing pastes, water-washable oils and greases [40]. Crude glycerine is also applied as an additive to low-boiling coolants and brake fluids [41] and its use as a component of heavier fuels is also becoming increasingly popular [42]. Waste glycerol can also be processed by thermochemical conversion [43], oxidation [44], hydrogenolysis [45], aqueous phase reforming [46], electroconversion [47] and thermal catalysis [48] to obtain economically valuable products. Some of these isolated technologies overlap to a certain extent, as the definitions of the processes are very broad, e.g., thermochemical conversion or thermal catalysis [23]. Many scientific publications confirm that catalysts play a key role in thermochemical processes to ensure the speed and ultimate efficiency of waste glycerol processing operations [49]. In the case of cast glycerol, many multidirectional thermal catalysis pathways have been defined, which means that many catalysts must be used depending on the process carried out and the expected end products [50]. This is a very broad branch of experimental work. From the point of view of efficient processing and final valorisation of waste glycerol, it is necessary to catalytically activate and increase the reactivity of the chemical bonds present in this compound, depending on the type of product desired. The possibility of increasing the reactivity of the bonds has a direct influence on the choice and properties of the catalyst and the processing conditions used [51]. Suitable bifunctional catalysts with metal and acid—base sites are used to cleave C-C and C-O bonds, including catalysts based on metal complexes or Rh, Pd and Pt nanoparticles [52]. To increase the reactivity of C-H or O-H bonds, active metal centres are used, with noble metals such as Pt, Ru and Pd being the most effective [53]. In recent years, alternative solutions have been sought by developing the possibility of replacing noble metals with transition metals such as Cu, Ni and Co [54].

Less-explored possibilities for the utilisation of waste glycerol concern the development and production of new materials for the construction of high-quality end products. The use of glycerol as a precursor for the production of carbon materials, e.g., activated carbon, is promising but little-known [55]. Glycerol-based carbon materials are obtained in one step by the partial in situ carbonisation and sulphonation of glycerol with sulphuric acid. Glycerol-based activated carbon materials, on the other hand, are produced in two steps, namely by the partial carbonisation and sulphonation of glycerol in the presence of sulphuric acid, followed by chemical or thermal activation of the glycerol-based carbon material [56]. The possibilities of utilising waste glycerol for the production of liquid fuels and other value-added products are shown in Figure 2.

The production of valuable products from waste glycerol is an important issue that determines the improvement of the economic viability of biofuel production and corresponds to the model of a waste-free, emission-free circular economy. Although waste glycerol derives mainly from the production of liquid biofuels, it can paradoxically become an immediate or intermediate substrate for their biosynthesis [57]. Currently, effective and efficient technological solutions are being sought for the profitable production of liquid biofuels from glycerol and other value-added products [58]. The main objective of this work is to characterise the mechanisms and evaluate the efficiency of existing methods for microbiological valorisation of waste glycerol into liquid biofuels, including biodiesel, bioethanol and biobutanol, as well as to identify future trends in the production of value-added products. In addition, it presents the results of a bibliographic analysis of publications related to the production of liquid fuels and economically valuable products from glycerol, evaluates the progress of research and application work and identifies areas of future research.

2. Bibliographical Analysis, Research Directions and Scientific Potential

Technologies for converting glycerol into liquid fuels and other products with commercial potential have spurred a growing interest in recent years. This is evidenced by the results of analyses of the resources available in the main databases and scientific publications. In the present study, the existing trends in the research topic were assessed based on statistics derived from searches of selected terms. The analysis included the following keywords: “glycerol to bioethanol”, “glycerol to biobutanol”, “glycerol to bio-oil”, “glycerol to value-added products”, “waste glycerol valorisation” and “liquid fuels from glycerol” over the years 2010–2023 (Figure 3). Taking into account the number of references found in the Google Scholar, Scopus, Scilit and Science Direct databases, it is clear that the topic is still newsworthy, and the growing number of reports related to glycerol waste-processing technologies proves the continued interest of many scientists, research institutions and companies in this topic around the world. The Google Scholar database features the highest number of mentions on the topic of “glycerol to bio-oil”, which increased from 6460 in 2010 to 25,200 in 2022, and the fewest number of these on the topic of “waste glycerol valorisation”, which increased from 105 in 2010 to 2130 in 2022. In the other databases, the greatest changes were found regarding the mentions of “glycerol to value-added products”. Between 2010 and 2023, their number increased from 11 to 130 in Scopus, from 29 to 217 in Scilit and from 5991 to 14,444 in Science Direct. No mentions of “glycerol to biobutanol” were found in the Scopus or Scilit databases.

Next, the data retrieved for the selected keywords, namely “liquid fuels from glycerol” and “value-added products from glycerol”, were analysed in terms of the form of published works, i.e., original research articles and review articles, in the Google Scholar (Figure 4), Scopus (Figure 5) and Science Direct (Figure 6) databases. Considering the last 13 years of research on glycerol conversion to liquid fuels, the number of papers published by type according to the Google Scholar database was comparable, as 49% were original research articles and 51% were reviews (Figure 4). In the Scopus database, 94% of the papers were published as original research articles and only 6% as reviews (Figure 5). In contrast, 77% of publications were original research publications and 23% of publications were reviews in the Science Direct database (Figure 6). The selection of papers on the valorisation of glycerol into value-added products was as follows: original research articles accounted for 59%, 81% and 91%, and reviews for 41%, 19% and 9% in the Google Scholar (Figure 4), Scopus (Figure 5) and Science Direct (Figure 6) databases, respectively.

3. Alcoholic Fermentation

Various yeast and bacterial strains can be used for the alcoholic fermentation of glycerol, including Citrobacter freundii [59], Clostridium butyricum [60], Enterobacter aerogenes [61], Enterobacter agglomerans [62], Klebsiella pneumoniae [63] and Lactobacillus reuteri [64]. However, most of these do not convert it into butanol. An analysis of the literature shows that the best-studied organism for this purpose is the Gram-positive anaerobic and non-pathogenic bacterium Clostridium pasteurianum [65]. During anaerobic fermentation, the overall redox balance in the cell is maintained by switching between different metabolic pathways, leading to the formation of different products and their reducing counterparts. The highly reduced nature of glycerol leads to the production of twice as many reducing equivalents compared to the degradation of lignocellulosic sugars, such as xylose or glucose [66]. These additional reducing equivalents give glycerol the inherent advantage of a higher theoretical product yield for reduced chemicals and fuels. The regulation of glycolysis and NAD⁺ and NADH₂ levels in the cell is mainly carried out by NADH-ferredoxin oxidoreductase, which can produce or oxidise NADH₂ (depending on cellular conditions). Acetyl-CoA is an obligatory activator of NADH-ferredoxin reductase activity, and NADH₂ is a competitive inhibitor of ferredoxin–NAD⁺ reductase activity [67]. It is particularly interesting that, according to Johnson and Rehmann [67], NADH-ferredoxin oxidoreductase was found to act reversibly in C. pasteurianum and C. acetobutylicum. In addition, glycolysis is a pH-dependent process. C. pasteurianum has been shown to maintain stable intracellular NAD and NADH₂ concentrations during different growth phases thanks to this regulation [68]. In published works, the increase in butanol yield was attributed to a higher rate of electron transfer to NAD by the NAD–ferredoxin oxidoreductase and, consequently, to the increased availability of the reduced form of NADH, which is necessary for butanol synthesis [69]. This phenomenon was explained either by hydrogenase inhibition or by the use of the mediator as an additional substrate for the NAD–ferredoxin oxidoreductase synthesis [69]. A simplified representation of the glycerol metabolism of C. pasteurianum, with particular emphasis on the end products, is shown in Figure 7 [66].

