Bioprocess optimization for lactic and succinic acid production from a pulp and paper industry side stream.

The effective and cheap production of platform chemicals is a crucial step towards the transition to a bio-based economy. In this work, biotechnological methods using sustainable, cheap, and readily available raw materials bring bio-economy and industrial microbiology together: Microbial production of two platform chemicals is demonstrated [lactic (LA) and succinic acid (SA)] from a non-expensive side stream of pulp and paper industry (fibre sludge) proposing a sustainable way to valorize it towards economically important monomers for bioplastics formation. This work showed a promising new route for their microbial production which can pave the way for new market expectations within the circular economy principles. Fibre sludge was enzymatically hydrolysed for 72 h to generate a glucose rich hydrolysate (100 g·L-1 glucose content) to serve as fermentation medium for Bacillus coagulans A 541, A162 strains and Actinobacillus succinogenis B1, as well as Basfia succiniciproducens B2. All microorganisms were investigated in batch fermentations, showing the ability to produce either lactic or succinic acid, respectively. The highest yield and productivities for lactic production were 0.99 g·g-1 and 3.75 g·L-1·h-1 whereas the succinic acid production stabilized at 0.77 g·g-1 and 1.16 g·L-1·h-1.

Production of lactic acid from pasta wastes using a biorefinery approach.

A total of 398 kt of pasta waste (PW), generated during the production process of pasta, were produced in 2021. Due to its chemical composition and practically zero cost, PW has already been studied as a raw material for the production of lactic acid (LA) through fermentations. The main objective of this article was to improve the economic viability of the process by replacing commercial enzymes, necessary for starch hydrolysis in PW, with raw enzymes also produced from wastes. Enzyme synthesis was achieved through solid-state fermentation (SsF) of wheat bran by Aspergillus awamori or Aspergillus oryzae at various moisture contents. The maximum amylase activity (52 U/g dry solid) was achieved after 2 days of fermentation with A. awamori at 60% of moisture content. After that, the enzymes were used to hydrolyse PW, reaching 76 g/L of total sugars, 65 g/L of glucose and a yield of 0.72 gglu/gds with the enzymes produced by A. awamori. Subsequently, the hydrolysate was fermented into LA using Bacillus coagulans A559, yielding 52 g/L and 49 g/L with and without yeast extract, respectively. Remarkably, compared to the process with commercial enzymes, a higher LA yield was reached when enzymes produced by SsF were added (0.80 gLA/gglu). Furthermore, the productivities between the two processes were similar (around 3.9 g/L/h) which highlights that yeast extract is not necessary when using enzymes produced by SsF.

Towards efficient production of highly optically pure d-lactic acid from lignocellulosic hydrolysates using newly isolated lactic acid bacteria.

This study presents the production of D-lactic acid with high enantiomeric purity using lignocellulosic hydrolysates from newly isolated lactic acid bacterial (LAB) strains. Six strains, 4 heterofermentative and 2 homofermentative, were investigated for their ability to grow and produce lactic acid on sugar beet pulp (SBP) hydrolysates, containing a mixture of hexose and pentose sugars. Among the strains tested, three were isolates designated as A250, A257 and A15, all of which belonged to the genus Leuconostoc. Only strain A250 could be reliably identified as Leuconostoc pseudomesenteroides based on cluster analysis of Maldi-ToF spectra. All strains produced D-lactic acid in the presence of SBP hydrolysates, but with varying optical purities. The homofermentative strains achieved higher D-lactic acid optical purities, but without assimilating the pentose sugars. Co-cultivation of the homofermentative strain Lactobacillus coryniformis subsp. torquens DSM 20005 together with the heterofermentative isolate A250 led to the production of 21.7 g/L D-lactic acid with 99.3 % optical purity. This strategy enabled the complete sugar utilization of the substrate. Nanofiltration of the SBP hydrolysate enhanced the enantiomeric purity of the D-lactic acid produced from the isolates A250 and A15 by about 5 %. The highest D-lactic acid concentration (40 g/L) was achieved in fed-batch cultures of A250 isolate with nanofiltered SBP, where optical purity was 99.4 %. The results of this study underline the feasibility of a novel isolate as an efficient D-lactic acid producer using lignocellulosic hydrolysates.

Chemical and Enzymatic Synthesis of Biobased Xylo-Oligosaccharides and Fermentable Sugars from Wheat Straw for Food Applications.

