Regulation Of Mixed Microbiome And Nutritional Structure To Enhance Lactic Acid Production From Kitchen Waste:Effectiveness And Mechanism

Lactic acid?LA?is a natural organic acid that has various applications in food,pharmaceutical,and other fields.Due to the deficiency of conventional LA fermentation methods,such as expensive raw materials,complex sterilization operation and high energy consumption,utilizing organic waste as raw material to produce lactic acid by fermentation has attracted increasing attention.Food waste?FW?and waste activated sludge?WAS?are two representative organic wastes with considerable output,while unstable and prone to corruption.Inappropriate treatment will pose a serious threat to the ecological environment and human health.Fermentation of FW,containing a large fraction of carbohydrate but relatively few fermentative microorganisms,showed a low efficiency for LA production.Although WAS contains a complex of various microorganisms favorable for fermentation,the proteinaceous substrate from WAS with a low fraction of carbohydrate was unsuitable for LA production.Therefore,co-fermentation of FW and WAS can offset the disadvantage during single fermentation feedstock.Up to date,the main concern towards the co-fermentation of FW and WAS still focuses on enriching volatile fatty acids?VFA?,hydrogen and methane,etc.However,the enhancement of LA production is rarely documented.To facilitate high-efficiency LA production during co-fermentation,the short-term batch fermentation,long-term repeated batch fermentation,and semi-continuous fermentation modes were respectively investigated.Then,the mechanistic of LA promotion was discerned through regulating mixed microbiome from WAS and balancing nutrition structure with addition of ammonia.The main conclusions are as follows:?1?Under the mode of short-term batch fermentation,the optimal conditions and impact factors of LA production by co-fermentation of FW and WAS were explored.The results showed that the optimal conditions to produce total LA were temperature 20°C at pH 11,temperature 35°C at pH and temperature 50°C at pH 7,and the corresponding LA yields were 0.45,0.67 and 0.55 g COD/g TCOD,respectively.Response surface methodology?RSM?confirmed that temperature and pH had an interactive effect on the production of total lactate and L-lactate,while temperature had a weak impact on the production of D-lactate.Mechanistic studies suggested that:1)the efficiency of solubilization and hydrolysis increased with the increase of temperature and pH;2)the consumption of soluble carbohydrate showed the highest rate at mesophilic temperature,but relatively lower at ambient temperature,thermophilic temperature,and strong alkaline conditions;3)VFA production was lower during strong alkaline fermentation,which was beneficial to the stable accumulation of LA;4)the relative activity of n-LDH related to lactate production was significantly improved under the above three optimal conditions;5)through high-throughput sequencing analysis,lactic acid bacteria were enriched under the conditions of temperature 20°C with pH 11?Enterococcus?,temperature 35°C with pH 9?Clostridium,Dysgonomas,Streptococcus,and Alkalidhilus?and temperature 50°C with pH 7?Clostridium and Caloramator?.?2?Afterward,the efficiency of LA production during the long-term fermentation of WAS and FW was investigated.The results of semi-continuous fermentation showed that the LA production was less than 1 g COD/L in the stable phase,due to its conversion into VFA?17.74±1.25 mg/L,mainly composed of acetic acid,propionic acid,and n-butyric acid?.The concentration of soluble carbohydrate was as low as 0.58±0.20 g COD/L,which limited the production of LA.The results of repeated batch fermentation?4 days/batch,8 batches?showed that,in one cycle,LA was rapidly produced within 2-3 days?20.79-35.50 g COD/L?,but the yield fluctuated greatly between batches.After 3 days of fermentation,the concentration of LA decreased rapidly with VFA accumulation4.50-17.56 g COD/L?mainly composed of acetic acid and propionic acid?.The optical activity?OA?of L-lactate was low?35.18-64.43%?.High-throughput sequencing demonstrated that in the long-term fermentation mode,WAS inoculum introduced lactic acid consuming bacteria into the system,resulting in the consumption and destabilization of LA.Thereby,it was essential to investigate the critical factor facilitating LA production through co-fermentation