3.1. Production of Bioethanol

Due to its popularity as a fuel, ethanol is a widely sought-after fermentation product. Klebsiella pneumoniae GEM167 [70], Kluyvera cryocrescens S26 [71], Pachysolen tannophilus CBS4044 [72], Escherichia coli and Enterobacter aerogenes [73] have been used for its production. Oh et al. [70] irradiated K. pneumoniae to produce a mutant strain that boosted ethanol production to 20.5 g/L. Liu et al. [71] achieved a total ethanol production of 28.1 g/L using P. tannophilus CBS4044 yeast, which was still very low compared to the bacteria-based production. The process was found to not be sensitive to the variability of crude glycerol batches depending on the feedstocks used to produce biodiesel. Oxygen transfer rate (OTR) was a key factor in ethanol production, with a lower OTR having a positive effect on the final yield [72]. Meyer et al. [73] found that once non-glycerol organic matter had been removed from crude glycerol, it could be fermented by E. aerogenes to ethanol at room temperature, which reduced energy costs.

Considerable efforts have also been made to genetically modify certain strains to increase their glycerol conversion rates. For example, Loaces et al. [74] achieved an ethanol production rate of 0.39 g/(h OD L) using E. coli with heterologous gene expression and improved glycerol conversion. Kata et al. [75] exploited the expression of PDC1 and ADH1 genes in the thermotolerant yeast Ogataea (Hansenula) polymorpha and carried out fermentation at relatively high temperatures (45–48 °C), which led to an increase in the fermentation rate. The wild strain O. polymorpha produces negligible amounts of ethanol from glycerol, namely 0.8 g/L. The overexpression of PDC1, encoding pyruvate decarboxylase, increased ethanol production to 3.1 g/L, while simultaneous overexpression of PDC1 and ADH1 (encoding alcohol dehydrogenase) led to a further increase in ethanol production from glycerol. In addition, increasing the fermentation temperature to 45 °C stimulated ethanol production from glycerol (which was used as the sole carbon source) to 5.0 g/L, which exceeded the previously reported data for methylotrophic yeast strains [75].

Thapa et al. [76] created a mutant strain of E. aerogenes SUMI014 that was able to block the formation of lactic acid and thus increase ethanol production. Under optimal fermentation conditions (34 °C, pH 7.5, 78 h), the production of bioethanol by the mutant strain was 34.54 g/L, which is 1.5 times that ensured by the wild type (13.09 g/L). The subsequent overexpression of the alcohol dehydrogenase (adhE) gene in the mutant strain increased bioethanol production to 38.32 g/L. The combination of gene deletion and overexpression resulted in a bioethanol production efficiency of 0.48 g/g using glycerine at a concentration of 80 g/L [76].

Ethanol is not the only product of glycerol fermentation; hydrogen is often produced at the same time. Maru et al. [77] carried out dark fermentation with a co-culture of Escherichia coli CECT432 and Enterobacter sp. spH1. The maximum efficiency of H₂ and ethanol production was achieved at 1.53 and 1.21 mol/mol glycerol, respectively [77]. Yazdani and Gonzalez [78] modified E. coli strains to convert glycerol to ethanol more efficiently, with the added benefits of hydrogen and formate production. The maximum production of ethanol and hydrogen was 4.65 mmol/L/h. In turn, the maximum formate production was 3.18 mmmol/L/h, with a simultaneous ethanol production of 3.58 mmmol/L/h [78]. Valle et al. [79] developed an experimental E. coli single mutant method to identify strains with increased ethanol and/or H₂ production compared to the wild-type strain. In an initial screening of 150 single mutants, 12 novel strains (gnd, tdcE, rpiA nanE, tdcB, deoB, sucB, cpsG, frmA, glgC, fumA and gadB) were found to ensure an increased yield of at least one of the target products [79]. In turn, Cofré et al. [80] pointed out the possibility of intensifying the ethanol/hydrogen production by Escherichia coli MG1655 on a pilot scale. The ethanol concentration obtained was 8.5 ± 1.70 g/L and 6.3 ± 0.62 mmol/mol crude glycerol, indicating the possibility of increasing the scale of the process [80].

Varrone et al. [81] carried out an energetic and economic evaluation of an innovative process for the bioconversion of crude glycerol into ethanol and hydrogen. The experiments carried out by these researchers showed that it was possible to obtain at least 26 g/L of ethanol together with 9 L of hydrogen without the addition of nutrients. They found that, with 26 g/L ethanol and a retention time of up to 120 h, the calculated energy costs would be approximately EUR 0.019/kWhth and EUR 0.057/kWhel, taking into account the contribution of hydrogen and bioethanol. In addition, the cost of bioethanol would be only EUR 0.21/L, even without taking into account possible revenues from hydrogen. These results are very promising and indicate that the process has a reasonable chance of becoming economically viable [81]. A summary of the research results on bioethanol production can be found in

Table 1. Use of crude glycerol for the production of bioethanol.

