Biorefinery based on lignocellulosic biomass

Plants are the main source of lignocellulosic materials such as cellulose, hemicellulose and lignin, together with trace amounts of starch, extractives, ash and proteins. Plant age, biomass, tissue and local environmental variables affect the concentrations of these chemicals. Lignin, which is found in plant biomass, is an important polymer that covalently binds cellulose and hemicellulose and consists of phenyl-propane units linked by non-hydrolysable bonds (54, 55). The ether linkages (β-O-4) and chemical functional groups of lignin make it resistant to hydrolysis or microbial hydrolysis (56, 57). However, its abundance in plant biomass makes it a sustainable and promising carbon source and a viable feedstock. Lignin production, predicted to be between 5 and 36·10⁸ tonnes per year, is a suitable carbon source for the ecologically friendly and cost-effective production of biopolymers.

The conversion of lignin to biopolymers involves two basic steps. First, the lignin polymer is broken down into constituent parts and depolymerised to yield a low molecular mass of lignin. The second stage is the production of bio-based products and opening lignin rings in microorganisms through the β-ketoadipate pathway. The molecular mass of lignin should be low enough to allow microbial bioconversion, and aromatic rings with a number lower than five should be able to pass through the cell wall and be digested by microorganisms (58).

The initial step in the bioconversion of lignocellulosic waste into bioplastics is to hydrolyse the lignocellulosic components from waste into fermentable sugars. The second step is detoxification of the hydrolysate and the removal of any inhibitory compounds generated during hydrolysis (59). Bacterial species such as Pseudomonas can form biopolymers by joining aromatic and aliphatic monomers in a series and valorizing various chemical compounds (60). Furthermore, different investigations have explored the possibilities of using Rastlonia and Bacillus sp. to bioconvert lignin into microplastics (61). Several studies have used P. putida KT440 to produce PHAs from lignocellulosic waste. The main advantage of using lignocellulosic waste over other substrates is their low carbohydrate content. One disadvantage of using lignocellulosic material as a carbon source is that it reduces microbial cell growth.

Lignocellulose, which contains lignin (20–30 %), cellulose (40–50 %) and hemicelluloses (20–50 %), is considered the most abundant and viable carbon source. Hemicellulose and cellulose are important sources of edible sugars, but lignin is not degradable due to its rich aromatic structure (62). Cellulose consists of β-1,4-glycosidic linkages of d-glucose and is neither digestible by humans nor soluble in water due to its large molecular mass and crystalline structure. Hemicellulose is an amorphous polysaccharide consisting of several pentose sugars, including xylose, galactose, rhamnose, mannose and arabinose. As a result, cellulose and hemicelluloses are separated from lignin by a process known as delignification, which provides a high sugar yield for microbes to produce PHA. The basic method for degrading solid biowaste into simpler sugar monomers is to pretreat it with cellulase enzymes and then steam it with diluted acids (63). Pseudomonas cepacia synthesised 48–56 % of poly-3-hydroxybutyrate with a yield of 0.11 g per g of xylose. However, the engineered E. coli carrying phbC genes of Ralstonia eutropha was able to synthesise 74 % PHB with a yield of 0.226 g per g of xylose and was thus superior to the indigenous strain.

However, genetically modified E. coli carrying Ralstonia eutropha phb can produce 74 % PHB with a yield of 0.226 g per of xylose, outperforming the indigenous strain (64, 65). It has also been investigated whether hemicellulose hydrolysates can be used directly as a sugar combination. Furthermore, the results showed that R. eutropha can ferment small amounts of hemicellulose hydrolysates, primarily for cell development, using fed-batch fermentation. Recently, the growth conditions of R. eutropha 5119 have been improved to produce PHA using lignocellulosic biomass hydrolysates (LBHs), such as Miscanthus biomass hydrolysate (MBH), barley biomass hydrolysate (BBH) and pine biomass hydrolysate (PBH), as effective carbon substrates. In addition, indigenous yeast species like Wickerhamomyces anomalus VIT-NN01, Candida tropicalis BPU1 (66) and marine Pichia kudriavzevii VIT-NN02 (67) have been reported to produce PHA using sugarcane molasses with palm oil, banana peel hydrolysate and orange peel, respectively, as the main carbon substrate.