Xylo-oligosaccharides are sugar oligomers with 2~7 xylose units considered non-digestible fibers that can be produced from biodegradable and low-cost biomass like wheat straw. An integrated approach consisting of hydrothermal pretreatment, alkaline treatment, enzymatic treatment and the combinations thereof was applied to overcome the recalcitrance structure of the wheat straw and allow selective fractioning into fermentable sugars and xylo-oligosaccharides. The hydrolysates and processed solids were chemically characterized by High-performance liquid chromatography and Ion chromatography, and the results were expressed as function of the severity factor and statistically interpreted. The concentration of fermentable sugars (glucose, xylose, arabinose) was the highest after the combination of alkaline and enzymatic treatment with xylanase (18 g/L sugars), while xylo-oligosaccharides (xylotriose and xylotetraose) were released in lower amounts (1.33 g/L) after the same treatment. Refining experiments were carried out to obtain a purified fraction by using anion and cation exchange chromatography. The polymer adsorber resin MN-502 showed efficient removal of salts, phenols and furan derivatives. However, the xylo-oligosaccharides yields were also slightly reduced. Although still requiring further optimization of the treatments to obtain higher purified oligomer yields, the results provide information on the production of xylo-oligosaccharides and fermentable sugars from wheat straw for potential use in food applications.

Integration of Solid State and Submerged Fermentations for the Valorization of Organic Municipal Solid Waste.

Solid state fermentation (SsF) is recognized as a suitable process for the production of enzymes using organic residues as substrates. However, only a few studies have integrated an evaluation of the feasibility of applying enzymes produced by SsF into subsequent hydrolyses followed by the production of target compounds, e.g., lactic acid (LA), through submerged-liquid fermentations (SmF). In this study, wheat bran (WB) was used as the substrate for the production of enzymes via SsF by Aspergillus awamori DSM No. 63272. Following optimization, cellulase and glucoamylase activities were 73.63 ± 5.47 FPU/gds and 107.10 ± 2.63 U/gdb after 7 days and 5 days of fermentation, respectively. Enzymes were then used for the hydrolysis of the organic fraction of municipal solid waste (OFMSW). During hydrolysis, glucose increased considerably with a final value of 19.77 ± 1.56 g/L. Subsequently, hydrolysates were fermented in SmF by Bacillus coagulans A166 increasing the LA concentration by 15.59 g/L. The data reported in this study provides an example of how SsF and SmF technologies can be combined for the valorization of WB and OFMSW.

Biorefinery Concept Employing Bacillus coagulans: LX-Lignin and L-(+)-Lactic Acid from Lignocellulose.

A new biorefinery concept is proposed that integrates the novel LX-Pretreatment with the fermentative production of L-(+)-lactic acid. Lignocellulose was chosen as a substrate that does not compete with the provision of food or feed. Furthermore, it contains lignin, a promising new chemical building material which is the largest renewable source for aromatic compounds. Two substrates were investigated: rye straw (RS) as a residue from agriculture, as well as the fibrous digestate of an anaerobic biogas plant operated with energy corn (DCS). Besides the prior production of biogas from energy corn, chemically exploitable LX-Lignin was produced from both sources, creating a product with a low carbohydrate and ash content (90.3% and 88.2% of acid insoluble lignin). Regarding the cellulose fraction of the biomass, enzymatic hydrolysis and fermentation experiments were conducted, comparing a separate (SHF), simultaneous (SSF) and prehydrolyzed simultaneous saccharification and fermentation (PSSF) approach. For this purpose, thermophilic B. coagulans 14-300 was utilized, reaching 38.0 g L-1 LA in 32 h SSF from pretreated RS and 18.3 g L-1 LA in 30 h PSSF from pretreated DCS with optical purities of 99%.

Volumetric oxygen transfer coefficient as fermentation control parameter to manipulate the production of either acetoin or D-2,3-butanediol using bakery waste.

The formation of either acetoin or D-2,3-butanediol (D-BDO) by Bacillus amyloliquefaciens cultivated on bakery waste hydrolysates has been evaluated in bioreactor cultures by varying the volumetric oxygen transfer coefficient (kLa). The highest D-BDO production (55.2 g L-1) was attained in batch fermentations with kLa value of 64 h-1. Batch fermentations performed at 203 h-1 led to the highest productivity (2.16 g L-1h-1) and acetoin production (47.4 g L-1). The utilization of bakery waste hydrolysate in fed-batch cultures conducted at kLa of 110 h-1 led to combined production of acetoin, meso-BDO and D-BDO (103.9 g L-1). Higher kLa value (200 h-1) resulted to 65.9 g L-1 acetoin with 1.57 g L-1h-1 productivity. It has been demonstrated that the kLa value may divert the bacterial metabolism towards high acetoin or D-BDO production during fermentation carried out in crude bakery waste hydrolysates.

High L(+)-lactic acid productivity in continuous fermentations using bakery waste and lucerne green juice as renewable substrates.