Substrate	Experimental Details	Glycerol Concentration	Production/Yield	Ref.
E. aerogenes SUMI014	100 mL serum bottle, 34 °C, pH 7.5, 78 h, 200 rpm	60 g/L	34.54 g/L	[76]
E. aerogenes SUMI2008		60 g/L	38.32 g/L	[76]
E. aerogenes ATCC 29007		60 g/L	13.09 g/L	[76]
Enterobacter aerogenes HU-101	Cylindrical glass column reactor, 37 °C, pH 6.8	1.7 g/L	0.96 g/L (ethanol), 1.12 g/L (hydrogen), 0.2 g/L (acetate), 0.2 g/L (1,3-PDO), 0.14 g/L (formate)	[61]
		3.3 g/L	0.83 g/L (ethanol), 0.9 g/L (hydrogen), 0.1 g/L (acetate), 0.22 g/L (1,3-PDO), 0.05 g/L (lactate), 0.2 g/L (formate)	[61]
		10 g/L	0.67 g/L (ethanol), 0.71 g/L (hydrogen), 0.09 g/L (acetate), 0.12 g/L (1,3-PDO), 0.11 g/L (lactate), 0.19 g/L (formate)	[61]
		25 g/L	0.56 g/L (ethanol), 0.71 g/L (hydrogen), 0.06 g/L (acetate), 0.17 g/L (1,3-PDO), 0.17 g/L (lactate)	[61]
E. aerogenes ATCC 13048	125 mL serum bottles, 37 °C, 120 rpm	20 g/L	12.8 g/L	[82]
E. coli LY180	1.2 L fermenters, 37 °C, pH 7.0, 24 h, 150 rpm	50% v/v	75 g/L	[74]
E. coli EH05	Multi-fermentation system with six 300 mL working volume vessels, 37.5 °C, pH 6.3–7.5, 300 rpm	20 g/L	20.7 g/L	[83]
Hansenula polymorpha DL1-L	250 mL flasks	2% v/v	2.74 g/L	[84]
K. cryocrescens	2.5 L stirring bioreactor, 30 °C, pH 7.0, 500 rpm	25 g/L	27 g/L	[71]
Klebsiella oxytoca M5al	7.5 L stirring bioreactor, 37 °C, pH 7.0, 300 rpm	60 g/L	12.26 g/L (ethanol), 13.31 g/L (acetate), 39.14 g/L (1,3-PDO), 16.73 g/L (lactate), 5.27 g/L (butanediol), 3.77 g/L (succinic acid)	[85]
K. pneumoniae GEM167	5 L stirred-vessel system, 37 °C, 200 rpm	20 g/L	20.5 g/L	[70]
O. polymorpha with genes of PDC1 and ADH1	300 mL Erlenmeyer flasks, 45 °C, 140 rpm	150 g/L	5.0 g/L	[75]
Enterobacter spH1 and E. coli CECT432	1.2 L jacketed bioreactor, 37 °C, 72 h, 200 rpm	289.7 mmol/L	220.77 mmol/L (ethanol), 278.7 mmol/L (hydrogen)	[77]
Saccharomyces cerevisiae YPH499	batch	20 g/L	0.14 g/g glycerol	[86]
Microbial mixed culture	3 L bioreactor, 37 °C, pH 8.0, 120 rpm	20 g/L	26 g/L (ethanol), 9 L/L fermenter (hydrogen)	[81]

.

3.2. Production of Biobutanol

Butanol and 1,3-propanediol (1,3-PDO) are also commonly produced via glycerol fermentation with Clostridium pasteurianum. Butanol is a new-generation biofuel and is gaining increasing attention due to its properties, such as higher energy density and lower volatility than ethanol. Khanna et al. [87] produced butanol, 1,3-propanediol and ethanol from glycerol using Clostridium pasteurianum MTCC 116 and found that cross-linked cells performed significantly better than the non-cross-linked ones and that the use of crude glycerol had no adverse effect on cell morphology. The use of 25 g/L crude glycerol produced the maximum yield of n-butanol (0.23 g/g), and 5 g/L crude glycerol formed the maximum amount of 1,3-PDO (0.61 g/g). Only traces of ethanol were obtained in all variants [87]. Gallardo et al. [65] investigated the effect of crude glycerol concentration on butanol yield during fermentation of crude glycerol by C. pasteurianum DSM 525. The effect of adding acetate and butyrate to the culture medium was also evaluated. There was a clear influence of crude glycerol concentration on the efficiency of butanol production. The butanol and 1,3-PDO pathways were observed to compete with each other, and the butanol pathway prevailed at higher substrate concentrations (up to 35 g/L). The addition of butyrate to the culture medium resulted in a 45% higher butanol titre, lower 1,3-PDO production and a shorter fermentation time. The addition of acetate also increased the butanol titre, but the fermentation was longer. Although it was not possible to increase the glycerol consumption above 32 g/L and simultaneously increase the concentrations of NH₄Cl and FeCl₂, similar results were obtained as after the addition of butyrate to the medium: a 35% higher butanol yield at the expense of 1,3-PDO and shorter fermentation [65].

Johnson and Rehmann [67] converted crude glycerol into butanol using Clostridium pasteurianum. They showed that, as the pH of the process decreased, the rate of cell growth and CO₂ production decreased, resulting in slower fermentation, a longer duration of butanol production and its higher production rate. The maximum butanol yield of 0.29 g/g glycerol (0.36 mol/mol) was achieved at pHs of 4.7 and 5.5 [67]. Lin et al. [88] added butyrate as a fermentation precursor using Clostridium pasteurianum CH4 and combined this with in situ butanol removal by vacuum membrane distillation (VMD). The addition of 6 g butyrate/L led to an increase in butanol production rate from 0.24 to 0.34 mol butanol/mol glycerol. The coupling of VMD and butyrate strategies led to an increase in butanol production to 0.39 mol butanol/mol glycerol (29.8 g/L) [88]. A summary of research conducted on the conversion of crude glycerol to biobutanol by fermentation can be found in

Table 2. Use of crude glycerol for the production of biobutanol.