Spent coffee grounds

Considering that coffee is one of the most popular consumer commodities in the world market today, it is not surprising that the coffee industry generates a large amount of waste through the production process. Furthermore, the coffee manufacturing sector is one of the fastest growing in the current food market. Around 6 million tonnes of waste coffee grounds are produced annually (67).

The age of the coffee plants, their location, the environment in which they grow, and the quality of the soil affect the chemical composition of spent coffee grounds. Coffee contains nitrogenous compounds (amino acids, a building block of proteins), lipids (free fatty acids, triglycerides and sterols), crude fibre (cellulose, hemicellulose, lignin, mono, oligo- and polysaccharides) and a variety of minerals. The traces of bioactive compounds in spent coffee grounds, such as polyphenols, diterpenes and alkaloids (trigonelline, caffeine), contribute to their antibacterial, anticarcinogenic and antioxidant properties (68-72).

Cellulose (7–9 %), hemicellulose (32–42 g/L), lignin (0–26 g/L protein (10–18 g/L), lipids (2–24 g/L), caffeine (0–2 g/L) and ash (1–2 %) are the main constituents of spent coffee grounds. Several pretreatment methods can dissolve the carbohydrates in spent coffee grounds. The primary by-products of the hydrolysis of spent coffee grounds are galactose, mannose and arabinose (73-75). Bacillus subtilis, Bacillus firmus, Pseudomonas fluorescens, Burkholderia cepacia, Burkholderia sacchari, Novosphingobium nitrogenfigens Y88 and Halomonas halophila are among the microorganisms that can break down the monosaccharides in spent coffee grounds to produce PHA (76-82). In a study by Kovalcik et al. (82), spent coffee ground hydrolysate, which is rich in carbohydrates (mannose, galactose and arabinose), was used to produce PHA with Halomonas halophila CCM 3662. In their study, dried spent coffee grounds were defatted, phenolic compounds were adsorbed onto Amberlite XAD4 and finally acid/alkali-treated to obtain spent coffee ground hydrolysate rich in sugars, which was later used to obtain PHB titres of 0.95 g/L with a yield on dry cell mass basis of 27 %.

According to a study in which C. necator was compared with different oils and the oil from spent coffee grounds, the oil extracted from spent coffee grounds produced significantly more effective PHB (83). The dry cell mass increased to 55 g/L and the PHB content to 89.10 % when the oil from spent coffee grounds was used in fed-batch mode. Because the oil from spent coffee grounds had a higher free fatty acid content than the other oils tested, it formed significantly more PHB. The main disadvantage of the oil extracted from spent coffee grounds is that it foams, although this can be reduced by using other oils that act as both carbon sources and anti-foaming agents. In another study, a recombinant bacterial strain called Cupriavidus necator DSM 428 was used, which utilised the oil from spent coffee grounds as a substrate and a fed-batch fermentation process to produce carbon dioxide. The final product had a PHB concentration on dry biomass basis of 10.7 g/L. An overview of a study on the agricultural waste is shown in Fig. 1.

Fig. 1

Production of bioplastics from agricultural waste (Canvas was used as the primary design tool)