The biotechnological production of platform chemicals from renewable resources is in the scientific spotlight, as researchers seek to develop environmentally friendly and cost-efficient processes to compete with the petroleum-based ones. Lactic acid (LA) is an established platform chemical, registering an important market share, mainly owing to the increasing demand for polylactic acid. This study investigated the feasibility to produce LA from bakery waste hydrolysates and lucerne green juice (LGJ) as inexpensive substrates, using a Bacillus coagulans strain. A final LA concentration of 62.2 g/L, with a productivity of 2.59 g/(L.h) and a conversion yield of 0.57 g LA/ g bakery waste was achieved in batch fermentation mode. LA productivity reached 11.28 g/(L.h), using a continuous fermentation system coupled with cell retention membranes at a dilution rate of 0.2 h-1. The results indicate that bakery waste hydrolysates and LGJ can be utilized for the production of highly optical pure L(+)-LA.

Batch and Continuous Lactic Acid Fermentation Based on A Multi-Substrate Approach.

The utilisation of waste materials and industrial residues became a priority within the bioeconomy concept and the production of biobased chemicals. The aim of this study was to evaluate the feasibility to continuously produce L-lactic acid from different renewable substrates, in a multi-substrate strategy mode. Based on batch experiments observations, Bacillus coagulans A534 strain was able to continuously metabolise acid whey, sugar beet molasses, sugar bread, alfalfa press green juice and tapioca starch. Additionally, reference experiments showed its behaviour in standard medium. Continuous fermentations indicated that the highest productivity was achieved when molasses was employed with a value of 10.34 g·L-1·h-1, while the lactic acid to sugar conversion yield was 0.86 g·g-1. This study demonstrated that LA can be efficiently produced in continuous mode regardless the substrate, which is a huge advantage in comparison to other platform chemicals.

Assessment of different Bacillus coagulans strains for l-lactic acid production from defined media and gardening hydrolysates: Effect of lignocellulosic inhibitors.

Cellulose valorisation has been successfully addressed for years. However, the use of hemicellulosic hydrolysates is limited due to the presence of C5-sugars and inhibitors formed during pretreatment. Bacillus coagulans is one of the few bacteria able to utilize both C6- and C5-sugars to produce l-lactic acid, but its susceptibility to the lignocellulosic inhibitors needs further investigation. For such a purpose, the tolerance of different B. coagulans strains to increasing concentrations of inhibitors is studied. The isolated A162 strain reached the highest l-lactic acid productivity in all cases (up to 2.4 g L-1  h-1), even in presence of 5 g L-1 of furans and phenols. Remarkably, most of furans and phenolic aldehydes were removed from defined media and hemicellulosic gardening hydrolysate after fermentation with A162. Considering the high productivities and the biodetoxifying effect attained, A162 could be pointed out as a great candidate for valorisation of mixed sugars from hemicellulosic hydrolysates with high inhibitors concentration, promoting the implementation of lignocellulosic biorefineries.

Production of Lactic Acid from Carob, Banana and Sugarcane Lignocellulose Biomass.

Lignocellulosic biomass from agricultural residues is a promising feedstock for lactic acid (LA) production. The aim of the current study was to investigate the production of LA from different lignocellulosic biomass. The LA production from banana peduncles using strain Bacillus coagulans with yeast extract resulted in 26.6 g LA·L-1, and yield of 0.90 g LA·g-1 sugars. The sugarcane fermentation with yeast extract resulted in 46.5 g LA·L-1, and yield of 0.88 g LA·g-1 sugars. Carob showed that addition of yeast extract resulted in higher productivity of 3.2 g LA·L-1·h-1 compared to without yeast extract where1.95 g LA·L-1·h-1 was obtained. Interestingly, similar LA production was obtained by the end where 54.8 and 51.4 g·L-1 were obtained with and without yeast extract, respectively. A pilot scale of 35 L using carob biomass fermentation without yeast extract resulted in yield of 0.84 g LA·g-1 sugars, and productivity of 2.30 g LA·L-1·h-1 which indicate a very promising process for future industrial production of LA.

A Simple Biorefinery Concept to Produce 2G-Lactic Acid from Sugar Beet Pulp (SBP): A High-Value Target Approach to Valorize a Waste Stream.

Lactic acid is a high-value molecule with a vast number of applications. Its production in the biorefineries model is a possibility for this sector to aggregate value to its production chain. Thus, this investigation presents a biorefinery model based on the traditional sugar beet industry proposing an approach to produce lactic acid from a waste stream. Sugar beet is used to produce sugar and ethanol, and the remaining pulp is sent to animal feed. Using Bacillus coagulans in a continuous fermentation, 2781.01 g of lactic acid was produced from 3916.91 g of sugars from hydrolyzed sugar beet pulp, with a maximum productivity of 18.06 g L-1h-1. Without interfering in the sugar production, ethanol, or lactic acid, it is also possible to produce pectin and phenolic compounds in the biorefinery. The lactic acid produced was purified by a bipolar membrane electrodialysis and the recovery reached 788.80 g/L with 98% w/w purity.