Substrate	Experimental Details	Glycerol Concentration	Production/Yield	Ref.
C. pasteurianum CH4	2 L bioreactor, 37 °C, pH 5.5, 100 rpm	100 g/L	0.24 mol/mol crude glycerol (butanol)	[88]
	2 L bioreactor, 37 °C, pH 5.5, 100 rpm, 6 g/L butyrate as a precursor	100 g/L	0.34 mol/mol crude glycerol (butanol)	[88]
	2 L bioreactor, 37 °C, pH 5.5, 100 rpm, 6 g/L butyrate as a precursor, vacuum membrane distillation (VMD) during the cultivation	100 g/L	0.39 mol/mol crude glycerol (butanol)	[88]
C. pasteurianum MTCC 116 (free cells)	250 mL custom fabricated anaerobic flasks, 30 °C, 24 h, 150 rpm	5 g/L	0.08 g/L (butanol), 0.25 g/L (1,3-PDO), 0.04 g/L (ethanol)	[87]
		10 g/L	0.01 g/L (butanol), 0.28 g/L (1,3-PDO), 0.02 g/L (ethanol)	[87]
		15 g/L	0.08 g/L (butanol), 0.11 g/L (1,3-PDO), 0.01 g/L (ethanol)	[87]
C. pasteurianum MTCC 116 cells (immobilized cells)	250 mL custom fabricated anaerobic flasks, 30 °C, 24 h, 150 rpm	5 g/L	0.07 g/L (butanol), 0.31 g/L (1,3-PDO), 0.06 g/L (ethanol)	[87]
		10 g/L	0.01 g/L (butanol), 0.26 g/L (1,3-PDO), 0.13 g/L (ethanol)	[87]
		15 g/L	0.14 g/L (butanol), 0.17 g/L (1,3-PDO), 0.04 g/L (ethanol)	[87]
C. pasteurianum DSM 525	160 mL serum bottles, 37 °C, pH 6.8 ± 0.2	5 g/L	0.17 g/L (butanol), 1.8 g/L (1,3-PDO), 0.06 g/L (ethanol), 0.74 g/L (acetic acid), 0.87 g/L (butyric acid), 0.33 g/L (lactic acid)	[65]
		10 g/L	0.77 ± 0.19 g/L (butanol), 2.71 ± 0.21 g/L (1,3-PDO), 0.18 ± 0.01 g/L (ethanol), 0.99 ± 0.05 g/L (acetic acid), 0.94 ± 0.05 g/L (butyric acid), 0.51 ± 0.07 g/L (lactic acid)	[65]
		15 g/L	2.21 ± 0.11 g/L (butanol), 4.8 ± 0.16 g/L (1,3-PDO), 0.34 g/L (ethanol), 1.52 ± 0.12 g/L (acetic acid), 1.63 ± 0.05 g/L (butyric acid), 1.61 ± 0.05 g/L (lactic acid)	[65]
		20 g/L	2.42 ± 0.1 g/L (butanol), 5.89 ± 0.23 g/L (1,3-PDO), 0.26 g/L (ethanol), 1.66 ± 0.1 g/L (acetic acid), 1.83 ± 0.17 g/L (butyric acid), 0.84 ± 0.1 g/L (lactic acid)	[65]
		35 g/L	6.71 ± 0.43 g/L (butanol), 6.86 ± 0.51 g/L (1,3-PDO), 0.59 ± 0.05 g/L (ethanol), 0.83 ± 0.08 g/L (acetic acid), 0.43 ± 0.08 g/L (butyric acid), 0.73 ± 0.18 g/L (lactic acid)	[65]
		50 g/L	6.73 ± 0.39 g/L (butanol), 6.26 ± 0.27 g/L (1,3-PDO), 0.68 ± 0.04 g/L (ethanol), 0.69 ± 0.03 g/L (acetic acid), 0.21 ± 0.03 g/L (butyric acid), 1.24 ± 0.04 g/L (lactic acid)	[65]
C. pasteurianum ATCC 6013	35 °C, pH 7.0	5 g/L	0.04 mol/mol glycerol consumed (butanol), 0.02 mol/mol glycerol consumed (1,3-PDO), 0.46 mol/mol glycerol consumed (ethanol), 0.04 mol/mol glycerol consumed (acetate), 0.07 mol/mol glycerol consumed (butyrate)	[89]
		25 g/L	0.37 mol/mol glycerol consumed (butanol), 0.078 mol/mol glycerol consumed (1,3-PDO), 0.13 mol/mol glycerol consumed (ethanol), 0.057 mol/mol glycerol consumed (acetate), 0.056 mol/mol glycerol consumed (butyrate)	[89]
C. pasteurianum DSM 525	1 L bioreactor, 35 °C, pH 6.0	29.5 g/L	17.1 mol/100 mol glycerol (butanol), 26.4 mol/100 mol glycerol (1,3-PDO), 1.5 mol/100 mol glycerol (ethanol), 3.2 mol/100 mol glycerol (acetate), 13.1 mol/100 mol glycerol (butyrate), 1.3 mol/100 mol glycerol (lactate)	[90]
		54.2 g/L	27 mol/100 mol glycerol (butanol), 10.5 mol/100 mol glycerol (1,3-PDO), 2.5 mol/100 mol glycerol (ethanol), 1.2 mol/100 mol glycerol (acetate), 3.9 mol/100 mol glycerol (butyrate)	[90]
		83.7 g/L	18 mol/100 mol glycerol (butanol), 23.4 mol/100 mol glycerol (1,3-PDO), 3.6 mol/100 mol glycerol (ethanol), 3.6 mol/100 mol glycerol (acetate), 3.9 mol/100 mol glycerol (butyrate), 3.2 mol/100 mol glycerol (lactate)	[90]
		114.6 g/L	28.1 mol/100 mol glycerol (butanol), 10.5 mol/100 mol glycerol (1,3-PDO), 4.2 mol/100 mol glycerol (ethanol), 4.2 mol/100 mol glycerol (acetate), 3.3 mol/100 mol glycerol (butyrate), 1.2 mol/100 mol glycerol (lactate)	[90]

.

4. Production of Bio-Oil

To date, many studies have been described in the literature on the search for cultivation methods to increase the production of microbial lipids based on waste glycerol. Ample studies have focused on the cultivation of microalgae from the Thraustochytriacae family on industrial waste or low-grade raw materials, such as empty palm fruit bunches [91], breadcrumbs [92], brewer’s yeast after the fermentation process [93], okra powder [94], coconut water [95], sweet sorghum juice [96], beer and potato processing residues [97]. Ensuring adequate growth of these microorganisms and, above all, expanding the scale of cultivation and production of biomass for commercial purposes requires the proper selection and optimisation of many factors, which include the physicochemical parameters of the culture, the properties of the waste substrate used, the availability and cost of the selected substrate, the presence of potential growth inhibitors, the cultivation efficiency, the investment costs and the selection of the appropriate microalgae strain. For this reason, more and more advanced experimental work is warranted in this area. The production of bio-oil and polyunsaturated fatty acids (PUFA) based on glycerol is also aided by certain species of microalgae, such as Nitzschia closterium or Crypthecodinium cohnii, which have the ability to accumulate lipids, especially docosahexaenoic acid (DHA) [98].

Other organisms that are able to grow on waste glycerol, accumulate lipids and produce PUFAs are the microalgae/fungi Skeletonema costatum, Pythium ultimum, Pythium irregulare, Mortierella alpine, Chlorella stigmatophora, Nannochloropsis salina and various species of Codium sp. [99,100]. The diagram of lipid production is shown in Figure 8. Triglycerides (TAGs) are the major storage lipids in microalgae, and the glycerolipid pathway relies mainly on TAG formation via the Kennedy pathway, which exists in both the endoplasmic reticulum and chloroplasts. The TAG synthesis pathway comprises three main steps. Firstly, acyl coenzyme A (acyl-CoA) is formed: glycerol-sn-3-phosphate acyltransferase (GPAT), glycerol-3-phosphate (G3P) and lysophosphatidate (LPA) acylated. LPA is then further condensed with another acyl-CoA catalysed by lysophosphatidic acid acyltransferase (LPAAT) to produce phosphatidate (PA). Phosphatidic acid phosphatase (PAP) then dephosphorylates PA to form diacylglycerol (DAG). Finally, TAG is synthesised by diacylglycerol acyltransferase (DGAT). Although lipid content is not significantly increased by the expression of acetyl-CoA carboxylase (ACCase) and fatty acid synthetase (FAS) genes, one can still hope to increase lipid production by genetically engineered key enzymes of the TAG pathway [101].

A study addressing the evaluation and selection of technological parameters of the bio-oil production process by microalgae of the genus Schizochytrium sp., using glycerol as waste from biodiesel production, has shown that the most important parameters affecting the concentration of biomass and lipids in the cells of Schizochytrium sp. are temperature and glycerol concentration in the culture medium, as well as oxygen concentration and peptone concentration in the bioreactor [40]. It has also been demonstrated that a temperature of 26 °C promotes the production of fatty substances in the cells, while higher values stimulate an increase in the growth rate of the biomass. Similar conclusions were reached by Wen and Chen [102], according to whom temperature is the most important parameter determining the concentration of cells in the culture medium and the accumulation of lipid compounds in the biomass obtained. The value at which the highest biomass concentrations can be achieved depends on the type of microalgae strain tested and the environment the strain was isolated from. The production of fatty acids in the cells of Chlorella minutissima, Pythium irregulare and Crypthecodinium cohnii always takes place at a lower temperature compared to the value of this parameter, which is the most suitable for increasing the biomass concentration in the technological system [103]. According to Richmond and Soeder [104], the phenomenon of increasing the bio-oil concentration in the cells of the tested microalgae at low culture temperatures is related to the activation of a defence mechanism in the microorganisms, which consists of improving the elasticity of the cell membranes. In addition, the phenomenon described above can be explained by the fact that the availability of intracellular molecular oxygen is increased at low temperatures, which activates oxygen-dependent enzymes responsible for the process of desaturation and elongation of PUFA in the cells [105].