Cheese whey

The global dairy industry faces the challenge of finding a cost-effective and environmentally responsible method of eliminating waste whey, a by-product of cheese and yoghurt. Large dairy producers can use sweet whey (pH=6–7) to produce whey protein and lactose powder. Nevertheless, the dairy industry faces significant challenges due to the presence of lactic acid in acid whey, which is produced in cottage cheese and Greek yoghurt. Dairy producers must pay for the removal of more than 4 million tonnes of acid whey produced in the United States, most of which is either disposed of in wastewater treatment plants or on fields (87). Each year, American dairy farms dispose of more than 4 million tonnes of acid whey; most of this waste ends up in wastewater treatment plants or on fields. As a result, acid whey causes logistical, financial and environmental problems for the dairy industry. Lactose (38–49 g/L), lactate (5.1–8 g/L), lipids (about 1.1 g/L), proteins (4.2–10 g/L) and mineral salts (2.6–5.1 g/L) are the typical components of acid whey. The production of PHAs from whey lactose and lactate would benefit the environment and offer a cheap, renewable feedstock that would not compete with edible goods. Although acid whey can potentially be used as a feedstock for bioplastics, several processing issues make it challenging to develop efficient techniques. For instance, two of the main carbon sources, lactose and lactic acid, are inaccessible to natural microbes. Lactose must be pretreated in acid whey to produce sugars with a higher carbon-to-nitrogen ratio (C/N) and promote fermentation, which is problematic. PHB production increases with higher C/N ratio and some industrially relevant bacteria cannot ferment lactose. The nitrogen retention of whey and acid whey reduces the C/N ratio. A higher C/N ratio (14:1) tends to promote biomass growth, but at the same time, the cells do not store as much PHB as they would with a lower C/N ratio (e.g. 2:1, resulting in a higher PHB content but a lower total yield). Therefore, for a higher PHB yield, a C/N ratio of 14:1 is better. If higher PHB content in the cells is needed, a lower C/N ratio (2:1 with bovine serum albumin (BSA)) is better.

For some microorganisms to successfully convert whey to PHAs, lactose must be broken down into monosaccharides by hydrolysis. The second main impediment to the use of acid whey is the low pH created by lactic acid, which inhibits most microorganisms that produce PHA. The inhibition induced by lactic acid reduces productivity, economic viability and the ability to fully utilise carbon in the production of PHA from acid whey (Fig. 2). In addition, keeping the fermentation pH˃7 and the PHA synthase enzymes active is difficult due to the formation of acidic intermediates and the low initial pH of acid whey.

Fig. 2

Production of polyhydroxyalkanoates (PHA) from acid whey (Canvas was used as the primary design tool)

The first step in evaluating the effects of different conditions on PHB synthesis in E. coli LSBJ with plasmid pBBRSTQKAB using synthetic acid whey was to determine the carbon and nitrogen supply, the initial pH and the addition of minerals and salt. This made it possible to determine if and when a decrease in the pH blocked the PHB biosynthetic pathway and what effect a stable pH has on PHB production. Finally, to maximise PHB production, the amounts of carbon sources, initial pH, minerals and salts in raw acid whey must be adjusted (88). Although recombinant E. coli LSBJ produced high PHB yields on various substrates and some E. coli strains are acid tolerant, it was hypothesised that this would be a viable method for converting acid whey to PHB. A high C/N ratio can, in principle, lead to changes in metabolic flow and increase the concentration of PHB. The production of PHB could be increased by the addition of salts and minerals. In summary, the results will show that the creation of a sustainable system that utilises waste with negative costs to produce biodegradable plastic precursors is possible.

Another cost-effective biotechnological method for PHA synthesis is the use of mixed microbial consortia (MMC), which eliminates the sterilisation phase. MMC is described as a microbial community of unknown composition that is capable of carrying out certain intra- and extracellular processes. As a result, the activated sludge produced in wastewater treatment plants is classified as MMC. The use of mixed microbial cultures is usually carried out in a series of stages, depending on the substrate type.

The first phase is acidogenic (anaerobic) fermentation, which produces volatile fatty acids (lactic, acetic and propionic acid) from carbon-rich wastewater. Following the production of volatile fatty acids, the culture was enriched mostly under aerobic dynamic feeding conditions in a sequential batch reactor to yield biomass with PHA accumulation potential (89). Another stage is PHA formation (accumulation) in biomass to maximise PHA content (90). The PHA was then removed and purified in the final stage (91). This production procedure can be used for complicated substrates such as olive mill effluent, cheese whey and other food waste to produce more homogeneous PHA (92).

Sugar cane

Polyhydroxyalkanoates, known as PHAs, are an alternative to petroleum-based polymers. Microbes that thrive in an unbalanced nutrient environment turn PHAs into biodegradable and biocompatible bioplastics for energy storage. PHAs are divided into numerous types based on their chemical composition. Polyhydroxybutyrate (PHB) and 3-hydroxybutyrate (3HB) are two homopolymers consisting of a single monomer. Polypropylene (PP) and polystyrene (PS) are petroleum polymers, and PHB plastics have comparable structures; they are both hard and brittle. Nevertheless, the production cost of PHAs is still higher than for polymers derived from petroleum. The production method has some pitfalls that can increase PHA costs.