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid.

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO2 -eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.

Recent Advances in d-Lactic Acid Production from Renewable Resources: Case Studies on Agro-Industrial Waste Streams.

The production of biodegradable polymers as alternatives to petroleum-based plastics has gained significant attention in the past years. To this end, polylactic acid (PLA) constitutes a promising alternative, finding various applications from food packaging to pharmaceuticals. Recent studies have shown that d-lactic acid plays a vital role in the production of heat-resistant PLA. At the same time, the utilization of renewable resources is imperative in order to decrease the production cost. This review aims to provide a synopsis of the current state of the art regarding d-lactic acid production via fermentation, focusing on the exploitation of waste and byproduct streams. An overview of potential downstream separation schemes is also given. Additionally, three case studies are presented and discussed, reporting the obtained results utilizing acid whey, coffee mucilage and hydrolysate from rice husks as alternative feedstocks for d-lactic acid production.

Biotechnological Production of Organic Acids from Renewable Resources.

Biotechnological processes are promising alternatives to petrochemical routes for overcoming the challenges of resource depletion in the future in a sustainable way. The strategies of white biotechnology allow the utilization of inexpensive and renewable resources for the production of a broad range of bio-based compounds. Renewable resources, such as agricultural residues or residues from food production, are produced in large amounts have been shown to be promising carbon and/or nitrogen sources. This chapter focuses on the biotechnological production of lactic acid, acrylic acid, succinic acid, muconic acid, and lactobionic acid from renewable residues, these products being used as monomers for bio-based material and/or as food supplements. These five acids have high economic values and the potential to overcome the "valley of death" between laboratory/pilot scale and commercial/industrial scale. This chapter also provides an overview of the production strategies, including microbial strain development, used to convert renewable resources into value-added products.

Membrane Technologies for Lactic Acid Separation from Fermentation Broths Derived from Renewable Resources.

Lactic acid (LA) was produced on a pilot scale using a defined medium with glucose, acid whey, sugar bread and crust bread. The fermentation broths were then subjected to micro- and nanofiltration. Microfiltration efficiently separated the microbial cells. The highest average permeate flow flux was achieved for the defined medium (263.3 L/m²/h) and the lowest for the crust bread-based medium (103.8 L/m²/h). No LA losses were observed during microfiltration of the acid whey, whilst the highest retention of LA was 21.5% for crust bread. Nanofiltration led to high rejections of residual sugars, proteins and ions (sulphate, magnesium, calcium), with a low retention of LA. Unconverted sugar rejections were 100% and 63% for crust bread and sugar bread media respectively, with corresponding LA losses of 22.4% and 2.5%. The membrane retained more than 50% of the ions and proteins present in all media and more than 60% of phosphorus. The average flux was highly affected by the nature of the medium as well as by the final concentration of LA and sugars. The results of this study indicate that micro- and nanofiltration could be industrially employed as primary separation steps for the biotechnologically produced LA.

Co-fermentation of the main sugar types from a beechwood organosolv hydrolysate by several strains of Bacillus coagulans results in effective lactic acid production.

Bacillus coagulans is an interesting facultative anaerobic microorganism for biotechnological production of lactic acid that arouses interest. To determine the efficiency of biotechnological production of lactic acid from lignocellulosic feedstock hydrolysates, five Bacillus coagulans strains were grown in lignocellulose organosolv hydrolysate from ethanol/water-pulped beechwood. Parameter estimation based on a Monod-type model was used to derive the basic key parameters for a performance evaluation of the batch process. Three of the Bacillus coagulans strains, including DSM No. 2314, were able to produce lactate, primarily via uptake of glucose and xylose. Two other strains were identified as having the ability of utilizing cellobiose to a high degree, but they also had a lower affinity to xylose. The lactate yield concentration varied from 79.4 ±â€¯2.1 g/L to 93.7 ±â€¯1.4 g/L (85.4 ±â€¯4.7 % of consumed carbohydrates) from the diluted organosolv hydrolysate.

Detoxification of sugarcane-derived hemicellulosic hydrolysate using a lactic acid producing strain.

Furfural and HMF are known for a negative impact in different bioprocesses, including lactic acid fermentation. There are already some methods described to remove these inhibitory compounds from the hydrolysates. However, these methods also reduce the yield of sugars from the hydrolysis and increase the process costs. In this work, the detoxification of sugarcane-derived hemicellulosic hydrolysate was performed by Lactobacillus plantarum during the fermentation time. At the end of the fermentation, a decrease of 98% of furfural and 86% of HMF and was observed, with a final lactic acid titer of 34.5 g/L. The simultaneous fermentation and bio-detoxification simplify the process and reduce operational costs, leading to economic competitiveness of second-generation feedstock for lactic acid production.

From lignin to nylon: Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida.

Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L-1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L-1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.