1- Introduction

2- Materials and methods

3- Results and discussion

4- Conclusion

References

Abstract

The daily output of orange residue from the orange juice production enterprise in the Three Gorges Reservoir Area (TGRA) of China is approximately 100 t/d, which seriously pollutes the environment of the TGRA. The key challenge of handling this waste is maximizing profitability. In this study, high-protein feed with low levels of crude fiber and pectin was produced by solid fermentation of orange waste. The synergistic and antagonistic effects of microorganisms on one another significantly influenced the quality of single cell protein (SCP) feed. The added true protein (ATP) content increased gradually as pectin degradation (PD) and crude fiber degradation (CFD) were enhanced because of the synergistic effects. However, ATP decreased because of antagonistic effects as PD and CFD were increased beyond certain values. Aspergillus oryzae (A. oryzae) and Trichoderma koningii (T. koningii) mutually promoted each other, but the growth of Candida tropicalis was inhibited by A. oryzae and T. koningii as polygalacturonase and carboxymethyl cellulase accumulated. Synergistic and antagonistic effects existed simultaneously during microorganic fermentation of orange wastes. In large-scale fermentation, ATP, PD, and CFD were increased by 14.20%, 15.80%, and 9.15%, respectively, in comparison with the flask test. The profit achieved by reusing orange waste in the Chongshou Agricultural Park as SCP feed was calculated to be 48500 USD per year, whereas the cost of disposing of the orange waste was 7560 USD. This study provides insight into how microorganic synergistic and antagonistic effects influence the quality of SCP feed and provides a potential route for recycling agricultural waste into valuable materials.

Introduction

China is the foremost orange producer in the world. In 2014, the orange planting area of China was 2.211
1010 m2 , and the annual output was 2.75
1010 kg (Shan, 2014). Oranges are consumed worldwide in the forms of pulp, peel and juice. Approximately 8
109 -2
1010 kg of solid and liquid residue is produced as waste during orange processing every year (Tripodo et al., 2004; Rezzadori et al., 2012). In most instances, this enormous amount of waste is scattered on soil in areas adjacent to the production facility, incinerated, placed in a land fill, or used as raw material for animal feed production (Martín et al., 2010; Patsalou et al., 2017). However, incineration of orange waste was found to lead to highly polluted surface and ground water with altered chemical oxygen demand and biological oxygen demand, as well as polluted soil (Rezzadori et al., 2012; Braddock, 1995). The term SCP refers to dead, dry cells of microorganisms such as yeast, bacteria, fungi and algae, which are utilized as a protein supplement in foods for humans or animal feed (Ofodile et al., 2011; Mahmood, 2012). SCP is made up of carbohydrates, fats, vitamins, minerals, and other cellular constituents (Mondal et al., 2012), and it can be produced from relatively inexpensive material or waste material (Santamaría-Fernandez et al., 2017).

Recycling ethanol fermentation stillage from food waste could help to resolve the pollution problems, but it would lead to the accumulation of lactic acid (LA), which had a negative effect on ethanol fermentation. In this study, bipolar membrane electrodialysis was used to recover LA as well as other fatty acids in the stillage, and the corresponding parameters that affected this process were investigated. Pretreatment via centrifugation and ultrafiltration reduced the turbidity successfully. Experiments on simulated and real stillage showed that organic acids such as formic acid and acetic acid had a slight effect on the separation of LA, but the existence of chloride ions could form strong anion competition with lactate ions. Simulation experiments with sodium chloride reduced the recovery of LA from 98% to 92%. The recovery rate of LA from real stillage could reach 71.2%. After electrodialysis, the stillage can be reused in ethanol fermentation, the fermentation time was shortened to 24 h, and the ethanol yield increased.