To reduce the cost of producing PHA, one of several readily available and inexpensive raw materials can be used. Sugarcane molasses is a highly exploited carbon source. A by-product of sugar refining, sugarcane molasses contains a high concentration of sugars such as sucrose, fructose and glucose. It also contains trace amounts of essential elements for cell formation, including organic acids, amino acids, vitamins and minerals.

Halomonas sp. are bacteria that produce PHB. The rod-shaped bacterium Halomonas is Gram-negative. This strain is halotolerant, which means it can thrive in environments with NaCl mass per volume ratios ranging from 0.1 to 32.5 %. To avoid cross-contamination, media with high NaCl amounts should be used when growing Halomonas. Halomonas is therefore a suitable strain for PHB production. Based on its dry cell mass, this strain can accumulate 7-80 % PHB. Furthermore, unlike other strains, it can metabolise sucrose independently, which means it does not require hydrolysis to utilise it. These advantages make Halomonas sp. a good bacterium for PHA production. It may be possible to determine if Halomonas sp. can produce PHB from cheap substrates such as sugarcane molasses.

The most successful strain of Halomonas for metabolising sugarcane molasses and producing PHB was found to be Halomona scerina YK44. The cultivation conditions were adapted to increase PHB production. The NaCl mass per volume ratio was 2 % and the pH was 7. With 8.65 g/L dry cell mass, 6.55 g/L PHB and 76.55 % PHB, the strain YK44 was the best producer of PHA at 4 %. The addition of a cost-effective nitrogen source improved cell proliferation and PHB accumulation; this is paramount for microbes because the C/N ratio influences PHB production (93). Additionally, the inhibitory effect of sugarcane molasses was discovered. By comparing the PHB film produced from sugarcane molasses to a film produced from simple sugars and petroleum-derived polymers such as polyethelene terephthalate (PET), polybutylene terephthalate (PBT), PP and PS were able to confirm its application and investigate its physical and thermal properties. This made it possible to evaluate the potential for substitution.

Sewage

According to the International Water Association's 2018 wastewater report, industrialised nations can dispose of 70 % of their sewage, while poor countries only manage 8 % (101). The growing amount of sewage sludge produced by various effluent treatment plants is a cause of great concern, as it is difficult to dispose of it responsibly.

Algal growth media have improved the models for algal production, because they can grow in urban environments or on non-land structures such as columns, and purify this water. Microalgae cultivated in nutrient-free wastewater have a high protein content, making them a viable candidate for conversion into a low-impact bioplastic (102).

Arthrospira platensis and Chlorella sp. are two examples of microalgae with a high protein content, which ranges, on dry mass basis, from 46 to 63 %. Blends with petroleum plastics or bioplastics are the primary focus of research into the use of microalgal biomass in the development of bioplastics (103). The development of microalgae in wastewater has several advantages over standard plant and seed cultivation methods, including the elimination of soil and the use of non-potable water as a growth medium. Their CO₂ fixation rates are faster than those of other, more complex species, and they can reportedly triple their biomass in just a few hours (104). Because of their small size and high protein content, they can be converted directly into polymers without previous treatment, increasing the cost-effectiveness of large-scale production while reducing waste (105).

Domestic peel

Fruit and vegetable waste has a high potential for the production of microbial bioplastics such as PHA. In one study, for example, C. necator and P. citronellolis were used in co-culture to produce poly(3-hydroxybutyrate [P(3HB)] and PHA, with apple pulp waste serving as the sole carbon source for the bacteria (106). The researchers discovered that the combination of P(3HB) and PHA lead to comparable quantitative results to develop flexible and elastic films. Pseudomonas citronellolis produced 1.2 g/L of PHA when grown on the soluble fraction of apple pulp.