Kujawska et al. [40] found that the use of 150 g/L of crude glycerol led to the highest concentration of microalgal biomass, reaching 67.55 g/L, and to an increase in the concentration of lipids, including DHA, to 17.25 g/L in the cells of Schizochytrium sp. Other researchers also found that the type and concentration of the external carbon source had a significant impact on the microalgae cultivation process and lipid content [106]. According to their reports, the carbon source influences both the growth of biomass and potentially the synthesis of lipids, and cultures in which glycerol served as the carbon source were characterised by higher yields compared to those aided by glucose, coconut oil, waste from the brewing industry or wastewater from the production of soy milk. It is also emphasised that the processes carried out with glucose as a carbon source have not yet been optimised [40].

The dry mass of the Schizochytrium sp. genus microalgae contains 14–20% nitrogen (m/m), which is mainly incorporated into the structure of proteins and nucleic acids. To cover the cells’ demand for this element, compounds representing its source, such as ammonium sulphate, peptone, corn liquor or their mixtures, are added to the culture medium. From a technological point of view, it is important that each strain favours a different nitrogen source, which influences rapid cell growth and lipid accumulation [107]. The study by Kujawska et al. [40] confirmed that, to increase the concentration of Schizochytrium sp. cells in the culture medium, it was necessary to add peptone at a concentration of 9.99 g/L. However, a concentration of 2.21 g/L was proven sufficient for the lipid accumulation stage. This observation is consistent with the hypothesis that the formation of new cells and the increase in biomass require providing compounds that are incorporated into the structure of primary metabolites, such as proteins, nucleic acids or enzymes [108]. What is noteworthy is that peptone belongs to the so-called comprehensive nitrogen sources. This means that it contains proteins, peptides and free amino acids, as well as has low contents of sugars, fats, inorganic ions, vitamins and growth factors. It not only supplies nitrogen to the cell biomass, but also aids its general development [109].

It has been proven that fed batch cultures are very effective, as they enable a significant increase in the biomass of the microorganisms and ensure a high lipid content in the cells [110]. This culture variant was used to produce lipids and DHA through Crypthecodinium cohnii and Aurantiochytrium sp. KRS101 microalgae using acetic acid [111] and glucose as external carbon sources, respectively [112]. Using waste glycerol as an external carbon source, three culture variants (batch culture, fed-batch culture and continuous culture) were tested for their potential to promote a high increase in the biomass of Schizochytrium sp. cells and cause the accumulation of a high concentration of lipids and thus of DHA acid [100]. The most effective technological solution proved to be batch cultivation with current supply. During the 120 h process, the biomass concentration reached 103.44 ± 1.50 g/L and the biomass increase was 0.86 ± 0.12 g/L·h. Lipids were accumulated in the microalgae cells at a final concentration of 48.85 ± 0.81 g/L, with DHA accounting for 45% m/m (21.98 ± 0.36 g/L) [100]. Chen and Walker [113] proved that the Chlorella protothecoides strain was capable of growing in a batch culture on media with crude glycerol as the sole carbon source, reaching the final biomass concentration of 23.5 g/L and final lipids of 14.6 g/L after 6 days of culture. By using a semi-continuous culture strategy, the lipid production rate could be increased to 3 g/L·d [113].

Talbierz et al. [114] investigated the effect of ethyl methanesulfonate (EMS) on the growth rate and intracellular lipid accumulation in Schizochytrium limacinum microalga heterotrophically cultivated on waste glycerol as a carbon source. The strain S. limacinum E20, which was produced by incubating the reference strain in EMS for 20 min, showed the best results in biomass production (0.054 gsm/L·h) and intracellular bio-oil accumulation (0.021 g/L·h). The following parameters proved to be optimal for biomass growth of S. limacinum E20: a temperature of 27.3 °C, a glycerol concentration of 249.0 g/L, oxygen in the culture at 26% and a yeast extract concentration of 45.0 g/L. The optimal parameters for lipid production in the S. limacinum E20 culture were as follows: a temperature of 24.2 °C, a glycerol concentration of 223.0 g/L, oxygen in the culture at 10% and a yeast extract concentration of 10.0 g/L. Due to the different conditions of biomass growth and intracellular lipid accumulation, it is recommended to use a two-stage culture process, which resulted in a lipid synthesis rate of 0.41 g/L·h [114].

Any impurity contained in waste glycerol will affect the cultivation of microalgae that utilise this compound as their main source of carbon. Impurities in the form of free fatty acids are used by microalgae to synthesise longer polyunsaturated fatty acids such as DHA. Methanol, on the other hand, has a negative effect on both the growth and production of PUFA. The soap contained in the waste glycerol affects cell growth and fatty acid composition; however, it may be removed by adding a strong acid to the reaction medium. The results of a study carried out by Pyle et al. [115] show that the composition of technical glycerine varies depending on the manufacturer and that the main impurities are methanol, soaps, calcium, phosphorus, potassium, sodium, silicon and zinc. It has also been found that methanol and soaps have a particularly negative impact on the growth rate of S. limacinum microalgae biomass, and therefore should be removed from technical glycerine to increase process efficiency [115]. Another study has confirmed that high methanol concentrations in the culture, exceeding 50 g/L, lead to a statistically significant slowdown in the growth of S. limacinum and a decrease in lipid concentrations. At a crude glycerol dose of 23 g/L, the lipid content was 65.8 ± 1.3%; however, the highest concentration of lipids reaching 73.3 ± 4.9% was achieved at a crude glycerol dose of 35 g/L [116].

In contrast, Rattanapoltee et al. [117] showed that impurities in the technical crude glycerine, e.g., soap and methanol, had only a minor influence on the final biomass concentration and the final content of valuable fermentation products, such as lipids. According to Pyle et al. [115], during the thermal sterilisation process (autoclaving at 121 °C, 20 min), methanol is removed from the medium composed based on technical glycerol. Strains of certain microalgae, such as S. limacinum, are able to grow on different carbon sources and accumulate lipids, as confirmed by the work of Yokochi et al. [118], who demonstrated the ability of this microalgal species to grow on nine carbon sources, including glucose, fructose, oleic acid and glycerol. The highest biomass concentration of 16 gTS/L was obtained with oleic acid, while the highest DHA concentration of 1.1 g/L was obtained with the glycerol-based medium [118]. The results of the research related to the use of glycerol for the production of lipids are listed in

Table 3. Use of crude glycerol for lipid production.