Introduction

For the sustainable development of society, the recycling of waste has become more and more important. Food waste (FW) accounts for a large proportion of municipal solid waste (15%–63%) [1]. It has high organic, volatile solid (85%–95%), and moisture content (75%–85%) and is easily degraded and deteriorated. There are many ways of reusing the waste, such as composting and production of pig feed, biomethane, biodiesel, ethanol, and lactic acid (LA) [2]. The use of FW to produce ethanol can take full advantage of the rich nutrients in FW and obtain fuel. However, a large quantity of stillage will be produced; one way for dealing it is stillage recycle fermentation [3]. This method can reduce pollution and utilize proteins, amino acids, and various metal ions in stillage [4]. In our previous study, LA was determined to be the major accumulated by-product along with stillage recyling. A significant inhibition phenomenon was observed after 5 times recycling. Although the addition of calcium carbonate could alleviate the influence of LA on ethanol fermentation, if we can separate the accumulated LA in the stillage, it will provide better economic benefits. LA is an important natural organic acid. It has received increasing attention as one of the most important building blocks for the production of polylactic acid [5,6]. At present, the main methods of LA production are microbial fermentation and chemical synthesis. Biological fermentation is inexpensive, utilizes a wide range of raw materials, and produces optically pure L- or D-lactic acid, which make it superior to chemical synthesis. More attention has also been paid to the utilization of renewable carbohydrates as a carbon source for LA fermentation [7,8]. However, the major cost of LA production is due to its downstream separation and purification steps [9]. As LA exists in the form of lactate salt in the fermentation broth, acidification and removal of impurities are required for future separation [10]. The extraction methods of LA include precipitation, solution extraction, adsorption, distillation, and nanofiltration [11]. Electrodialysis has the advantages of diversity, sophisticated functions, and technological compatibility [12,13]. Danner et al. used monopolar electrodialysis to extract LA from grass silages and obtained press extraction yields of 31–96 g of lactate per kilogram of silage dry matter (g LA/kg DM) [14]. Chen used conventional electrodialysis (CED) to remove 80% lactate from acid whey [15]. Lopez-Garzon used bipolar membrane electrodialysis (BMED) to recycle LA and other carboxylic acids in the fermentation broth [16].

In recent years, forest breeding programs have increased Eucalyptus production for commercial purposes; however, high-performing Eucalyptus clones have had problems with propagation, especially when rooting. Nevertheless, studies have shown that inoculation of microorganisms producing indole-3- acetic acid (IAA) is an especially productive procedure to help these clones breed. In this study, we evaluated the production of IAA and analogues in 16 microbial strains. For the first time, a high IAA production was described in the strain Aspergillus flavipes (ATCC® 16814™), and A. flavipes was shown by LC-MS/MS to produce IAA through a tryptophan-dependent biosynthetic pathway. A. flavipes reached the highest IAA production when cultivated under solid-state fermentation in an optimized medium composed of soybean bran, water and tryptophan. We mixed the fermentation products in solid form (SF) and liquid form (LF) with the substrate Carolina I® and then planted the cuttings of the hybrid Eucalyptus grandis x E. urophylla (clone IPB2). In fact, treatments with 40e120 mg kg
1 of SF increased the adventitious rooting rate, root length and both root fresh and dry mass, while 120 mg kg
1 of LF increased root length and dry mass. Additionally, there was no toxicity on fibroblasts (NIH/3t3), and, therefore, the plant biostimulant was confirmed as a novel, non-toxic, and eco-friendly solution.

Introduction

Clonal plantation of Eucalyptus based on vegetative propagation is successful mainly due to well-developed forest breeding programs. However, vegetative propagation efficiency is prejudiced in some species due to specific endogenous and exogenous factors of the mother plant, alterations of the root system architecture and topophysis effects that influence the rooting potential (Peralta et al., 2012). The clone IPB2 of the hybrid Eucalyptus grandis x Eucalyptus urophylla from ArborGen is classified as a clone SuperTree®. Such clones are selected for a higher volume of wood, disease resistance, trunk straightness, and wood quality characteristics (ArborGen, 2018). Despite the advantages in the field, the clone has difficulties in rooting, demanding research to promote the adventitious roots induction and development. The inoculation of some classes of microorganisms can assist in overcoming this difficulty, stimulating adventitious root growth, increasing the absorption of nutrients and water, the host-plant biomass and the tolerance to stresses such as drought and diseases (Sukumar et al., 2013). When a substance can improve the plant nutritional efficiency and abiotic stress and/or influence the quality of the crop, regardless of its nutritional status, it is denominated as a plant biostimulant (du Jardin, 2015). The commercial products are currently divided into five classes: microbial inoculants, humic acids, fulvic acids, amino acids and seaweed extracts (Calvo et al., 2014). The commercial biostimulants can contain one or more microorganisms and/or substances (du Jardin, 2015).