Another study used Bacillus sp. to produce PHA from hydrolysed apple pomace fatty acids as a carbon source (107). The production of biodegradable bioplastic films can be facilitated by using banana peel, which is high in starch and readily available in large quantities. The most important parts that are discarded are banana peels and cellulosic fibres. That study provides a new viewpoint on converting waste biomass into lucrative products. The new component of that study was the use of waste biomass to produce a bioplastic film suitable for the packaging of dry goods. The use of cellulose fibre as a filler improved the physical, mechanical and thermal properties of the bioplastic film. Although the peel and the pseudostems are wasted, they could be recycled and used to make the packaging for dry goods. To do this, the following subactivities were completed (108): firstly, the availability of starch, a complex carbohydrate, can be determined by analysing the proximate composition of the banana peel. The starch can then be extracted and characterised. The optimal parameters can be determined to maximise yield. Fibre from banana pseudostem is extracted and characterised. Water absorption rate, tensile strength and elongation at break of the produced bioplastic film are improved. The physicochemical parameters of the synthesised plastic film are determined, including solubility, transparency, thickness and density. The best film is then analysed using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), which allowed a clear comparison of the bioplastic film with and without cellulosic fibre.

Process for the production of bioplastics from macroalgae

Macroalgae are collected by various approaches, including mechanical harvesting and handpicking (118, 119). Collecting macroalgae from the beach by hand is very tedious. This strategy is effective for localised work but impractical for mass production (119). In contrast, mechanical harvesting involves gathering macroalgae from water using boats equipped with specialised equipment.

The extraction of polysaccharides from macroalgae is an important step in the production of bioplastics from these plants. Extracts from brown macroalgae include alginate and laminarin, while carrageenan is obtained from red macroalgae and ulvan from green macroalgae (120-122).

Alginate films are usually produced by casting and solvent evaporation (123). Unlike films made with alginate polymers ionically crosslinked with Ca²⁺ ions, sodium alginate films have lower mechanical properties, barrier properties and water resistance (124, 125). The most common ionic crosslinking processes are external, internal, interfacial and direct mixing of the crosslinking agent (126). Covalent crosslinking with substances like citric acid and ferulic acid has improved thermal stability, elasticity and transparency of alginate films (127).

Macroalgal bioplastics are produced using methods such as extrusion blow moulding, thermoreversible gelling, casting and compression moulding. Plasticizers such as glycerol are widely used to increase malleability (128). The polysaccharide powder is dissolved by heating and stirring the liquid. The powder is dissolved in water, then poured into a mould and left to cool and harden. The physical properties of hydrogel films can be modified by ionic or covalent crosslinking of polymers such as alginate and carrageenan with multivalent cations such as calcium. By blending different algal polysaccharides, the properties of bioplastics can be modified for various applications, from food packaging to medical equipment (129, 130).

Production process of bioplastics with microalgae

Bioplastics derived from microalgae are the result of a fascinating and complex technology that uses the power of these bacteria to make environmentally friendly products. Selecting microalgae, cultivating them, harvesting them, extracting their lipids, synthesizing bioplastics, characterising them, and finally, utilising their waste are all common processes in the production of bioplastics from microalgae. The method starts with the careful selection of microalgal strains known for their high biomass and lipid concentration. These strains are the main source of bioplastic precursors. Controlled habitats, such as ponds or specialised bioreactors, provide optimal conditions for the development of microalgae. The light intensity, temperature and nutrient contents are all closely controlled to ensure the maximum potential.

When the microalgal biomass reaches a certain level, they are harvested. The microalgae are removed from the growth medium by various methods, including centrifugation, filtration and flocculation. Lipid extraction is the process of obtaining usable lipids from microalgae. These lipids serve as raw ingredients in the production of bioplastics. The lipids are converted into bioplastics using various polymerization processes. Polyhydroxyalkanoates (PHAs) are a common type of biodegradable polymer produced from these lipids.

Haloferax mediterranei utilises carbohydrates as a carbon source to produce PHAs. Simulants of hydrolysates from seven different macroalgal biomasses were produced and PHA synthesis was investigated. The medium containing green macroalgae had the highest biomass concentration and PHA content. Growing Haloferax mediterranei in 25 % Ulva sp. hydrolysate at 42 °C and an initial pH=7.2 resulted in the highest cell dry mass and PHA concentration of (3.8±0.2) and (2.2±0.1) g/L, respectively. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was the primary PHA constituent.