Damaged Wheat Grain Fermentation Yields Biodegradable Plastic Under Optimized Acid Hydrolysis

Agricultural waste streams represent an abundant and renewable reservoir of carbon, offering an avenue to bypass refined sugars in the microbial production of biodegradable alternatives to petroleum-derived polymers. In a comprehensive laboratory investigation, researchers evaluated the bioconversion of damaged wheat grain obtained from a silage facility in Karaj, Iran, into poly(3-hydroxybutyrate), commonly designated as PHB. This intracellular microbial polyester consists of repeating 3-hydroxybutyrate units linked by ester bonds, exhibiting mechanical properties comparable to conventional petrochemical plastics while remaining susceptible to microbial breakdown in soil, water, and compost. Because broad industrial adoption of polyhydroxyalkanoates has been historically constrained by high raw-material costs, utilizing compromised, non-food grains provides an integrated valorization pathway. The experimental process combined dilute hydrochloric acid hydrolysis to break down complex cereal polysaccharides into fermentable reducing sugars, followed by batch fermentation using an equimolar consortium of three lactic acid bacteria: Lactobacillus plantarum PTCC 1058, Lactobacillus murinus PTCC 322, and Lactobacillus casei PTCC 1608.

Before chemical processing, the raw damaged wheat grain was thoroughly washed with distilled water, dried at 60 degrees Celsius for 18 hours, milled to a particle size between 500 and 800 micrometers, and subjected to compositional profiling following standard Association of Official Analytical Chemists protocols. Proximate analysis established that the dry-milled grain comprised 13.86 percent moisture, 50.97 percent carbohydrates, 24.34 percent crude fiber, 5.91 percent ash, 3.99 percent crude protein, and 0.99 percent crude fat. When compared to previously documented spoiled wheat samples, the current material exhibited lower protein levels alongside distinct fiber and mineral contents, reflecting variations in grain damage severity, post-harvest handling, and environmental storage history. Elemental quantification conducted via inductively coupled plasma optical emission spectroscopy identified major concentrations of potassium at 4153.48 micrograms per gram, phosphorus at 3254.78 micrograms per gram, magnesium at 1191.18 micrograms per gram, and calcium at 512.91 micrograms per gram, alongside essential cellular trace elements including iron at 43.09 micrograms per gram, zinc at 34.10 micrograms per gram, and manganese at 33.17 micrograms per gram. Concurrently, potentially toxic heavy metals remained at low levels, recording 0.17 micrograms per gram of lead, 0.05 micrograms per gram of mercury, 0.01 micrograms per gram of cadmium, and 2.36 micrograms per gram of nickel, establishing a suitable baseline profile for bioprocess inputs.

Transforming the solid lignocellulosic and starchy matrix of damaged wheat into an assimilable broth required systematically optimizing dilute acid treatment to maximize sugar release while suppressing inhibitory degradation byproducts. Across twenty-seven distinct pretreatment runs designated HDWG1 through HDWG27, the investigative team tested three hydrochloric acid concentrations of 2, 6, and 10 percent weight by volume, three acid-to-sample ratios of 10, 15, and 20 percent volume by weight, three substrate loadings of 10, 20, and 30 percent weight by weight, and four reaction periods ranging from 15 to 60 minutes at a constant temperature of 60 degrees Celsius. In starch-rich cereals, hydronium-catalyzed cleavage breaks the alpha-glucosidic bonds of amylose and amylopectin polymers into soluble glucose and linear oligosaccharides. However, excessive thermal and acidic severity accelerates secondary degradation, wherein liberated pentose sugars dehydrate into furfural while hexoses convert into 5-hydroxymethylfurfural, which subsequently breaks down into formic and levulinic acids. High-performance liquid chromatography coupled with refractive index detection quantified these inhibitory compounds alongside residual acetic acid to determine the exact chemical severity of each treatment.

Among the experimental regimes, treatment HDWG4 emerged as the optimal hydrolysate formulation, employing 6 percent hydrochloric acid, 20 percent substrate loading, and a 20 percent acid-to-sample ratio over a 45-minute residence time. This condition generated 336.66 milligrams of glucose equivalents per gram of dry grain weight while preserving 4.67 percent protein content and restricting inhibitor formation to 2.36 milligrams of acetic acid, 6.95 milligrams of formic acid, 0.49 milligrams of furfural, and 1.17 milligrams of 5-hydroxymethylfurfural per gram of biomass. Conversely, the excessive severity of treatment HDWG26 provoked extensive sugar degradation, yielding only 172.85 milligrams per gram of reducing sugars and 3.81 percent protein, while simultaneously driving inhibitor concentrations to peak values of 7.49 milligrams per gram of acetic acid, 16.18 milligrams per gram of formic acid, 1.51 milligrams per gram of furfural, and 3.22 milligrams per gram of 5-hydroxymethylfurfural. The resulting HDWG4 liquor was subsequently neutralized with 1 normal sodium hydroxide and clarified by centrifugation to prepare the fermentation growth medium.