Substrate	Experimental Details	Glycerol Concentration	Production/Yield	Ref.
Schizochytrium sp.	Biostat B Twin (Sartorius Stedim, Göttingen, Germany) bioreactor with a working capacity of 2 L, 27 °C, oxygen concentration 50%, initial peptone concentration 10 g/L, pH 6.5, volumetric airflow rate 0.3 Lair/min·Lreact., salinity 17.5 PSU, initial yeast extract concentration 0.4 g/L, 175 rpm	150 g/L	48.85 ± 0.81 g/L	[100]
Schizochytrium limacinum SR-21 (ATCC MYA-1381)	250 mL Erlenmeyer flasks, 20 °C, 170 rpm, glycerol derived from soybean oil by Virginia Biodiesel Refinery (West Point, VA, USA)	75 g/L	43.24 ± 1.28%	[115]
	250 mL Erlenmeyer flasks, 20 °C, 170 rpm, glycerol from a chicken fat and soybean oil mixture by Virginia Biodiesel	75 g/L	50.57 ± 1.32%
	250 mL Erlenmeyer flasks, 20 °C, 170 rpm, glycerol from canola oil by Seattle Biodiesel LLC (Seattle, WA, USA)	75 g/L	46.71 ± 1.01%
Schizochytrium sp. ATCC	27 °C, peptone concentration 10 g/L, oxygen mass transfer rate kLa, 150 1/h, salinity 17.5 psu, pH 6.5, yeast extract concentration 0.4 g/L, 185 rpm, and inoculum DCW 5.0 g/L	150 g/L	69.44 ± 0.76 g/L	[119]
Schizochytrium limacinum E20	Biostat B Twin (Sartorius Stedim) bioreactor with a working capacity of 2 L, 26 °C, oxygen concentration 30%, pH 6.5 ± 0.1	223.0 g/L	48 ± 1.2%	[114]
Schizochytrium limacinum C			42 ± 0.9%
Schizochytrium limacinum SR21	250 mL Erlenmeyer flasks, pH 7.0, 170 rpm	23 g/L	65.8 ± 1.3%	[116]
Scenedesmus incrassulatus PPAY1	3 L tubular bioreactors, 28–32 °C	5 g/L	31.50 ± 0.71%; 0.51 ± 0.01 g/dm3	[117]
		10 g/L	38.49 ± 0.26%; 0.74 ± 0.02 g/dm3
		20 g/L	44.64 ± 0.19%; 1.26 ± 0.01 g/dm3
		30 g/L	50.25 ± 0.35%; 1.22 ± 0.02 g/dm3
		5 g/L	38.03 ± 0.35%; 1.08 ± 0.02 g/dm3
		10 g/L	47.15 ± 0.49%; 1.46 ± 0.03 g/dm3
		20 g/L	58.27 ± 0.05%; 2.45 ± 0.01 g/dm3
		30 g/L	52.01 ± 0.01%; 2.94 ± 0.03 g/dm3
Trichosporon fermentans	250 mL conical flask, 28 °C, pH 6.0, C/N 60, inoculum concentration 10%	50 g/L	5.2 g/L	[120]
Trichosporon cutaneum	250 mL conical flask, 30 °C, pH 6.0, C/N 60, inoculum concentration 10%	70 g/L	5.6 g/L
Rhodosporidiobolus fluvialis DMKU-RK253	500 mL Erlenmeyer flask, 30 °C, pH 7.0, two-stage cultivation	57 g/L	27.81 ± 1.86 g/L	[121]
Yarrowia lipolytica CCMA 0357	500 mL flasks, 28 °C, 120 h, 150 rpm	100 g/L	70% w/w	[122]
Trichosporon oleaginosus ATCC 20905	15 L fermenter, 28 °C, pH 6.5, 300–400 rpm	25 g/L	32.0% w/w (9.35 g/L)	[123]
		50 g/L	33.6% (10.13 g/L)
		100 g/L	33.3% (9.13 g/L)
		150 g/L	33.1% (9.03 g/L)
Trichosporon oleaginosus ATCC 20905	15 L fermenter, batch, 30 °C, pH 5 ± 0.1, 500 rpm, C/N 20 w/w	45 mL	22.98% w/w (3.08 g/L)	[124]
	15 L fermenter, batch, 30 °C, pH 5 ± 0.1, 500 rpm, C/N 30 w/w	67 mL	47.5% w/w (11.26 g/L)
	15 L fermenter, batch, 30 °C, pH 5 ± 0.1, 500 rpm, C/N 45 w/w	101 mL	48.95% w/w (12.14 g/L)
	15 L fermenter, batch, 30 °C, pH 5 ± 0.1, 500 rpm, C/N 60 w/w	133 mL	52.02% w/w (10.04 g/L)
	15 L fermenter, fed-batch, 30 °C, pH 5 ± 0.1, 500 rpm, C/N 45 w/w	101 mL	49.89% w/w (21.87 g/L)

.

5. Production of Other Bio-Based Products

5.1. Production of Industrial-Value Organic Products

Glycerol can serve as a carbon source for the Clostridium sp. genus bacteria that are able to produce 1,3-propanediol, which is used in composites, adhesives, laminates, ultraviolet (UV)-cured powders and coatings, innovative aliphatic polyesters, copolyesters, solvents and antifreezes [125,126]. Himmi et al. [127] investigated batch fermentation with C. butyricum and found that it was potent enough to convert waste glycerol into 1,3-propanediol. According to a study by Cardona et al. [128], the strain Klebsiella pneumoniae was also able to grow on waste glycerol and produce 1,3-propanediol. Another substance produced by microorganisms on waste glycerol is dihydroxyacetone. It is a non-toxic simple sugar consisting of three carbon atoms that is used in the cosmetics industry [129]. It may be produced by immobilised strains of Acetobacter xylinum upon glycerol oxidation [130] and synthesised by Gluconobacter oxydans, which uses glycerol as a carbon source [131]. Dihydroxyacetone is mainly used for the production of self-tanning lotions, and the skin colour achieved after using such cosmetics is the result of a chemical reaction with the amino acids contained in the stratum corneum of the epidermis [132]. Fermentation carried out by Actinobacillus succinogenes with glycerol as a carbon source produces succinic acid [133]. This acid is also effectively produced by the Basfia succiniciproducens species, with a yield of 1.2 g per 1 g of crude glycerol, and the whole process is characterised by high stability and low production costs [134]. Glycerol is also used by Anaerobiospirillum succiniciproducens to produce succinic acid via anaerobic fermentation [135]. Succinic acid is mainly used for the production of derivatives (succinic anhydride, succinimide and N-bromosuccinimide (NBS)) and also in the food industry as an acidity regulator and flavour enhancer (as a food additive E363) [136]. There are reports describing the possibility of using crude glycerol for the biosynthesis of citric acid by the yeast Yarrowia lipolytica. The process is similar to that carried out with a conventional substrate with the addition of sugar [137]. Citric acid is used as an acidity regulator and antioxidant in foods and as an acidic cleaning agent in various cleaning processes [138].