Solvent recovery and reutilization in bioprocess have great potential to reduce production cost and environmental harm. However, solvent recovery process is often energy-intensive and costly. In this study, we developed an integrated bioprocess for fermentative production of protopanaxadiol (PPD) from ethanol waste recycled in down-stream extraction process, achieving solvent recovery and reutilization in a cost-effective manner. In this integrated process, PPD isolation and purification were achieved by foam separation and resin chromatography, respectively; ethanol solution used as PPD extractant and chromatography eluent was recycled to be reused directly as yeast carbon source for PPD biosynthesis. Notably, in 3 batches of 5 L-fermentation productions, the recycled ethanol could compensate 81.3% of the ethanol used in fermentation. Since the lost PPD during chromatography elution was returned into the next-batch fermentation together with the recycled ethanol, the overall yield of 3- batch production (85.78%) was higher than the yield of single batch production (~75%). This study demonstrates a promising integrated bioprocess for triterpene compounds production from ethanol waste.

Introduction

Protopanaxadiol (PPD), a major bioactive component of Panax ginseng, is regarded as promising high-value pharmaceutical compound for tumor inhibition and depression treatment (Li et al., 2006; Popovich and Kitts, 2004; Hui et al., 2012b). Traditional PPD production relies on phytoextraction that is followed by hydrolysis or enzymolysis, and is a labor-intensive and highly polluting process. Synthetic biology technology provides an alternative method for producing PPD in engineered Saccharomyces cerevisiae (S. cerevisiae) (Xu et al., 2013). In recent years, considerable progress has been achieved for engineering yeast for high production of PPD (Dai et al., 2013; Zhao et al., 2016, 2017). However, the down-stream bioprocess of PPD isolation and purification has not yet been studied. The down-stream bioprocess is known to be relatively solvent intensive. It has been reported that for the production of 1 kg of commercially available active pharmaceutical ingredients a median value of 45 kg of material needs to be used, of which about 50% has been organic solvents (Henderson et al., 2007). Recovery of waste solvent through crystallization or membrane separation can offer significant benefits with regards to reduced storage and waste costs as well as increased compliance with environmental legislation. However, these recovery processes are highly energy-intensive and costly (Constable et al., 2007). Therefore, it is of great interest to find a new way to reutilize the wasted organic solvent to be recycled during down-stream bioprocess.

تقاضای مصرف کنندگان برای محصولات طبیعی و نیاز به فرآیندهای سازگار با محیط زیست، توسعه فرایندهای نوآورانه را برای سنتز طعم از طریق بیوتکنولوژی توسعه می دهد. در این راستا، کشت مخلوط مخمر صنعتی برای تخمیر حالت جامد پوست پرتقال (OP) انتخاب و مورد بررسی قرار گرفت. برای این منظور، از اتوکلاو استریل OP برای حذف D -limonene استفاده شد و میکروفلور طبیعی با توجه به قابلیت انقباض مخمر، مصرف مواد مغذی و توانایی سلول برای تولید ترکیبات طعم دهنده مورد بررسی قرار گرفت. OP غیر استریل نیز برای پیگیری جوانب مثبت و منفی به فرآیند استریلیزاسیون استفاده شد. سلول های مخمر عملکرد رشد بهتری را در شرایط فرآیند استریلیزه ، نسبت به موارد غیر استریلیزه نشان دادند. در اولین مورد، سنتز پیشرفته استرهای “میوه ای” بعد از 72 ساعت به ترتیب (48.7، 25.2، 9.3، 6.3 و 4.5) میلی گرم بر کیلوگرم از OP برای تهیه ایزوآمیل استات، اتیل دودکانوات، دکانوات، اکتانوات و فنیل اتیل استات تخمیر نمودند. سلولهای مخمر، سنتز اتیل هگزانوات (mg / kg 154.24 کیلوگرم در 48 ساعت) را سرعت بخشیدند. در این فرآیند، ممکن است تبدیل زیستی ترکیبات طعم دهنده به وسیله مخمر در نظر گرفته شود. فرایند پیشنهادی منجر به بازدهی صنعتی بالای استرهای مهم فرار طعم (کل 250 ~ میلی گرم بر کیلوگرم OP) می شود که می تواند به یک پالاینده زیستی پایدار برای ارزش گذاری مواد زائد OP به کار گرفته شود.