of FW and WAS.Elucidating the knowledge gap might stabilize the functional microbiome and sustain high-quality products in the long-term fermentation.?3?To address the above problems,initially,FW and WAS were mixed in batch fermentation for the accumulation of strains.After that,repeated batch fermentation was operated using different substrates:1)sole FW as the control group without the effect of WAS?Ctrl group?;2)supplementing autoclaved WAS as the organic nitrogen source to eliminate the influence of mixed microbiome in WAS?Org-N group?;3)supplementing ammonia nitrogen as the critical factor of WAS?Am-N group?.The results manifested that when dosing 300 mg/L of ammonia nitrogen in Am-N group,the LA production increased from 11.63±2.16 g COD/L?Ctrl?to 24.49±3.56 g COD/L?Am-N?without consumption,while the OA of L-LA?76.79±18.39%?was 5-fold higher than that of Ctrl group.The LA production in Org-N group was also elevated to 20.94±3.21 g COD/L,but decreased rapidly with VFA accumulation 9.71±3.47 g COD/L?mainly composed of acetic acid?.This was mainly attributable to the fact that protein-based substrates can promote VFA production significantly.Fermentation metabolism exhibited that the addition of ammonia nitrogen and autoclaved WAS enhanced solubilization,hydrolysis,glycolysis,and acidification,but the addition of ammonia nitrogen reduced the activity of i-LDH and facilitate the accumulation of LA significantly.Overall,the production of LA in Am-N group was relatively stable.High-throughput sequencing demonstrated that the addition of nitrogen source increased the relative abundance of LA producing bacteria with 19.4%of Ctrl group,86.6%of Am-N group and 80.0%of Org-N group.Simultaneously,Carbohydrate Metabolism pathway and Membrane Transport were also boosted,leading to the promotion of LA production.Notably,the addition of ammonia nitrogen improved the enrichment of the L-LA producing bacteria?i.e.,Streptococcus,Lactococcus,Enterococcus,and Corynebacterium?as well as the relative abundance of ldhL-encoding gene,and reduced the relative abundance of ldhD-encoding gene.Moreover,a stable reducing environment remained by ammonium addition,and the oxidation-reduction potential?ORP?ranged from-428±41 mV to-333±30 mV in Am-N group compared with large fluctuation in Ctrl group?from-427±21 mV to-200±28 mV?.Ammonium addition also increased the intracellular NADH levels and NAD⁺/NADH ratio,which favors the reduction of pyruvate to lactate.¹⁵N isotopic tracer technique showed that 47%of ¹⁵NH₄⁺-N underwent assimilation.?4?As mentioned above,the co-fermentation of WAS and FW was unable to accumulate LA under the semi-continuous fermentation mode.So,the method of using ammonia nitrogen to strengthen the nutritional balance of FW is proposed.Under three optimal LA fermentation parameters?temperature 20°C at pH 11,temperature 35°C at pH 9,and temperature 50°C at pH7?,the LA production from FW added with ammonia nitrogen was compared on the long-term fermentation.Then,the feasibility of recovering LA production from nitrogen-deficient control?Ctrl?reactors was evaluated by supplementing ammonia nitrogen.The effects of free ammonia?NH₃-N,FAN?and ammonia nitrogen?NH₄⁺-N?on the fermentation process were also elucidated.When ammonia nitrogen was added initially,the concentration of lactate was 23.92±1.41 g COD/L?temperature 35°C with pH 9?and 14.85±2.74 g COD/L?temperature 50°C with pH 7?respectively,which were significantly higher than that in the un-amended group and alkaline fermentation under ambient temperature at pH 11.The production of LA could be properly recovered after adding ammonia nitrogen in the nitrogen-deficient reactors.The appropriate concentrations of FAN?79.64-94.51 mg/L?and NH₄⁺-N?71.8-117.5 mg/L?under the mesophilic condition at pH 9,accelerated solubilization,hydrolysis,glycolysis,and acidification,the same as under the thermophilic condition at pH 7.Nevertheless,the high concentration of FAN inhibited the metabolism of lactate under the ambient temperature at pH 7.High-throughput sequencing showed that under the mesophilic condition at pH 9,ammonia nitrogen addition selectively enriched the microbes responsible for LA accumulation,i.e.Bavariicoccus,Enterococcus,Bifidobacterium,and Corynebacterium,etc.The relative abundance of metabolic pathways associated with carbohydrate transport and LA production was also enhanced,thereby increasing the LA production efficiency of the system.