5.2. Carotenoids

An important group of compounds produced by microalgae in glycerol-based processes are carotenoids. Only a few of them are commercially utilised: β-carotene, astaxanthin and canthaxanthin [139]. Carotenoids have recently spurred increasing interest due to their growing demand in the cosmetic industry, where they are used in masks, balms and self-tanning lotions. For instance, β-carotene is a precursor of vitamin A, which improves the condition of the skin and supports natural protection against UV radiation. They are also applied in the food industry as colorants [140] and substances with antioxidative properties that are added to animal feedstuffs [141]. Astaxanthin has been commercialised as a functional food additive potent to protect against carcinogenic compounds, boost the immune system and treat some diseases [142]. It is synthesized mainly by Phaffia rhodozyma [143] and Sporobolomyces ruberrimus [144], which use glycerol as the sole carbon source. Other pigments, such as β-carotene, have been extracted from Blakeslea trispora using crude glycerol [145] and from the green alga Chlamydomonas acidophila, which has recently been described as a viable alternative for its production [146]. Carotenoids are also derived from Rhodotorula glutinis using pure and crude glycerol [147].

5.3. Production of Biopolymers

Volova et al. [148] showed that glycerol can be used to produce polyhydroxyalkanoate (PHA) polymer, which is a complex of naturally occurring bacteria and polyesters that has been recognised as a substitute for non-biodegradable petrochemical polymers. It is used as a biodegradable plastic, both as a thermoplastic and as an elastomer (with a melting point from 40 to 180 °C), and for the manufacture of implants and artificial tissue [149]. Polyhydroxybutyrate (PHB) is the best-known biodegradable polymer from the PHA group, used to manufacture packaging for shampoos and cosmetics [150]. It slowly decomposes into water and carbon dioxide under the influence of bacteria found in soil, wastewater or sludge, especially when exposed to anaerobic conditions. Studies on the use of crude glycerol for PHB production by the strains Paracoccus denitrificans and Cupriavidus necator JMP 134 showed that all polymers obtained were similar to those produced using glucose [151]. Waste glycerol was also used as the sole carbon source to produce phytase in the culture of Pichia pastoris recombinants possessing a constitutively expressed pGAP vector [152]. In turn, Gluconobacter sp. CHM43 can be used to produce glyceric acid from crude glycerol [153] and Staphylococcus caseolyticus EX17 to produce lipase-resistant solvents from crude glycerol [154], whereas Ustilago maydis has also been shown to be a good biocatalyst for the conversion of crude glycerol into glycolipid biosurfactants and other useful products [155]. Another use of crude glycerol is protein production by the fungus Rhizopus microsporus var. oligosporus. The biomass obtained has a high threonine content and can be used for commercial purposes [156]. A considerable number of microorganisms are able to produce polyene fatty acids (PUFAs), like, for example, Mortierella alpina fungus, which can synthesise arachidonic acid using glycerol as a carbon source [157].

5.4. Production of Health-Promoting Products

The strain Rhodosporidium sp. DR37 is able to synthesise squalene and accumulate it in the lipid fraction, using glycerol as a carbon source [158]. It is the most important factor in reducing the risk of cancer development compared to other dietary constituents and also a good chemopreventive agent. Squalene is also used in the cosmetics industry [159]. Kośmider et al. [160] used Propionibacterium freudenreichii ssp. Shermanii to produce vitamin B12 from crude glycerol. Vitamin B12 is one of the most important vitamins and is frequently used in medicine and the food industry. Its deficiency causes neuropathy, diseases of the nervous system and pernicious anaemia [161]. Glycerol has also been used to aid trehalose production by Propionibacterium freudenreichii ssp. Shermanii 1 [162]. Due to its unique properties, trehalose has a wide range of applications. It is half as sweet as sucrose and suppresses insulin secretion, which makes an ideal alternative for the production of foods for diabetics. Trehalose is also an ingredient of eye drops (to treat dry eyes), tissue storage solutions, organs, enzymes and vaccines. It is also used in cosmetic creams and balms to retain moisture or improve stability during storage. Attempts have also been made to use trehalose to treat osteoporosis and neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases [163].

Due to its characteristics and properties, pure glycerol can be converted into valuable products such as glycerol carbonate, new polymers and other derivatives obtained in oxidation and chemical reduction processes [164]. However, the presence of methanol, unreacted or partially reacted fats, esters, free fatty acids and neutralised catalysts make it difficult to process. The purification of crude glycerol for cosmetic or pharmaceutical purposes is not economically viable, especially when considering small- or medium-sized biodiesel producers [165]. The list of bioactive substances produced from glycerol can be found in

Table 4. Bioactive substances produced by microorganisms with waste glycerol.

Microorganism	Product	Ref.
Clostridium sp.	1,3-Propanediol	[125,126]
Clostridium butyricum CNCM 1211		[127]
Klebsiella pneumoniae DSM-2026		[128]
Acetobacter xylinum	1,3-Dihydroxyacetone	[130]
Gluconobacter oxydans		[131]
Actinobacillus succinogenes ATCC 55618™	Succinic acid	[133]
Basfia succiniciproducens		[134]
Anaerobiospirillum succiniciproducens DSMZ 6400		[135]
Phaffia rhodozyma BPAX-A1	Astaxanthin	[143]
Phaffia rhodozyma CBS 6938		[166]
Sporobolomyces ruberrimus H110		[144]
Blakeslea trispora ATCC 14271	β-Carotene	[145]
Rhodotorula glutinis		[135]
Chlamydomonas acidophila		[146]
Yarrowia lipolytica	Citric acid	[137]
Cupriavidus eutrophus B-10646	Polyhydroxyalkanoates (PHA)	[148]
Paracoccus denitrificans i Cupriavidus necator JMP 134	Poly(3-hydroxybutyrate) (PHB)	[151]
Pichia pastoris	Phytase	[152]
Gluconobacter sp. CHM43	Glyceric acid	[153]
Staphylococcus caseolyticus EX17	Organic solvent tolerant lipase	[154]
Ustilago maydis	Glycolipid biosurfactants	[155]
Rhizopus microsporus var. oligosporus	Protein	[156]
Mortierella alpina NRRL-A-10995	Arachidonic acid	[157]
Rhodosporidium sp. DR37	Squalene	[158]
Propionibacterium freudenreichii ssp. shermanii	Vitamin B12	[160]
Propionibacterium freudenreichii ssp. shermanii 1	Trehalose	[161]

.

6. Summary and Directions for Future Research

Considering the rapidly changing environmental awareness of populations, the introduction of ambitious legal regulations, programmes and packages related to waste management, the development of clean energy sources and the implementation of circular economy assumptions, the search for technologies to convert waste glycerol into economically valuable products seems fully justified. It should be noted that, in most cases, research in this area has not yet progressed beyond the laboratory scale; hence, it is still at the initial stage of technological readiness. The possibility of its practical implementation in the future depends on many technological, legal, political and economic factors. In the optimal scenario, the implementation of innovative technologies for the recovery of energy and other economically valuable products from waste should be economically competitive compared to other alternative or conventional production systems. Given the limited progress in research and the low efficiency of biological conversion, it should be concluded that such a scenario is very unlikely in the near future, in particular with regard to the production of bioethanol and biobutanol.