Fermentation was conducted using a formulated multispecies bacterial community consisting of equal culture volumes of Lactobacillus plantarum, Lactobacillus murinus, and Lactobacillus casei, designed to leverage metabolic complementarity and cross-protection under acidic and chemical stress. In this consortium, Lactobacillus plantarum and Lactobacillus casei contributed documented capabilities for intracellular polyhydroxyalkanoate accumulation and flexible carbohydrate uptake across hexose and pentose substrates through glycolytic and phosphoketolase routes, backed by established tolerance toward furfural and phenolic derivatives. Lactobacillus murinus provided rapid acidification dynamics, structural floc stability, and physiological buffering against environmental stress. Working as an integrated unit, the microbial community channeled surplus carbon and reducing equivalents into intracellular polyhydroxybutyrate granules, utilizing the polymer as a protective metabolic redox sink. Because the bacterial consortium was evaluated exclusively as a combined, cooperative inoculant, the researchers noted that the specific metabolic contribution, growth rate, and polymer accumulation attributable to each independent strain were not separately isolated or individually quantified during the fermentation trials.

The neutralized HDWG4 hydrolysate, diluted to a nominal 10 percent weight-by-volume substrate loading containing approximately 33.6 grams per liter of reducing sugars, was adjusted to an initial neutral pH of 7.0 to prevent premature medium acidification. The team executed thirty experimental fermentations, designated FH1 through FH30, in 250-milliliter Erlenmeyer flasks across three inoculum levels of 3, 6, and 9 percent volume by volume, two cultivation temperatures of 30 and 60 degrees Celsius, and five incubation durations spanning 24, 48, 72, 96, and 120 hours under continuous agitation at 200 revolutions per minute. Statistical evaluation via one-way analysis of variance and Duncan multiple range tests confirmed that incubation temperature, cell density, and cultivation duration exerted statistically significant effects on biopolymer yield. The maximum polyhydroxybutyrate titer of 1.85 grams per liter, accompanied by an accumulation level of 18.5 percent relative to dry cell weight, was achieved under treatment FH12, which utilized a 6 percent inoculum level at 30 degrees Celsius over a 96-hour incubation interval, closely followed by treatment FH11 at 1.81 grams per liter and FH28 at 1.78 grams per liter.

The experimental observations highlighted precise physiological boundaries governing polymer biosynthesis within the lactic acid bacteria community. When the starting inoculum was lowered to 3 percent in treatment FH1, the resulting polymer concentration dropped to 1.35 grams per liter, reflecting an insufficient initial cell density that delayed substrate uptake and prolonged the lag phase prior to intracellular accumulation. Conversely, increasing the bacterial inoculum to 9 percent in treatment FH2 lowered polymer synthesis to 1.67 grams per liter, a reduction plausibly caused by rapid nutrient exhaustion, intensified intercellular competition, and the accelerated channeling of available carbon into cell maintenance or excess organic acid generation rather than polyester storage. Temperature proved equally decisive, as elevating the incubation from 30 to 60 degrees Celsius precipitated a 48 to 59 percent decrease in total polymer yield across corresponding treatments, with the highest thermal stress yield reaching only 1.63 grams per liter in treatment FH27. This pronounced reduction aligned with the mesophilic physiology of Lactobacillus species, wherein thermal stress impairs membrane integrity, disrupts enzyme kinetics, and accelerates adverse medium acidification.

For example, within the 6 percent inoculum group cultivated at 30 degrees Celsius, polyhydroxybutyrate concentration fell sharply from the peak of 1.85 grams per liter in treatment FH12 to 0.86 grams per liter in treatment FH14 after 120 hours of cultivation. This post-peak decline is biologically consistent with the depletion of assimilable extracellular reducing sugars, compelling the starved bacterial cells to mobilize and enzymatically depolymerize their intracellular polyhydroxybutyrate reserves as an internal carbon and energy source for basal survival. However, because residual sugars, dynamic cell dry weights, and organic acid titers were not continuously tracked across every sampling interval, researchers emphasized that polymer turnover and carbon shifting toward lactic acid remain plausible physiological explanations rather than directly verified kinetic pathways.