After analysing the results of previous experimental works assessing the possibility of producing bioethanol from waste glycerol, it must be concluded that the road to industrial implementation of this type of solution is very long. This is mainly due to the low technological efficiency of glycerol biotransformation into this liquid biofuel. The yields obtained in the tests carried out on a laboratory scale are several times lower than those obtained when using typical alcoholic fermentation substrates. The main direction of the scientists’ research is to develop effective genetic modification techniques to improve the rate and ultimate effects of converting waste glycerol into bioethanol. However, it should be noted that the tests carried out are often based on the use of mutagenic factors responsible for spontaneous mutations, which are difficult to repeat and apply systematically in large-scale plants. In addition, in many cases, genetic engineering techniques raise ethical concerns, which often effectively limits the possibility of their implementation in industrial applications. Unfortunately, factors related to the modification of process parameters and technological conditions have a much smaller impact on the production efficiency achieved, which would be much easier for operators to handle and simpler from a practical point of view. Although the alcoholic fermentation process produces gaseous hydrogen in addition to bioethanol and although simple economic analyses of the process, taking into account the entire process and an in-depth analysis of investment and operating costs, appear encouraging, the implementation of this type of solution is not justified from a technological, economic and environmental point of view. Further research should focus on testing the best selected technological variants on a larger scale. The verification of the final results obtained should be carried out in continuous bioreactors, the functioning of which most closely corresponds to operating conditions. This will allow an unbiased and reliable assessment of the actual technological impact. Continuous research is also expected to enable the collection of the appropriate amount of data and determine the observed variations necessary to produce a reliable life cycle analysis that includes both the energy and economic impact of the process and the full environmental impact of the introduction of this type of technology.

Research on the use of waste glycerol as a feedstock for the production of biobutanol is still less advanced. The interest in this issue is related to the fact that butanol is being treated by many researchers as a new-generation biofuel and because it has unique and valuable properties, such as a higher energy density and lower volatility than ethanol. So far, very few papers have addressed this topic, which is currently not being intensively researched. One microorganism that has been tested in this respect is the Clostridium pasteurianum bacterium, whose efficiency has been investigated on a laboratory scale. Considering the fact that this process is much less universal compared to the production of bioethanol, it seems that this line of research could prove to be a dead end that will never lead to an increase in the technological progress of this method and its possible implementation.

Research into the development and implementation of technologies for the production of lipids for biodiesel production by heterotrophic strains of microalgae is of much greater and continuing interest. Many species of microalgae featuring mixotrophic or purely heterotrophic pathways of metabolism have been tested in this area of performance research using waste glycerol. The efficiency of the processes used was also determined, both in investigation carried out under laboratory conditions with batch reactors and in continuously operated systems. The optimisation of this technology was also carried out using numerous statistical methods and techniques. Based on experimental work with several variants, empirical models were also developed to estimate the ultimate technological impact of the process of converting glycerol to bio-oil and further transesterification to biodiesel. The analytical results obtained on a small scale were verified several times in pilot plants operated under fractionated technical conditions. Based on the facts presented above, this technology for neutralising waste glycerol appears to be the most advanced and has the greatest chance of being used on an industrial scale. It should be emphasised that all important technological parameters and process conditions are well known and verified and that the intensification of metabolic transformations of microalgae in the context of molecular engineering is considered possible.

There are many possibilities to produce other economically valuable bioproducts based on waste glycerol. The feasibility of its biotechnological transformation into many substances that can be used in the food, pharmaceutical, fitness and beauty industries has been broadly demonstrated. In most cases, however, the efficiency of the production of value-added products is not satisfactory and the end products are largely contaminated with other by-products. This fact, in many cases, reduces their market value and the possibility of their wide utilisation. It is, therefore, necessary to continue research in this area in order to increase technological progress in this type of solution.

The greatest challenges for researchers and practitioners involved in the processing of waste glycerol concern, on the one hand, increasing the technological efficiency of the process and, on the other hand, reducing unit production costs. The market value of all products must be confirmed not only by the convergence of the ecological, environmental or climate policy measures implemented, but above all by the economic competitiveness and profitability of production. It should be emphasised that, despite the significant increase in interest in the production of liquid fuels and other value-added products based on waste glycerol and the observed increase in the number and scope of experimental work, the solutions tested and described are still at a low level of technological readiness level. Certainly, there is currently a lack of sufficient and properly validated and verified data to perform a credible and reliable life cycle analysis, including an energy, economic and environmental balance. Therefore, it is difficult to judge whether the solutions that are directly in line with the assumptions of the circular economy or “zero waste” policies have a chance to work in the real economic reality in the distant future and not just be the subject of even the most advanced scientific research.

7. Conclusions

Waste glycerol is a promising organic substrate that can be utilised in many ways. The factor that determines the possibility of its processing is primarily the economic aspect, which determines the implementation of the technology in practice. It has been shown that this organic substrate can be successfully used in dark fermentation processes, mainly to produce biogas with a high methane content. This is one of the least technologically advanced methods of waste glycerol energetic processing, which has therefore been implemented on an industrial scale. Another group of dominant technologies leading to the production of gaseous energy carriers are thermochemical processes, including gasification and pyrolysis.

Much less space in the literature is devoted to other, alternative methods for waste glycerol bioconversion into liquid biofuels and other economically valuable end products. It should be emphasised that these technologies are primarily based on complex enzymatic conversions and the metabolism of various groups of microorganisms, mainly bacteria and microalgae. This means that specific environmental conditions and their high stability must be ensured during the ongoing biochemical processes. In many cases, the presence of impurities in the waste glycerol is the factor limiting the synthesis of economically valuable substances, which has a direct impact on the efficiency of the processes and the quality of the end products. In practise, the most difficult stage in the conversion of waste glycerine into liquid fuels and other economically valuable substances is its separation and recovery from the biomass of the microorganisms that enrich them. This is often associated with high operating costs and technological complications.

Although multi-faceted research into the bioconversion of glycerol into valuable end products is becoming increasingly important worldwide and is developing dynamically, in most cases, it has not yet progressed beyond the laboratory scale. Its actual potential for practical utilisation will only become apparent in the next phase of verification tests on a larger scale and in plants with a higher degree of technological maturity. Only in this phase will it be possible to reliably estimate the actual efficiency of the process as well as the investment and operating costs, which may ultimately lead to a decision in favour of a particular technological solution.

The main challenges and necessities to verify the possibility of practical implementation of methods for recovering valuable materials from waste glycerol are the need to improve the efficiency of production processes already on a laboratory scale, to carry out tests of the most promising and promising solutions on a larger scale to collect enough data for a reliable analysis of technological efficiency, energy balance and real assessment of environmental impact. In the future, legal regulations and economic support mechanisms may be required for breakthrough solutions implemented on an industrial scale.