To confirm the chemical identity, purity, and molecular structure of the biosynthesized polymer, the microbial biomass harvested from treatment FH12 was dried, digested with 40 percent sodium hypochlorite, washed sequentially with distilled water, acetone, and methanol, and extracted into chloroform. Nuclear magnetic resonance spectroscopy of the purified material dissolved in deuterated chloroform corroborated authentic polyhydroxybutyrate architecture. Proton spectra displayed characteristic aliphatic resonances at chemical shifts of 1.7 to 1.9 parts per million corresponding to methyl and methylene protons, alongside a distinct multiplet at 5.0 to 5.2 parts per million assigned to methine protons located at the ester backbone. Carbon-13 spectra confirmed structural integrity through diagnostic signals at approximately 63 parts per million for methylene carbon, 71 to 75 parts per million for central methine carbon, and approximately 180 parts per million representing the quaternary ester carbonyl carbon. Complementary Fourier-transform infrared spectroscopy revealed intense absorption bands at 1732 reciprocal centimeters corresponding to ester carbonyl stretching, distinct carbon-hydrogen vibrations at 3013 and 2951 reciprocal centimeters, and asymmetric carbon-oxygen-carbon fingerprint bands at 1174, 1009, and 921 reciprocal centimeters, matching commercial polymer benchmarks despite minor residual traces of media constituents.

Thermal and crystallographic analyses provided further verification of the physical properties governing the biosynthesized biopolymer. Thermogravimetric analysis revealed a clean, single-step thermal decomposition profile initiating at approximately 270 degrees Celsius and progressing through 340 degrees Celsius, representing non-radical beta-elimination and random ester linkage scission that degrades the polyester into crotonic acid and oligomeric fragments. Derivative thermogravimetry identified a maximum degradation rate peak at approximately 350 degrees Celsius with an overall mass loss of 55 percent, demonstrating higher thermal resistance than previously reported wheat-derived polyhydroxybutyrate materials that decomposed at 228.6 degrees Celsius. Differential thermal analysis confirmed an endothermic melting transition peak at 184 degrees Celsius followed by an exothermic decomposition event at 289 degrees Celsius. X-ray diffraction patterns exhibited sharp, well-resolved reflections at Bragg angles of 5.8, 13.51, 21.15, 26.42, and 30.60 degrees, with the prominent peak at 13.51 degrees corresponding to the characteristic orthorhombic crystalline lattice of authentic polyhydroxybutyrate. Scanning electron microscopy resolved a cohesive, granular surface architecture interspersed with microscopic voids, while ultraviolet-visible spectrophotometry demonstrated strong light attenuation between 230 and 329 nanometers with a minor absorption band at 405 nanometers, indicating potential ultraviolet-shielding characteristics relevant to bio-based packaging.

Notwithstanding these successful laboratory benchmarks, the authors carefully articulated several fundamental boundaries that constrain the immediate generalizability of their results. Although the 1.85 grams per liter polyhydroxybutyrate titer achieved from damaged wheat grain hydrolysate exceeded the 1.305 grams per liter reported for Bacillus species cultivated on enzymatically treated wheat, the yield was obtained under bench-scale shake-flask conditions without continuous dissolved oxygen regulation, automated pH maintenance, or closed-loop feeding controls. Furthermore, the final accumulation ratio of 18.5 percent of dry cell weight remains well below the levels typically attained by specialized industrial strains grown on refined commercial substrates. The research team underscored that formal techno-economic analyses, comprehensive life-cycle assessments, and rigorous contaminant tracking across extraction steps remain strictly unperformed, meaning that industrial scalability, environmental impact, and regulatory compliance cannot yet be established. Validating the viability of damaged cereal grains within the circular bioeconomy will therefore require extensive pilot-scale bioreactor optimization, strain improvement, and detailed downstream purification studies.

Subject of Research: Bioprocessing of damaged wheat grain into polyhydroxybutyrate using acid hydrolysis and bacterial fermentation

Article Title: Bioprocessing of Damaged Wheat Grain Into PHB Using Lactobacillus Fermentation Following Optimized Acid Hydrolysis

Article References: Boorboormoradi, Z., Eyvazzade, O., Rashidi, L., & Nateghi, L. (2026). Bioprocessing of Damaged Wheat Grain Into PHB Using Lactobacillus Fermentation Following Optimized Acid Hydrolysis. Food Science & Nutrition, 14(11), Article e72491. https://doi.org/10.1002/fsn3.72491

Image Credits: AI Generated

DOI: 10.1002/fsn3.72491

Keywords: Polyhydroxybutyrate, Damaged Wheat Grain, Lactobacillus Consortium, Acid Hydrolysis, Bioplastics, Waste Valorization, Fermentation, Circular Bioeconomy, Bioprocessing, Damaged, Wheat, Grain